**Meet the editor**

Professor E. M. Irusen, FCP(SA), Pulmonology, PhD, is a principal specialist and Clinical Head of the Pulmonology and Critical Care Division of the Department of Internal Medicine, Faculty of Health Sciences, University of Stellenbosch and Tygerberg Academic Hospital in Cape Town, South Africa. He is widely published and has tremendous experience in these speciality fields. His

PhD training in molecular immunology and biology related to asthmatic inflammation was undertaken under the supervision of Professors Fan Chung and Peter Barnes at the Royal Brompton Hospital and the Unit of Thoracic Medicine of the National Heart and Lung Institute at Imperial College. He has won numerous awards for his research, both local and international. He has served on the Council of SA Thoracic Society and is the immediate former president. He is currently the South African national representative in the European Respiratory Society, GOLD and on the international executive of the International COPD Coalition.

## Contents

#### **Preface XIII**

#### **Part 1 Airways Disease 1**


#### **Part 2 Neoplasia 127**

	- **Part 3 Immunity and Infection 297**

#### Chapter 19 **Inhibition of Adhesion and Invasion of**  *Pseudomonas aeruginosa* **to Lung Epithelial Cells: A Model of Cystic Fibrosis Infection 429**  Ayman M. Noreddin, Ghada Sawy, Walid Elkhatib, Ehab Noreddin and Atef Shibl

#### **Part 4 Paediatrics 441**

VI Contents

Chapter 9 **Neuroendocrine Tumours of the Lung 203**

Chapter 10 **Chemotherapy for Large Cell Neuroendocrine** 

Satoshi Yoda and Koichiro Asano

**of Small-Cell Lung Cancer 239**  Erkan Topkan and Cem Parlak

**for Pulmonary Malignancies 257** 

**in Mucosal Immunity of the Lung 299**  M.S. Paats, P.Th.W. van Hal, C.C. Baan, H.C. Hoogsteden, M.M. van der Eerden

Chapter 15 **Current Status of the Mollicute (Mycoplasma) Lung Disease:** 

Silvia Giono-Cerezo, Guadalupe Estrada-Gutiérrez, José Antonio Rivera-Tapia, Jorge Antonio Yáñez-Santos

Luz E. Cano, Ángel González, Damaris Lopera, Tonny W. Naranjo and Ángela Restrepo

Chapter 17 **Nocardia Infection in Lung Transplantation 393**  Pilar Morales, Ana Gil-Brusola and María Santos

**Pathogenesis, Diagnostics, Treatment and Prevention 331** 

**Clinical, Immunological and Histopathological Aspects 359**

**Past, Present and Future 275** Cristian Rapicetta, Sara Tenconi, Tommaso Ricchetti, Sally Maramotti

Chapter 11 **Radiation Therapy in Management** 

Chapter 12 **Lung Parenchyma Sparing Resection**

Chapter 13 **Surgery in Small-Cell Lung Cancer:** 

and Massimiliano Paci

**Part 3 Immunity and Infection 297** 

Chapter 14 **Interleukin-17 and T Helper 17 Cells**

and Francisco Javier Díaz-García

Chapter 16 **Pulmonary Paracoccidioidomycosis:**

Chapter 18 *In Vivo* **Models of Lung Disease 407** 

Tracey L. Bonfield

and R.W. Hendriks

Arpad Pereszlenyi

and L.M. Antón Aparicio

Guadalupe Aparicio Gallego, Vanessa Medina Villaamil,

**Carcinoma of the Lung: Should It Be Treated with the Same Strategy as Small Cell Lung Carcinoma? 231** Katsuhiko Naoki, Kenzo Soejima, Takashi Sato, Shinnosuke Ikemura, Hideki Terai, Ryosuke Satomi, Sohei Nakayama,

Ana Capdevila Puerta, Enrique Grande Pulido

	- **Part 5 Pulmonary Oedema: Cardiogenic and Non-Cardiogenic 523**

#### **Part 6 Miscellaneous 579**


#### Chapter 29 **The Pneumoconioses 625**  Nlandu Roger Ngatu, Ntumba Jean-Marie Kayembe, Benjamin Longo-Mbenza and Narufumi Suganuma


Ho Sung Lee, Jae Sung Choi, Ki Hyun Seo, Ju Ock Na, Yong Hoon Kim, Mi Hye Oh and Sung Shick Jou

## Preface

The developments in molecular medicine are leading to important insights with regard to lung diseases. Leading clinicians and scientists in the world have brought their considerable knowledge and experience, and focused research in their contributions to this book. Clinicians and researchers will learn about the most recent advances in a variety of lung diseases that will enable them to better understand respiratory disorders. The book presents state-of-the-art essays on airways disease, neoplastic diseases, and pediatric respiratory conditions. Additionally, aspects of immune regulation, respiratory infections, acute lung injury/ ARDS, pulmonary oedema, functional evaluation in respiratory disorders, and a variety of other conditions are also expounded upon. The compilation and availability of the book online has also ensured that information is as current as possible, which is an advantage over the printed editions.

The book will be invaluable for clinicians who appreciate the pathogenetic basis and current concepts in lung diseases and improve their diagnostic and therapeutic skills. Scientists will also gain something from the detailed applied molecular techniques and descriptions that could potentially open a plethora of new research avenues for exploration.

> **Professor E.M. Irusen** Department of Medicine, Faculty of Health Sciences, University of Stellenbosch, Western Cape Province, South Africa

**Part 1** 

**Airways Disease** 

## **Airway Smooth Muscle in Asthma Symptoms: Culprit but Maybe Innocent**

Ynuk Bossé1, Peter D. Paré1 and Yohan Bossé2 *1James Hogg Research Center, University of British Columbia, Vancouver 2Institut Universitaire de Cardiologie et de Pneumologie de Québec and Department of Molecular Medicine, Laval University, Quebec City Canada* 

## **1. Introduction**

The main function of smooth muscle found in either the airways or in other hollow organs is to contract. Once stimulated to contract, the smooth muscle strives to shorten. In turn, smooth muscle shortening narrows the lumen of the organ it surrounds. Contraction usually serves a physiological purpose, such as increasing arterial tension for vascular smooth muscle, micturition for the detrusor muscle or parturition for the uterus muscle. However, for the airway smooth muscle (ASM), the shortening narrows the airway lumen, which concomitantly increases the resistance to airflow. So it seems that every time ASM manifests its function it causes respiratory distress. This had raised the question of whether its existence is the problem (65, 162)?

One common respiratory disorder in which the symptoms are greatly engendered by ASM contraction is asthma. In fact, a proper asthma diagnosis involved testing the reversibility of airway obstruction with a bronchodilator, a drug relaxing ASM (usually a 2-adrenoceptor agonist). A positive test is indicated by complete or partial reversibility, which simultaneously confirms the implication of ASM shortening in asthma symptoms. However, we know that a judge will never blame the gun for a murder. She/he would rather blame the assassin that pulled the trigger. Thus, the ASM could simply be an obeisant effector tissue that responds to external cues that are asking it to contract. So despite being culprit in the elaboration of asthma symptoms it may still be 'innocent'.

Hence, for asthma symptoms (at least the one mediated by airway narrowing) to be provoked, contractile stimuli need to be present. There is no doubt about the increased expression of spasmogens (i.e., contractile agonists) into asthmatic airways. Histamine (251), leukotrienes (125), endothelin-1 (148), prostaglandin D2 (170), thromboxane A2 (249), adenosine (61), bradykinin (136), anaphylatoxin C3a and C5a (121), substance P (231) and others, are all inflammatory mediators capable of stimulating ASM contraction and were all shown to be overexpressed in asthmatic lungs. These spasmogens are secreted/synthesized following exposure to environmental asthma triggers, such as allergens, viruses, bacteria, fungi, air pollutants, exercise, aspirin and/or cold dry air. The nature of the environmental trigger involved obviously varies among asthmatics but all of them ultimately lead to a type of airway inflammation with inflammation-derived spasmogens. However, not everyone who gets inflammation into her/his airways because of a cold, because they are exposed to an allergen to which they are sensitized (atopic), because they do exercise, etc… gets asthma symptoms. So it might not be enough to have airway inflammation to get asthma symptoms. If you try to kill a moose with a pellet gun, you may have the gun and you may pull the trigger, but you are more likely to have no responsiveness. Saying that it may also be necessary to be responsive, or maybe hyperresponsive, to these inflammation-derived spasmogens to get asthma symptoms.

In fact, one of the pathognomonic feature of asthma is airway hyperresponsiveness (AHR). AHR is defined as an increased sensitivity and maximal narrowing in response to an inhalational challenge with a spasmogen (methacholine is the most commonly used). Whether AHR is a prerequisite to suffer from asthma or whether asthma is the cause of AHR is a contemporaneous debate and will be slightly addressed in this chapter. Some can argue that asthma can affect the degree of airway responsiveness and others would be just as right by arguing that AHR is a predisposing factor to be diagnosed with asthma. What is clear is that the degree of responsiveness is a good surrogate for the airway narrowing that will take place *in vivo* in response to endogenously produced spasmogens that are released either in normal state or during asthma exacerbation.

In a syndrome like asthma, understanding the factors involved in AHR may give important clues concerning the pathogenesis of asthma and the generation of asthmatic symptoms. As aforementioned, this is because the symptoms of asthma are caused, to a great extent, by airway narrowing induced by ASM shortening. Due to its unequivocal role in airway responsiveness, it is clear that the ASM plays an important role in AHR; without responsiveness there would be no hyperresponsiveness. However, whether the ASM is intrinsically different in asthma and responsible for AHR is still unclear. Several ASM dysfunctions, but also many other defects in non-muscle factors have been suggested to play a role in the manifestation of AHR. Whether these defects are genetically inherited or acquired as a result of disease processes is also a question of great interest. This chapter is an attempt to outline the current state of comprehension regarding the alterations in muscle and non-muscle factors that may contribute to the hyperresponsive phenotype seen in asthmatics.

## **2. Muscle factors**

Studying ASM mechanics involves more than measuring its force-generating capacity. Many other ASM contractile properties may play a role in determining the degree of airway responsiveness *in vivo*, such as shortening amount and velocity, stiffness, ability to relax and to tolerate and/or recover from the decline in contractility induced by length perturbations. The term 'contractility' in this chapter is vague and refers to any contractile properties. So a hypercontractile ASM phenotype can mean one or all of the following: the muscle is stronger (increased force-generating capacity), it shortens more and/or shortens faster, it is stiffer, it has an attenuated ability to relax either spontaneously or in response to bronchodilators, or has an increased ability to tolerate and/or to recover from a drop in contractility caused by length perturbations. In the following section, these contractile properties are discussed individually and the rationale for their respective involvement in determining the degree of airway responsiveness *in vivo* is described. The published evidences suggesting that alterations to some of these contractile properties contribute to AHR in asthma are also briefly reviewed. The premise, here, is that AHR would be due to an inherited ASM hypercontractility; not one that would be acquired due to defects in nonmuscle factors. Some of the factors discussed were addressed in a previous review (24).

It is worth-mentionning that the ASM has also been shown to proliferate, to migrate, to express adhesion molecules and receptors interacting with immune cells, as well as to synthesize extracellular matrix components, cytokines and chemokines. Most of these ASM functions were studied in monolayers of ASM cells in culture. More evidences are eagerly needed to confirm the existence of these ASM functions *in vivo*. However, if they happen *in vivo*, their relevance to asthma pathogenesis is unquestionable. These subjects have been reviewed lately and will not be addressed in the present chapter (18, 50, 72).

#### **2.1 Force**

4 Lung Diseases – Selected State of the Art Reviews

an allergen to which they are sensitized (atopic), because they do exercise, etc… gets asthma symptoms. So it might not be enough to have airway inflammation to get asthma symptoms. If you try to kill a moose with a pellet gun, you may have the gun and you may pull the trigger, but you are more likely to have no responsiveness. Saying that it may also be necessary to be responsive, or maybe hyperresponsive, to these inflammation-derived

In fact, one of the pathognomonic feature of asthma is airway hyperresponsiveness (AHR). AHR is defined as an increased sensitivity and maximal narrowing in response to an inhalational challenge with a spasmogen (methacholine is the most commonly used). Whether AHR is a prerequisite to suffer from asthma or whether asthma is the cause of AHR is a contemporaneous debate and will be slightly addressed in this chapter. Some can argue that asthma can affect the degree of airway responsiveness and others would be just as right by arguing that AHR is a predisposing factor to be diagnosed with asthma. What is clear is that the degree of responsiveness is a good surrogate for the airway narrowing that will take place *in vivo* in response to endogenously produced spasmogens that are released

In a syndrome like asthma, understanding the factors involved in AHR may give important clues concerning the pathogenesis of asthma and the generation of asthmatic symptoms. As aforementioned, this is because the symptoms of asthma are caused, to a great extent, by airway narrowing induced by ASM shortening. Due to its unequivocal role in airway responsiveness, it is clear that the ASM plays an important role in AHR; without responsiveness there would be no hyperresponsiveness. However, whether the ASM is intrinsically different in asthma and responsible for AHR is still unclear. Several ASM dysfunctions, but also many other defects in non-muscle factors have been suggested to play a role in the manifestation of AHR. Whether these defects are genetically inherited or acquired as a result of disease processes is also a question of great interest. This chapter is an attempt to outline the current state of comprehension regarding the alterations in muscle and non-muscle

Studying ASM mechanics involves more than measuring its force-generating capacity. Many other ASM contractile properties may play a role in determining the degree of airway responsiveness *in vivo*, such as shortening amount and velocity, stiffness, ability to relax and to tolerate and/or recover from the decline in contractility induced by length perturbations. The term 'contractility' in this chapter is vague and refers to any contractile properties. So a hypercontractile ASM phenotype can mean one or all of the following: the muscle is stronger (increased force-generating capacity), it shortens more and/or shortens faster, it is stiffer, it has an attenuated ability to relax either spontaneously or in response to bronchodilators, or has an increased ability to tolerate and/or to recover from a drop in contractility caused by length perturbations. In the following section, these contractile properties are discussed individually and the rationale for their respective involvement in determining the degree of airway responsiveness *in vivo* is described. The published evidences suggesting that alterations to some of these contractile properties contribute to AHR in asthma are also briefly reviewed. The premise, here, is that AHR would be due to an inherited ASM hypercontractility; not one that would be acquired due to defects in nonmuscle factors. Some of the factors discussed were addressed in a previous review (24).

factors that may contribute to the hyperresponsive phenotype seen in asthmatics.

spasmogens to get asthma symptoms.

**2. Muscle factors** 

either in normal state or during asthma exacerbation.

The load impeding ASM shortening is auxotonic; i.e., it increases progressively as the muscle shortens. It is thus logical that greater force would lead to more shortening and concomitantly more airway narrowing. That is the reason why the force-generating capacity of ASM is such an important determinant of airway responsiveness.

The force-generating capacity also matters because it influences other ASM contractile properties. The relationship between the load and the velocity can be fitted with an exponential decay equation; so that increasing the load decreases the shortening velocity exponentially. This implies that a stronger muscle would counteract a given load faster and would thus shorten faster. In a context where the muscle is subjected to contract under a progressively increasing load, as it occurs *in vivo*, a stronger muscle would also shorten further. This is because a muscle able to produce more force at any given length would allow the shortening to progress further before reaching a load equal to its force. A stronger muscle would also increase ASM stiffness, which, as discussed below (subsection 2.2), can have an important impact on *in vivo* airway responsiveness.

The force or stress, which is the force per cross-sectional area, produced by the ASM depends on the potency and the concentration of the contractile stimulus involved. The relationship between spasmogen concentration and ASM-force can be described by a sigmoidal equation. So, *in vivo*, the amount of spasmogen reaching the ASM is one of the main determinants of the force produced by the muscle. The force produced by the ASM is also dictated by its length. Longer muscle generally generates more force in response to a given contractile stimulus (86, 154, 259, 261). In fact, the decrease in ASM-force caused by length reduction is proportional to he magnitude of the length change (103). Hence, *in situ* factors affecting the operating length of the ASM can be of considerable importance in the understanding of AHR, but that will be discussed later in this chapter (subsection 4.1.5).

Regardless of the aforementioned factors, the force can also be determined by the muscle's intrinsic capacity to generate force. So for a given concentration of a chosen spasmogen and a given length, the stress produced by the muscle can be different. This has led some to suggest that ASM derived from asthmatics may produce more stress than ASM derived from non-asthmatics, and that might be the cause of AHR. This hypothesis has been tested by several groups now and, although still debatable, the bulk of evidence suggests that the stress-generating capacity of asthmatic and non-asthmatic ASM is the same (reviewed in (153)). Taken together, the force-generating capacity of the ASM is certainly an important determinant of airway responsiveness, but no data published thus far convincingly demonstrate that this contractile property is altered in asthma.

#### **2.2 Stiffness**

In the field of ASM the term 'stiffness' certainly has different connotations. By definition stiffness is the amount of force required to cause a given change in length. The stiffness of ASM can be either passive or active depending on whether the resistance to stretch stems from relaxed or activated components of ASM, respectively. Passive stiffness relies predominantly on the cell cytoskeleton. In fact, ablation of the cytoskeleton protein vimentin was shown to reduce passive stiffness by 3-fold (243). However, it came to experts' attention that the ASM is relatively compliant. The resting tension observed along the range of *in situ* operating lengths, even when the ASM is stretched to a level comparable to the one observed in a lung inflated to total lung capacity (TLC), is almost neglectable. On the other hand, the amount of tension generated by the same stretch in the presence of an active tone (i.e., in the presence of spasmogens) is disproportionally greater. For this reason, we focus here on ASM active stiffness, since it would be the principal component affecting airway responsiveness. Passive stiffness can also have a broader connotation if one considers the other components of the airway wall and the extracellular matrix (ECM) surrounding the ASM cells. These other passive elements obviously impact the overall stiffness of the airway wall and will be addressed later in this chapter (subsections 4.1.4 and 4.1.6).

Active stiffness is related to the level of ASM activation by spasmogenic stimuli. Its magnitude has always been thought to be dictated by the number of myosin heads bound to the actin filaments (i.e., number of cross-bridges). However, an emerging field in ASM suggests that other factors might play a part in active siffness. That is the level of interconnectivity between the ECM, the plasma membrane and the cell's cytoskeleton. These points of junction are excessively important for mechanotransduction efficiency; i.e., the transfer of individual resistive forces to the overall stiffness of the tissue during a stretch. Once considered as passive, recent studies rather suggested that the structures responsible for this interconnectivity can rearrange extensively upon ASM cell activation (reviewed in (264)). For example, Zhang and coworkers (263) have shown that -actinin translocates to the plasmalemma and binds to 1-integrin early after acetylcholine (ACh) activation, and blocking this interaction reduces active tension in response to ACh (263). This and other recent findings (105, 188, 265) confirmed that this interconnectivity between intracellular proteins, the plasma membrane proteins (integrins) and the ECM relies on dynamic processes that are activated by spasmogens. We shall henceforth consider this level of interconnectivity as an integral part of the active stiffness component of ASM.

ASM stiffness has captured the eyes of many scientists in the field recently because of its influence on airway responsiveness. To understand the link between stiffness and airway responsiveness, it is important to emphasize that ASM operates in a dynamic environment. The swings in transpulmonary pressure required for ventilation cause oscillating stresses on the airway wall, which, in turn, cause continuous variations of airway caliber. The effects of these oscillating strains (a strain is a change in length caused by a change in stress) on ASM mechanics are not small (Fredberg). In fact, in was predicted that the bronchodilating effect of oscillating strains at an amplitude that is thought to prevail *in vivo* due to tidal breathing is just as potent as high concentrations of a 2-adrenoceptor agonist (isoproterenol) to reverse airway constriction induced by different contractile stimuli (84). In fact, a single stretch of the airway wall at greater amplitude seems to be sufficient. Stretching the airway wall by taking a deep inspiration (DI) has been recognized as a powerful way to induce bronchodilatation and bronchoprotection (171, 222). Inversely, refraining from taking regular DIs has been shown to increase AHR in normal (i.e., non-asthmatic) subjects (117, 118, 221). In the same vein, cessation of tidal breathing during breath-holding caused a decrease in tracheal and central bronchial diameter (165). This later observation suggests that the bronchodilating effect of breathing is omnipresent *in vivo,* and that removing the

ASM can be either passive or active depending on whether the resistance to stretch stems from relaxed or activated components of ASM, respectively. Passive stiffness relies predominantly on the cell cytoskeleton. In fact, ablation of the cytoskeleton protein vimentin was shown to reduce passive stiffness by 3-fold (243). However, it came to experts' attention that the ASM is relatively compliant. The resting tension observed along the range of *in situ* operating lengths, even when the ASM is stretched to a level comparable to the one observed in a lung inflated to total lung capacity (TLC), is almost neglectable. On the other hand, the amount of tension generated by the same stretch in the presence of an active tone (i.e., in the presence of spasmogens) is disproportionally greater. For this reason, we focus here on ASM active stiffness, since it would be the principal component affecting airway responsiveness. Passive stiffness can also have a broader connotation if one considers the other components of the airway wall and the extracellular matrix (ECM) surrounding the ASM cells. These other passive elements obviously impact the overall stiffness of the airway

Active stiffness is related to the level of ASM activation by spasmogenic stimuli. Its magnitude has always been thought to be dictated by the number of myosin heads bound to the actin filaments (i.e., number of cross-bridges). However, an emerging field in ASM suggests that other factors might play a part in active siffness. That is the level of interconnectivity between the ECM, the plasma membrane and the cell's cytoskeleton. These points of junction are excessively important for mechanotransduction efficiency; i.e., the transfer of individual resistive forces to the overall stiffness of the tissue during a stretch. Once considered as passive, recent studies rather suggested that the structures responsible for this interconnectivity can rearrange extensively upon ASM cell activation (reviewed in (264)). For example, Zhang and coworkers (263) have shown that -actinin translocates to the plasmalemma and binds to 1-integrin early after acetylcholine (ACh) activation, and blocking this interaction reduces active tension in response to ACh (263). This and other recent findings (105, 188, 265) confirmed that this interconnectivity between intracellular proteins, the plasma membrane proteins (integrins) and the ECM relies on dynamic processes that are activated by spasmogens. We shall henceforth consider this level of

wall and will be addressed later in this chapter (subsections 4.1.4 and 4.1.6).

interconnectivity as an integral part of the active stiffness component of ASM.

ASM stiffness has captured the eyes of many scientists in the field recently because of its influence on airway responsiveness. To understand the link between stiffness and airway responsiveness, it is important to emphasize that ASM operates in a dynamic environment. The swings in transpulmonary pressure required for ventilation cause oscillating stresses on the airway wall, which, in turn, cause continuous variations of airway caliber. The effects of these oscillating strains (a strain is a change in length caused by a change in stress) on ASM mechanics are not small (Fredberg). In fact, in was predicted that the bronchodilating effect of oscillating strains at an amplitude that is thought to prevail *in vivo* due to tidal breathing is just as potent as high concentrations of a 2-adrenoceptor agonist (isoproterenol) to reverse airway constriction induced by different contractile stimuli (84). In fact, a single stretch of the airway wall at greater amplitude seems to be sufficient. Stretching the airway wall by taking a deep inspiration (DI) has been recognized as a powerful way to induce bronchodilatation and bronchoprotection (171, 222). Inversely, refraining from taking regular DIs has been shown to increase AHR in normal (i.e., non-asthmatic) subjects (117, 118, 221). In the same vein, cessation of tidal breathing during breath-holding caused a decrease in tracheal and central bronchial diameter (165). This later observation suggests that the bronchodilating effect of breathing is omnipresent *in vivo,* and that removing the oscillating airway wall strains caused by breathing allows the baseline level of ASM activation (tone) to constrict the airways. Finally, breathing at low lung volume has been shown to increase airway responsiveness (56), suggesting that not only the presence but also the amplitude of the airway wall strains induced by breathing impacts on the subsequent degree of airway narrowing provoked by a given spasmogenic challenge. Therefore, identifying the factors decreasing airway lumen expansion during a DI, or the factors limiting fluctuating strains of the airway wall during tidal breathing, such as passive and active ASM stiffness, are relevant to the understanding of airway narrowing and AHR.

The decrease in airway responsiveness induced by a DI has also been mimicked *ex vivo* on isolated porcine (178) and human (177) airways. In these studies, the liquid-filled airways were subjected to luminal volume changes reproducing the changes in transmural pressure occurring during tidal breathing (from 5 to 10 cmH2O) and DIs (from 5 to 30 cmH2O). The authors showed that the presence of DI simulations reduces the active pressure or the decrease in luminal volume produced by ASM in response to different concentrations of ACh.

Oscillations of airway caliber by breathing maneuvers seem to have a bronchodilating effect because they stretch the ASM. It was estimated that tidal breathing, sigh and DI, stretch the relaxed ASM by 4, 12 and 25% of its initial length, respectively (66). This is probably an overestimation since it was calculated based on changes in lung volume occurring during these breathing maneuvers while considering the lungs as isotropic material. Nevertheless, these length oscillations are known to decrease ASM stiffness, even during supra-maximal activation with ACh (66, 84, 128, 198). Importantly, the maximal force-generating capacity of ASM during or immediately following these oscillations is also reduced (66, 84, 85, 128, 186, 198, 242). The decline in isometric force that ensues length oscillations has been shown to be proportional to the amplitude and the duration of the stretch, but not to the frequency (242). These results demonstrated that the force produced by ASM in response to a given stimulus is greater in a static environment than a dynamic environment.

Oscillating strains at amplitude that is thought to prevail *in vivo* during breathing maneuvers also caused elongation of the contracted muscle (128, 130, 160). This later phenomenon is now referred to as force fluctuation-induced relengthening (FFIR). It also occurred in experimental settings more closely mimicking the *in situ* environment, where the ASM was subjected to an auxotonic load (186). Therefore, oscillating strains not only reduce the stiffness and the force-generating capacity of ASM in response to a given stimulus but also cause ASM relengthening. *In vivo*, this ASM relengthening will be translated into airway dilatation. The mechanisms underlying FFIR are not well understood but the length of the actin filaments seems to play a role (160).

Collectively, these studies have shown that the force (66, 84, 85, 128, 186, 198, 242) and the stiffness (66, 84, 128, 198) of ASM, as well as the length of the contracted ASM (128, 130, 160, 186), are affected by length oscillations (66, 84, 85, 128, 130, 160, 186, 198, 242) or simply by an acute stretch (242). Considering these phenomena together, one can envision the following *in vivo* vicious cycle. With exposure to spasmogens and the development of stiffer ASM, the same fluctuating stresses of breathing will cause less airway wall strains. This will allow the muscle to operate in a more static environment, where it will be able to produce more force and will be subjected to less FFIR. By producing more force, the muscle becomes stiffer, which further decreases the airway wall strains induced by breathing, and so on. Because the load impeding muscle shortening increases with the amount of airway narrowing, ASM shortening will eventually stop (when the force generated by the muscle is equal to the load opposing its contraction). However, the repetitive sequence of events described above allows greater ASM shortening and consequently greater airway narrowing for any level of ASM activation. The link between stiffer ASM and AHR is thus indirect and explained by the fact that stiffening of ASM reduces the magnitude of airway wall strains (i.e., ASM stretches) caused by breathing maneuvers. In conjunction, these studies also suggest that the bronchodilating and bronchoprotective effect of breathing maneuvers seen *in vivo* may be due to he fact that ASM contractile properties are malleable and affected by straining forces.

Having said that, the bronchodilating effect of tidal breathing does not make unanimity (reviewed in (176)). In systems that more closely imitate the *in vivo* situation, such as in a liquid-filled airway segment subjected to transmural pressure oscillations mimicking the swings in transpulmonary pressure occurring *in vivo* due to breathing maneuvers, only the DI (but not tidal breathing) was shown to attenuate the increase in pressure (178) or the reduction in luminal volume (177) caused by ACh stimulation. Nevertheless, it will be important to determine whether the stiffness of the ASM is different between asthmatics and nonasthmatics. So far, the only evidence to support the conjecture that an increased ASM stiffness causes AHR comes from a study using animal cells. The ASM cells derived from the inherently hyperresponsive Fisher rats were shown to exhibit a higher stiffening response to a panel of spasmogens compared to the cells derived from the hyporesponsive Lewis rats (6).

#### **2.3 Tolerance to oscillating stretches and rate of recovery following length perturbations**

In the previous subsection, we have seen that length perturbations can greatly affect ASM contractility. From now on, by length perturbations we meant any of the following: a length change, either elongation or length reduction; a single stretch or release with an immediate return to the initial length; oscillating strains (length oscillations); or oscillating stresses (force oscillations) that is sufficient to modulate ASM length. The ability to tolerate and recover from these length perturbations are thus important ASM contractile properties that may influence the degree of airway responsiveness. The ASM's ability to maintain its forcegenerating capacity during shortening could also be important in determining the degree of airway narrowing. It is well-known that the ASM becomes 'weaker' as it shortens. So that the instantaneous capacity to produce force during (or immediately after a) length reduction is inversely proportional to the magnitude of the length change (103). This suggests that the force-generating capacity of ASM at shorter lengths is an important factor determining the extent of airway narrowing; simply because it dictates the remaining force available to counteract the loads which limit further airway narrowing at these new shorter lengths. There is currently no data comparing the decline in force caused by given reductions of ASM length between asthmatics and non-asthmatics.

Since we just came to realize the potentially important role of these contractile properties in airway responsiveness, it is not surprising that not enough comparisons were made between asthmatic and non-asthmatic tissues. The only evidence that we are aware of comes from our group (Leslie *et al*., accepted in the European Respiratory Journal). In that study, tracheal ASM strips derived from asthmatics and non-asthmatics were isolated and their ability to tolerate length perturbations and to recover from them was studied *ex vivo*. We found that the decline in force caused by length perturbations was attenuated in asthmatic tissues. The length perturbations used were a 60% length oscillations for 10 min, which are way beyond the length changes that would occur *in vivo*. The physiologic meaning of this finding may thus be

equal to the load opposing its contraction). However, the repetitive sequence of events described above allows greater ASM shortening and consequently greater airway narrowing for any level of ASM activation. The link between stiffer ASM and AHR is thus indirect and explained by the fact that stiffening of ASM reduces the magnitude of airway wall strains (i.e., ASM stretches) caused by breathing maneuvers. In conjunction, these studies also suggest that the bronchodilating and bronchoprotective effect of breathing maneuvers seen *in vivo* may be due to he fact that ASM contractile properties are malleable and affected by

Having said that, the bronchodilating effect of tidal breathing does not make unanimity (reviewed in (176)). In systems that more closely imitate the *in vivo* situation, such as in a liquid-filled airway segment subjected to transmural pressure oscillations mimicking the swings in transpulmonary pressure occurring *in vivo* due to breathing maneuvers, only the DI (but not tidal breathing) was shown to attenuate the increase in pressure (178) or the reduction in luminal volume (177) caused by ACh stimulation. Nevertheless, it will be important to determine whether the stiffness of the ASM is different between asthmatics and nonasthmatics. So far, the only evidence to support the conjecture that an increased ASM stiffness causes AHR comes from a study using animal cells. The ASM cells derived from the inherently hyperresponsive Fisher rats were shown to exhibit a higher stiffening response to a panel of

spasmogens compared to the cells derived from the hyporesponsive Lewis rats (6).

**2.3 Tolerance to oscillating stretches and rate of recovery following length** 

ASM length between asthmatics and non-asthmatics.

In the previous subsection, we have seen that length perturbations can greatly affect ASM contractility. From now on, by length perturbations we meant any of the following: a length change, either elongation or length reduction; a single stretch or release with an immediate return to the initial length; oscillating strains (length oscillations); or oscillating stresses (force oscillations) that is sufficient to modulate ASM length. The ability to tolerate and recover from these length perturbations are thus important ASM contractile properties that may influence the degree of airway responsiveness. The ASM's ability to maintain its forcegenerating capacity during shortening could also be important in determining the degree of airway narrowing. It is well-known that the ASM becomes 'weaker' as it shortens. So that the instantaneous capacity to produce force during (or immediately after a) length reduction is inversely proportional to the magnitude of the length change (103). This suggests that the force-generating capacity of ASM at shorter lengths is an important factor determining the extent of airway narrowing; simply because it dictates the remaining force available to counteract the loads which limit further airway narrowing at these new shorter lengths. There is currently no data comparing the decline in force caused by given reductions of

Since we just came to realize the potentially important role of these contractile properties in airway responsiveness, it is not surprising that not enough comparisons were made between asthmatic and non-asthmatic tissues. The only evidence that we are aware of comes from our group (Leslie *et al*., accepted in the European Respiratory Journal). In that study, tracheal ASM strips derived from asthmatics and non-asthmatics were isolated and their ability to tolerate length perturbations and to recover from them was studied *ex vivo*. We found that the decline in force caused by length perturbations was attenuated in asthmatic tissues. The length perturbations used were a 60% length oscillations for 10 min, which are way beyond the length changes that would occur *in vivo*. The physiologic meaning of this finding may thus be

straining forces.

**perturbations** 

questioned. However, since all tissues were exposed to the same oscillating strains, it still means that there is an intrinsic difference between asthmatic and non-asthmatic ASM tissues in their ability to tolerate length oscillations. Interestingly, other ASM contractile properties were also compared in that study, such as the stress-generating capacity in response to electrical field stimulation (EFS), the velocity of shortening, the amount of shortening and the ability to relax. Among all the contractile properties tested, only the ability to tolerate length oscillations was clearly different between asthmatic and non-asthmatic ASM. These results suggested that the influence of disparate ASM in determining the different degree of airway responsiveness observed between asthmatics and non-asthmatics would only be manifested in certain circumstances… such as in a human that is breathing?? These results would need to be confirmed by other investigators.

The speed and the extent of force recovery following an initial decline in force induced by length perturbations could also contribute to the manifestation of AHR. In the aforementioned study using human tracheal ASM strips (Leslie *et al*., accepted in the European Respiratory Journal), no difference in the rate and extent of recovery was observed between asthmatic and non-asthmatic tissues. Since the decline in force induced by length oscillations was greater in non-asthmatic ASM, this implies that the force produced by the non-asthmatic ASM was lower during the entire recovery period (which was measured for 30 min). Also worth-mentioning is that this time period was sufficient for the asthmatic ASM to come back to its force before oscillations, which was not the case for non-asthmatic tissues. Other studies have used animal models to study the recovery of ASM-force following length oscillations. Wang and coworkers (241) measured the effect of length oscillations on isometric force-generating capacity of tracheal ASM strips from guinea pigs of different age groups. The force was assessed before and immediately after length oscillations, as well as at 6-min intervals thereafter to follow both the change and the recovery of force following oscillations. All age groups showed a similar decline in force immediately following length oscillations. However, whereas the force produced by tissues from older animals (3 week-old and adult) recovered to pre-oscillations levels over a time course of ~30 min, the force produced by the tissues from the youngest animals (1 week-old) rapidly rose above baseline (i.e., force before oscillations) and remained at this higher value for the entire time-window over which force recovery was measured. The increase in force over baseline induced by length oscillations was called 'force potentiation'. The molecular mechanisms underlying force potentiation are not well understood, but differential synthesis of prostaglandins seems to explain this age-dependent phenomenon in the guinea pig (44)).

A phenomenon closely related to force potentiation, which was dubbed the 'myogenic response', has also been suggested as a possible contributor to AHR in humans. Marthan and Woolcock (144) studied asthmatic patients in whom a DI induced a decrease in specific airway conductance. As discussed earlier, this paradoxical response is not uncommon in severe asthmatics and is an indicator of marked AHR (134). They found that nifedipine, a Ltype calcium channel blocker, prevented the decrease in specific airway conductance induced by the DI (144). They suggested that the stretch of the ASM caused by the DI provoked a calcium-dependent bronchoconstriction (myogenic response).

Taken together, it seems clear that the tolerance to the decrease in contractility induced by length perturbations and the ability to recover from them may play a role in determining the degree of airway responsiveness. However, more data are warranted to confirm that these contractile properties can discriminate normo-responsiveness from AHR. Studying these contractile properties also unveiled other phenomena, such as force potentiation and the myogenic response, which can also be significant in the understanding of AHR.

#### **2.4 Amount of shortening and velocity of shortening**

The amount of ASM shortening is of major importance because it ultimately determines the amount of airway narrowing. As discussed earlier, the amount (as well as the velocity) of shortening depends on the ASM-force relative to the load. Therefore, all the factors influencing ASM-force, such as the potency and the concentration of the contractile stimulus involved, the quantity of spasmogens reaching the ASM, the muscle's intrinsic capacity to generate force, and its length, as well as all the factors influencing the load impeding muscle shortening affect the amount and velocity of shortening. However, the intrinsic ability to shorten may also be different between asthmatics and non-asthmatics. So that under the same load and despite producing the same stress, the amount of shortening achieved may be different. Interestingly, this has been shown in isolated ASM cells (140). In that study, the authors showed that unloaded ASM cells derived from asthmatics shorten more at room temperature in response to EFS. However, this observation, which now has a decade old, still awaits confirmation. The underlying mechanisms involved are also unclear but decreased resistance to shortening due to reduction in either internal resistive load (214, 226) or stiffness (44) has been proposed.

The shortening velocity of ASM could also be a critical determinant of the amount of airway narrowing. Again, to comprehend the potential implication of ASM shortening velocity in determining the degree of airway responsiveness, it is important to understand that ASM operates in a dynamic environment. The load impeding its shortening is continuously fluctuating due to swings in transpulmonary pressure caused by ventilatory maneuvers (e.g., tidal breathing and DI). The amount of ASM shortening *in vivo* is determined by a balance between the rate of cross-bridge cycling on the actin filaments causing muscle shortening versus the rate and the magnitude of stretch-induced disruption of cross-bridges causing muscle elongation (67). A faster cycling rate of the cross-bridges with a commensurate increased velocity of shortening would lead to more shortening during exhalation, when the load opposing muscle shortening is lowering. A faster rate of cycling would also lead to more cross-bridges being attached at the end of expiration, rendering the ASM and the entire airway wall stiffer. In turn, the stiffer airway wall would be less vulnerable to the stress imposed by the subsequent inspiration; i.e., the airway wall would be exposed to the same stress of breathing but the strain of the airway wall and, consequently, the stretch of the ASM would be attenuated in an airway with stiffer ASM. The combination of more shortening and more cross-bridge attachments during exhalation with less stretch and less cross-bridge detachments during inhalation means that the ASM with an increased shortening velocity would eventually reach a new equilibrium where the size of the airway lumen would be smaller than with a slower ASM. In addition, the airway wall that has reached this equilibrium becomes more static. Since ASM operating in a static environment produces more force in response to the same stimulus (as discussed above), it is possible that a faster muscle would not only cause more shortening and more crossbridges during exhalation, but would also becomes stronger (i.e. able to produce more force for the same level of activation). By acquiring more force the ASM would then be able to narrow the airway further during the next exhalation and… the cycle can perpetuate itself. This vicious cycle is likely to happen in airways possessing ASM with faster shortening velocity and this is the rationale behind the idea that the speed of ASM contraction could be the cause of AHR.

Experimental evidences exist to support the hypothesis that a faster ASM velocity of shortening can contribute to AHR. The velocity of ASM shortening was shown to be greater in animal models in which there is innate AHR (reviewed in (132)). Similarly, there is greater maximal shortening velocity in human ASM cells derived from asthmatics (140). Whether this increased velocity of shortening is innate or acquired due to asthma in humans remains to be determined. Two mechanisms have been suggested to be responsible for the observed increased velocity of shortening in asthmatic ASM: 1-The increased expression of myosin light chain kinase (MLCK) (3, 16, 133); and 2-a preponderant expression of the faster cycling smooth muscle myosin heavy chain (smMHC) isoform B over the slower cycling smMHC isoform A (reviewed in (132)).

MLCK is a enzyme capable of phosphorylating the regulatory myosin light chain (rMLC), which is a necessary step required for actin-activation of myosin ATPase activity and the subsequent binding and pivotal of the cross-bridges on the actin filaments. The rationale is that faster rMLC phosphorylation caused by the increased amount of MLCK would lead to the activation of more cross-bridges and a faster onset and velocity of shortening at early timepoints following ASM stimulation. However, increased expression of MLCK in asthma is not a unanimous finding (147, 256). An alternate explanation for the increased velocity of shortening of asthmatic ASM is a differential expression of smMHC isoforms. The so called B isoform (also called the (+) insert isoform because of the presence of a 7-amino acid insert in the loop 1 of the protein) shows a greater rate of cross-bridge cycling *in vitro* (129). Its preponderant expression over the A isoform would likely increase the velocity of ASM shortening and, therefore, contributes to AHR. In accordance to this assertion, the ratio of the isoforms correlates with the level of airway responsiveness in rats; i.e., hyperresponsive animals expressed more of the B than the normo-responsive animals (73). The mRNA expression of the B isoform is also overexpressed in human asthmatics (133). Taken together, the amount and the velocity of shortening are potentially important factors determining the level of airway responsiveness. However, more data are needed to confirm that derangements in these contractile properties are involved in the manifestation of asthmatic AHR.

#### **2.5 Ability to relax**

10 Lung Diseases – Selected State of the Art Reviews

The amount of ASM shortening is of major importance because it ultimately determines the amount of airway narrowing. As discussed earlier, the amount (as well as the velocity) of shortening depends on the ASM-force relative to the load. Therefore, all the factors influencing ASM-force, such as the potency and the concentration of the contractile stimulus involved, the quantity of spasmogens reaching the ASM, the muscle's intrinsic capacity to generate force, and its length, as well as all the factors influencing the load impeding muscle shortening affect the amount and velocity of shortening. However, the intrinsic ability to shorten may also be different between asthmatics and non-asthmatics. So that under the same load and despite producing the same stress, the amount of shortening achieved may be different. Interestingly, this has been shown in isolated ASM cells (140). In that study, the authors showed that unloaded ASM cells derived from asthmatics shorten more at room temperature in response to EFS. However, this observation, which now has a decade old, still awaits confirmation. The underlying mechanisms involved are also unclear but decreased resistance to shortening due to reduction in either internal resistive load (214, 226) or stiffness (44) has been proposed. The shortening velocity of ASM could also be a critical determinant of the amount of airway narrowing. Again, to comprehend the potential implication of ASM shortening velocity in determining the degree of airway responsiveness, it is important to understand that ASM operates in a dynamic environment. The load impeding its shortening is continuously fluctuating due to swings in transpulmonary pressure caused by ventilatory maneuvers (e.g., tidal breathing and DI). The amount of ASM shortening *in vivo* is determined by a balance between the rate of cross-bridge cycling on the actin filaments causing muscle shortening versus the rate and the magnitude of stretch-induced disruption of cross-bridges causing muscle elongation (67). A faster cycling rate of the cross-bridges with a commensurate increased velocity of shortening would lead to more shortening during exhalation, when the load opposing muscle shortening is lowering. A faster rate of cycling would also lead to more cross-bridges being attached at the end of expiration, rendering the ASM and the entire airway wall stiffer. In turn, the stiffer airway wall would be less vulnerable to the stress imposed by the subsequent inspiration; i.e., the airway wall would be exposed to the same stress of breathing but the strain of the airway wall and, consequently, the stretch of the ASM would be attenuated in an airway with stiffer ASM. The combination of more shortening and more cross-bridge attachments during exhalation with less stretch and less cross-bridge detachments during inhalation means that the ASM with an increased shortening velocity would eventually reach a new equilibrium where the size of the airway lumen would be smaller than with a slower ASM. In addition, the airway wall that has reached this equilibrium becomes more static. Since ASM operating in a static environment produces more force in response to the same stimulus (as discussed above), it is possible that a faster muscle would not only cause more shortening and more crossbridges during exhalation, but would also becomes stronger (i.e. able to produce more force for the same level of activation). By acquiring more force the ASM would then be able to narrow the airway further during the next exhalation and… the cycle can perpetuate itself. This vicious cycle is likely to happen in airways possessing ASM with faster shortening velocity and this is the rationale behind the idea that the speed of ASM contraction could be

Experimental evidences exist to support the hypothesis that a faster ASM velocity of shortening can contribute to AHR. The velocity of ASM shortening was shown to be greater in animal models in which there is innate AHR (reviewed in (132)). Similarly, there is

**2.4 Amount of shortening and velocity of shortening** 

the cause of AHR.

ASM relaxation can also affect airway luminal diameter and the degree of airway responsiveness (reviewed in (69) and (44)). Just as stiffness, relaxation can have different connotations. It could refer to the relaxation either during or following the removal of the spasmogenic stimulation, as well as the relaxation induced by a relaxing agonist (bronchodilator). The time of onset, the rate and the extent of relaxation following stimulation with a relaxing agonist, or during or after the removal of the spasmogen, can also impact airway patency. The potential implication of impaired relaxation in asthma is clear. Incomplete or slower relaxation could keep the airways narrowed and, thus, prolong the respiratory distress experienced by asthmatics during an asthma attack. However, the mechanism by which impaired relaxation could contribute to AHR is not as obvious. It relies on the assumption that spontaneous relaxation occurs rapidly following airway narrowing induced by inhalational challenge with a spasmogen. Increased time for the muscle to relax following a spasmogenic stimulation certainly means that the airway luminal size will remain smaller for an extended period of time following a bronchoprovocative challenge and, consequently, will limit airflow during a forced expiratory maneuver (which is often used to assess airway responsiveness).

The following studies provide some evidences that impaired relaxation may play a role in determining the degree of airway responsiveness. Altered relaxation of ASM has been proposed to play a role in determining inherent differences in the degree of airway responsiveness observed between guinea pigs of different ages (reviewed in (44)). The absence or presence of spontaneous relaxation in response to continuous EFS has also been proposed to be a factor explaining innate differences of airway responsiveness seen in different strains of mice (44). The role of impaired relaxation of ASM in AHR seen in human asthmatics needs further investigations.

To reiterate, contractility is defined here as a vague term that may imply different contractile properties. So a hypercontractile ASM phenotype may imply one or all of the following: the muscle may be stronger (increased force-generating capacity), it may shorten more and/or shorten faster, it may be stiffer, it may have an attenuated ability to relax either spontaneously or in response to bronchodilators, or it may have an increased ability to tolerate or recover from a drop in force caused by length perturbations. As seen in this last chapter's section, there are currently evidences suggesting that some intrinsic defects of ASM can contribute to asthmatic AHR. This also suggests that being hyperresponsive could be a prerequisite, or at least a predisposing factor, to be diagnosed with athma. However, whether these intrinsic defects are genetically inherited or acquired as a result of other defects seen in asthmatics is still unknown. One might expect that if asthmatic AHR is due to inherited ASM hypercontractility, it should be encrypted into the genome? The next section shall explore this question.

#### **3. Interrogating the genome for asthma susceptibility genes**

Genetics has evolved tremendously in the last decades and is now endowed with powerful tools to ask questions on the etiology of complex human traits (77). Asthma and the degree of airway responsiveness are irrefutably complex traits. Based on genetic epidemiology studies, the heritability of asthma was estimated to be 40 to 60% (22). However, finding genes and genetic variants responsible for this important genetic component is still very challenging. Not only asthma is a complex trait because of its heterogeneous nature, but also because its manifestation relies on the exposure of an (or many) environmental trigger(s). Thus, a lot of noise in genetic analyses of asthma may stand from the fact that some nonasthmatics (control cohort) are carriers of susceptibility alleles, but are not asthmatics because they never encountered the environmental trigger(s). In addition, maybe more than a single complex trait is required to develop clinical manifestations of asthma. For example, both AHR and airway inflammation may be required to suffer from asthma. If true, the noise in genetic analyses may arise from people with airway inflammation (e.g., atopy) that are protected from asthma symptoms because they are normo- or hypo-responsive. In fact, ~50% of the population is atopic but most of the affected individuals are non-asthmatics. On the other hand, some people may be carrier of alleles that confer susceptibility to AHR, which also confer susceptibility to asthma, but will always be free of asthma symptoms because they will never develop airway inflammation. In fact, it is estimated that roughly 15% of the population is hyperresponsive. Some of these people are asymptomatic, will never be diagnosed with asthma, and are counted in the control (non-asthmatic) group in most genetic studies despite being carriers of alleles conferring susceptibility to asthma. Despite these challenges, recent progress in genetics of asthma was made by genome-wide

association studies (GWAS). These studies consist of testing hundreds of thousands of singlenucleotide polymorphisms (SNPs) distributed across the human genome for association with a disease in hundreds or thousands of individuals. This genomic approach was particularly successful to identify robustly replicated genetic variants involved in complex diseases and biological traits (142). Asthma and asthma-related phenotypes are no exception and a number of GWAS were performed in the field (83, 104, 163, 164, 223).

proposed to be a factor explaining innate differences of airway responsiveness seen in different strains of mice (44). The role of impaired relaxation of ASM in AHR seen in human

To reiterate, contractility is defined here as a vague term that may imply different contractile properties. So a hypercontractile ASM phenotype may imply one or all of the following: the muscle may be stronger (increased force-generating capacity), it may shorten more and/or shorten faster, it may be stiffer, it may have an attenuated ability to relax either spontaneously or in response to bronchodilators, or it may have an increased ability to tolerate or recover from a drop in force caused by length perturbations. As seen in this last chapter's section, there are currently evidences suggesting that some intrinsic defects of ASM can contribute to asthmatic AHR. This also suggests that being hyperresponsive could be a prerequisite, or at least a predisposing factor, to be diagnosed with athma. However, whether these intrinsic defects are genetically inherited or acquired as a result of other defects seen in asthmatics is still unknown. One might expect that if asthmatic AHR is due to inherited ASM hypercontractility, it should be encrypted into the genome? The next

Genetics has evolved tremendously in the last decades and is now endowed with powerful tools to ask questions on the etiology of complex human traits (77). Asthma and the degree of airway responsiveness are irrefutably complex traits. Based on genetic epidemiology studies, the heritability of asthma was estimated to be 40 to 60% (22). However, finding genes and genetic variants responsible for this important genetic component is still very challenging. Not only asthma is a complex trait because of its heterogeneous nature, but also because its manifestation relies on the exposure of an (or many) environmental trigger(s). Thus, a lot of noise in genetic analyses of asthma may stand from the fact that some nonasthmatics (control cohort) are carriers of susceptibility alleles, but are not asthmatics because they never encountered the environmental trigger(s). In addition, maybe more than a single complex trait is required to develop clinical manifestations of asthma. For example, both AHR and airway inflammation may be required to suffer from asthma. If true, the noise in genetic analyses may arise from people with airway inflammation (e.g., atopy) that are protected from asthma symptoms because they are normo- or hypo-responsive. In fact, ~50% of the population is atopic but most of the affected individuals are non-asthmatics. On the other hand, some people may be carrier of alleles that confer susceptibility to AHR, which also confer susceptibility to asthma, but will always be free of asthma symptoms because they will never develop airway inflammation. In fact, it is estimated that roughly 15% of the population is hyperresponsive. Some of these people are asymptomatic, will never be diagnosed with asthma, and are counted in the control (non-asthmatic) group in most genetic studies despite being carriers of alleles conferring susceptibility to asthma. Despite these challenges, recent progress in genetics of asthma was made by genome-wide association studies (GWAS). These studies consist of testing hundreds of thousands of singlenucleotide polymorphisms (SNPs) distributed across the human genome for association with a disease in hundreds or thousands of individuals. This genomic approach was particularly successful to identify robustly replicated genetic variants involved in complex diseases and biological traits (142). Asthma and asthma-related phenotypes are no exception and a number

**3. Interrogating the genome for asthma susceptibility genes** 

of GWAS were performed in the field (83, 104, 163, 164, 223).

asthmatics needs further investigations.

section shall explore this question.

Before the era of GWAS, more than 100 genes have been associated with asthma and related phenotypes (22, 183). Figure 1 shows these genes classified by their most likely pathobiological implication in asthma. Most of these genes were found using a candidate gene strategy (in blue), while fewer were found using genome-wide linkage studies (in green). Eleven genes were specifically studied for their possible involvement in bronchoconstriction and it is suspected that the risk conferred by genetic variants in these genes may act directly through ASM. However, for all these genes the functional demonstration remains to be made. In addition, candidate gene studies were plagued with

Fig. 1. Overview of asthma susceptibility genes. Genes illustrated are reported to be associated with asthma or asthma-related phenotypes in at least one published study. Each gene is categorized according to what we believe is its major role in the pathogenesis of asthma. In blue and green are genes identified using candidate gene and genome-wide linkage studies, respectively. The latest and more robust asthma susceptibility genes are illustrated in red and identified using a genome-wide association approach. Complete references for genes in blue and green can be found in Ober & Hoffjan (183). References for genes in red are in the text (see section 3). The shapes and subcellular localizations are taken from the Ingenuity System (http://www.ingenuity.com). Genes are labeled with official Entrez Gene symbols, and common alias names are shown in parentheses for some genes. The figure is updated from Bossé & Hudson (Annu Rev Med 2007; 58: 171-84).

inconsistency and genetic associations illustrated in Figure 1 require validation in large population samples.

In contrast to candidate gene studies, susceptibility genes derived from GWAS on asthma were confirmed in multiple and larger set of samples. These genes include ORMDL3, GSDMB, IL33, TSLP, IL18R1, IL1RL1, IL2RB, RAD50, MYB, SMAD3, CHI3L1, PDE4D, and DENND1B (illustrated in red in Figure 1). The specific cells and tissues that are molecularly altered by the risk variant in these genes are still unknown. Most of these genes are not known as regulators of airway responsiveness, suggesting that the genetic predisposition to suffer from asthma do not originate from genes altering ASM function. In fact, most of the GWAS-nominated asthma genes are expressed in the airway epithelium or in inflammatory cells, which is consistent with the growing body of evidences that asthma originates in subjects whose epithelium has altered response to environmental triggers and whose immune system is susceptible to the development of inflammation. The exception is the PDE4D gene found to be associated with asthma in multiple white and Hispanic populations (104). PDE4D is expressed in ASM and can potentially alter ASM contractile function, suggesting that this tissue, in addition to the epithelium and inflammatory cells, may influence susceptibility to asthma.

It should be noted that we cannot rule out the contribution of other GWAS-nominated genes acting on ASM. We know from our previous whole-genome gene expression experiment in bronchial smooth muscle cells (23) that approximately half of the genes in the human genome are expressed in non-stimulated ASM cells (nearly 10,000 genes). Obviously other genes might be inducible and expressed only in a 'sick' environment. However, at baseline, in a monolayer of ASM cells, many GWAS-nominated asthma genes are expressed including ORMDL3, GSDMB, IL33, TSLP, RAD50, SMAD3, CHI3L1, and DENND1B. Although what is known about the biological functions of these genes is not pointing toward ASM as the primary pathobiological target, their expression in this tissue suggests that they may play a role. The characterization of asthma susceptibility genes derived from GWAS is currently a priority in the field of genetics of asthma (182). The functions of these genes in ASM, if any, will need to be investigated, as are the consequences of the risk variants on these functions.

#### **4. Non-muscle factors**

In contrast to the above, AHR may also be secondary to the asthmatic syndrome. In fact, many causes of AHR in asthma are attributed to alterations in the lung environment; implying that the ASM can be absolutely normal but would lead to AHR upon activation because it operates in a 'bad' environment. Two scenarios here are envisaged: First, AHR may develop in asthmatic individuals because inflammatory or remodeling changes alter ASM contractile properties. In those instances, AHR still relies on a defect in ASM contractile properties, as discussed in section 2, but they are acquired as a result of an altered environment. Parenthetically, the phenomena that lead to an acquired increase in contractility will be called here an 'ASM behavior' (see Figure 2). Thus, a behavior is a normal ASM's ability to adapt to its surrounding environment. Some ASM behaviors are addressed in the non-muscle factors section of this chapter because their appearance is attributable to lung defects. This didactic distinction between ASM contractile properties and behaviors would become clearer along the remaining of this chapter. In the second scenario, certain causes of AHR are unrelated to genetically or acquired abnormalities of ASM but are rather due, exclusively, to lung alterations. In those instances, the abnormal milieu is sufficient to cause AHR in a setting where none of the ASM contractile properties are altered. In the following section, we will describe the non-muscle factors that are potentially involved in asthmatic AHR by affecting, or not, the contractile properties of ASM. Four broad themes will be discussed; that is remodeling, airway inflammation, ASM-tone and ventilation heterogeneity. Some of the factors discussed below were addressed in previous reviews (24, 26).

Fig. 2. Muscle and non-muscle mechanisms potentially involved in airway hyperresponsiveness (AHR) seen in asthmatic patients. Any alterations in airway smooth muscle (ASM) contractile properties, which can be innate or acquired as a result of lung defects, may participate in the development and manifestation of AHR. Lung defects can also contribute to AHR either directly or by fostering normal ASM behaviors, such as force adaptation. See text for further details.

## **4.1 Remodeling**

14 Lung Diseases – Selected State of the Art Reviews

inconsistency and genetic associations illustrated in Figure 1 require validation in large

In contrast to candidate gene studies, susceptibility genes derived from GWAS on asthma were confirmed in multiple and larger set of samples. These genes include ORMDL3, GSDMB, IL33, TSLP, IL18R1, IL1RL1, IL2RB, RAD50, MYB, SMAD3, CHI3L1, PDE4D, and DENND1B (illustrated in red in Figure 1). The specific cells and tissues that are molecularly altered by the risk variant in these genes are still unknown. Most of these genes are not known as regulators of airway responsiveness, suggesting that the genetic predisposition to suffer from asthma do not originate from genes altering ASM function. In fact, most of the GWAS-nominated asthma genes are expressed in the airway epithelium or in inflammatory cells, which is consistent with the growing body of evidences that asthma originates in subjects whose epithelium has altered response to environmental triggers and whose immune system is susceptible to the development of inflammation. The exception is the PDE4D gene found to be associated with asthma in multiple white and Hispanic populations (104). PDE4D is expressed in ASM and can potentially alter ASM contractile function, suggesting that this tissue, in addition to the epithelium and inflammatory cells,

It should be noted that we cannot rule out the contribution of other GWAS-nominated genes acting on ASM. We know from our previous whole-genome gene expression experiment in bronchial smooth muscle cells (23) that approximately half of the genes in the human genome are expressed in non-stimulated ASM cells (nearly 10,000 genes). Obviously other genes might be inducible and expressed only in a 'sick' environment. However, at baseline, in a monolayer of ASM cells, many GWAS-nominated asthma genes are expressed including ORMDL3, GSDMB, IL33, TSLP, RAD50, SMAD3, CHI3L1, and DENND1B. Although what is known about the biological functions of these genes is not pointing toward ASM as the primary pathobiological target, their expression in this tissue suggests that they may play a role. The characterization of asthma susceptibility genes derived from GWAS is currently a priority in the field of genetics of asthma (182). The functions of these genes in ASM, if any, will need to be investigated, as are the consequences of the risk variants on these functions.

In contrast to the above, AHR may also be secondary to the asthmatic syndrome. In fact, many causes of AHR in asthma are attributed to alterations in the lung environment; implying that the ASM can be absolutely normal but would lead to AHR upon activation because it operates in a 'bad' environment. Two scenarios here are envisaged: First, AHR may develop in asthmatic individuals because inflammatory or remodeling changes alter ASM contractile properties. In those instances, AHR still relies on a defect in ASM contractile properties, as discussed in section 2, but they are acquired as a result of an altered environment. Parenthetically, the phenomena that lead to an acquired increase in contractility will be called here an 'ASM behavior' (see Figure 2). Thus, a behavior is a normal ASM's ability to adapt to its surrounding environment. Some ASM behaviors are addressed in the non-muscle factors section of this chapter because their appearance is attributable to lung defects. This didactic distinction between ASM contractile properties and behaviors would become clearer along the remaining of this chapter. In the second scenario, certain causes of AHR are unrelated to genetically or acquired abnormalities of ASM but are rather due, exclusively, to lung alterations. In those instances, the abnormal milieu is sufficient to cause AHR in a setting

population samples.

may influence susceptibility to asthma.

**4. Non-muscle factors** 

The term 'remodeling' refers to any structural changes occurring in the lungs. It could be any alteration in the quantity, the distribution and the composition of the molecular, cellular and tissular constituents of the lungs. It can affect, or not, the physical and mechanical properties of the lung constituents. The term 'remodeling' is also usually reserved for structural changes that are permanent or fixed (relatively), such as the deposition of extra ECM components. So even if edema and inflammatory cell infiltrates fit into the definition of remodeling given above, these changes are not usually perceived as remodeling changes. They are rather the results of inflammatory processes that are transient and reversible in nature. Generally speaking, remodeling can designate any or all of the following: epithelial metaplasia, such as zone of denudation, deciliated areas and goblet cell hyperplasia; fibrosis of all the airway wall layers but especially the *lamina reticularis* (the third layer of the basal lamina); vascular alterations, such as vessel enlargement and angiogenesis; thickening of all the layers of the airway wall including the epithelium, the basal lamina, the lamina propria, the ASM and the adventitia; and parenchymal changes, such as emphysema and breaks of alveolar attachments on the outer edge of the airway wall (Figure 3). Lung remodeling, especially airway remodeling, has been reviewed by many in the past (25). In the present chapter, we focus on the structural changes that have been shown or suggested to alter the level of airway responsiveness.

Fig. 3. A cartoon of an airway half normal (on the left) and half asthmatic (on the right) to illustrate the various features of airway remodeling in asthma. Airway responsiveness can be inuenced by (1) an increase in volume of mucus and/or cellular debris in the airway lumen, (2) fewer but deeper folds in the mucosal membrane, (3) increased volume or altered surface tension of the liquid layer, (4) thickening and stiffening of the basement membrane (BM), (5) increased ASM mass embedded in an increased brous network, (6) decreased integrity and/or (7) decreased number of alveolar tethers to the adventitia contributing to decreased elastic recoil, (8) increased thickness of the lamina propria, (9) increased goblet cell number, (10) increased vascularity and/or vascular dilatation in the lamina propria and adventitia, (11) increased thickness of the adventitia, and (12) increased inammatory cell inltration throughout the airway wall. From Bossé *et al*. Annu Rev Physiol 2010; 72: 437-62.

#### **4.1.1 Epithelium integrity**

16 Lung Diseases – Selected State of the Art Reviews

remodeling can designate any or all of the following: epithelial metaplasia, such as zone of denudation, deciliated areas and goblet cell hyperplasia; fibrosis of all the airway wall layers but especially the *lamina reticularis* (the third layer of the basal lamina); vascular alterations, such as vessel enlargement and angiogenesis; thickening of all the layers of the airway wall including the epithelium, the basal lamina, the lamina propria, the ASM and the adventitia; and parenchymal changes, such as emphysema and breaks of alveolar attachments on the outer edge of the airway wall (Figure 3). Lung remodeling, especially airway remodeling, has been reviewed by many in the past (25). In the present chapter, we focus on the structural changes

Fig. 3. A cartoon of an airway half normal (on the left) and half asthmatic (on the right) to illustrate the various features of airway remodeling in asthma. Airway responsiveness can be inuenced by (1) an increase in volume of mucus and/or cellular debris in the airway lumen, (2) fewer but deeper folds in the mucosal membrane, (3) increased volume or altered surface tension of the liquid layer, (4) thickening and stiffening of the basement membrane (BM), (5) increased ASM mass embedded in an increased brous network, (6) decreased integrity and/or (7) decreased number of alveolar tethers to the adventitia contributing to decreased elastic recoil, (8) increased thickness of the lamina propria, (9) increased goblet cell number, (10) increased vascularity and/or vascular dilatation in the lamina propria and adventitia, (11) increased thickness of the adventitia, and (12) increased inammatory cell inltration throughout the airway wall. From Bossé *et al*. Annu Rev Physiol 2010; 72: 437-62.

that have been shown or suggested to alter the level of airway responsiveness.

Researchers working with isolated airway segments *ex vivo* have long recognized that the concentration of spasmogens required for a given response is orders of magnitude greater (30 to 300-fold) when a chosen spasmogen is delivered on the mucosal side (within the lumen) compared to when it is delivered on the adventitial side (179). Mechanical removal of the epithelium in bronchial segments of dogs has also been shown to increase ACh sensitivity by 100 to 716-fold (159, 187). These observations suggested that the epithelium acts as a cellular barrier and establishes a large concentration gradient across the airway wall. A breach in the integrity of the epithelium would disrupt its barrier function and, consequently, increases airway responsiveness by facilitating the delivery of spasmogens to the ASM. In fact, association between epithelial permeability and airway responsiveness in humans has been previously reported (108); albeit this finding is not unanimous (197). Increased leakiness of the epithelium can be due to cellular damage and desquamation, which can be induced, for example, by eosinophil mobilization and activation into the airways. Disruption of cell-cell connectivity, by interrupting E-cadherin adhesion for example, can equally facilitate the delivery of spasmogens to the ASM by increasing the paracellular conductance (254). Many asthma stressors, such as allergens, ozone, particulate matters, smoke and occupational triggers have been shown to affect airway epithelial integrity, which may underlie their link with AHR. Alterations in epithelium integrity by these asthma stressors can be mediated directly, or indirectly by fostering inflammatory cell infiltration into the epithelium or by increasing the release of endogenous proteases (187). In addition to act as a physical barrier, the airway epithelium can also play a direct role in controlling ASM contractility. The airway epithelium has been shown to produce several bronchoactive substances, such as lipooxygenase and cyclooxygenase products as well as nitric oxide. This subject was reviewed in the past (225) and will not be discussed in detail here. Briefly, the combined effect of these mediators seems to be bronchodilatory and may thus play a protective role against airway narrowing. Collectively, these observations suggest that any alteration in the lungs affecting the integrity of the epithelium or the release

of epithelium-derived bronchoactive substances may influence airway responsiveness.

#### **4.1.2 Vascular remodeling**

The number and size of blood vessels, as well as the vascular area, are increased in asthmatic airways (reviewed in (55)). Signs of dilation, congestion, endothelium leakiness and abnormal wall thickness of the airway vessels have also been noted, which all alter by different manner and different extent the normal function of these vessels. The space occupied by the vessels conspicuously increases airway wall thickness, but may also do so by changing its turgidity. It may also modify the mechanical properties of the airway wall and affect the pattern of airway wall folding during airway narrowing. The detrimental effects of these structural changes are discussed in the following subsections (4.1.3 and 4.1.4).

Chronic vascular alterations also have the potential to exacerbate edematous changes occurring in the face of inflammation (discussed in section 4.2.2). It may also alter the clearance of spasmogens into the airways, which can greatly affect the degree of airway responsiveness (151). Taken together, these results suggest that changes in number, size, structure and integrity of the vascular bed interwinding the airway wall can have direct or indirect repercussions on airway responsiveness.

#### **4.1.3 Increased amount of material inner the ASM layer**

As aforementioned, all the layers of the airway wall were shown to be thicker in asthma. An increased amount of material inner the ASM layer amplifies the luminal airway narrowing for any given degree of ASM shortening. This is because of a geometrical effect. Airway resistance is inversely related to the luminal radius at the fourth power. So small decreases in radius can cause huge increases in airflow resistance. This effect is more pronounced when the thickening encroaches the lumen (inwardly-directed remodeling) prior constriction. This is because the airway lumen will be smaller to begin with. Thus, the same amount of ASM shortening (or the same decrease in luminal radius) would increase further the resistance to airflow. However, this geometric effect would also occur if the thickening does not encroach the lumen (outwardly-directed remodeling) prior constriction. This is because the luminal area decreases as the ASM shortens, but the area of the material inner the ASM does not; assuming that this material is uncompressible. With a greater fraction of the total area occupied by uncompressible material to begin with, a thicker airway wall would exaggerate the changes in the luminal area during ASM shortening. In other words, for a given change in total area (i.e., area inner the ASM layer), if more area is occupied by uncompressible material, more changes in the compressible area (i.e., lumen) would have to occur. Importantly, filling the airway lumen by inflammatory cells, mucus accumulation or by plasma extravasation would have the same detrimental consequence.

*In vivo*, even if the material inner the ASM would be somewhat compressible, an increased amount of material inner the ASM layer would still contribute to AHR, unless the compressibility of this material is severely affected in asthma. Computation models of the human bronchial tree confirmed that thicker airway wall inner the ASM increases airflow resistance caused by any given degree of ASM activation (126). This effect may not be small. Wagers and coworkers have developed a computational model based on morphometric and functional data derived from a mouse model of allergic airway inflammation to address this question. They predicted that thickening of the airway wall combined with an increased propensity for airway closure (discussed later in subsection 4.4.2) in the 'asthmatic' mice were sufficient to explain entirely AHR, without the need of increasing ASM shortening (237). Taken together, any lung defect that increase the volume of material inner the ASM layer leads to more luminal narrowing for any given degree of ASM shortening. This is a good example showing that AHR may be present despite normal ASM contractility.

#### **4.1.4 Reduced ASM-load due to remodeling**

The load impeding muscle shortening is just as important as the force-generating capacity of the ASM. A weaker load will allow the ASM to shorten more but also to shorter faster for the same reasons as a stronger ASM would shorten more and faster against a given load (discussed in subsection 2.1).

The loads impeding ASM shortening originate from different airway wall and lung elements. One of the most important load is the parenchymal recoil; i.e., the radial tethering force offered by the parenchymal attachments on the outer edge (adventitia) of the airway wall. In fact, this load seems to be crucial for the maintenance of intraparenchymal airway patency. In condition where the ASM would be fully relaxed, this load is totally counterbalanced by the circumferential tension of the airway wall; an inwardly-directed force equal to the force of recoil but acting in opposite direction. To overcome a greater recoil, more strain within the airway wall would be required to reach a circumferential tension equal but opposite to the recoil, which would mean more airway dilatation. Therefore, greater

As aforementioned, all the layers of the airway wall were shown to be thicker in asthma. An increased amount of material inner the ASM layer amplifies the luminal airway narrowing for any given degree of ASM shortening. This is because of a geometrical effect. Airway resistance is inversely related to the luminal radius at the fourth power. So small decreases in radius can cause huge increases in airflow resistance. This effect is more pronounced when the thickening encroaches the lumen (inwardly-directed remodeling) prior constriction. This is because the airway lumen will be smaller to begin with. Thus, the same amount of ASM shortening (or the same decrease in luminal radius) would increase further the resistance to airflow. However, this geometric effect would also occur if the thickening does not encroach the lumen (outwardly-directed remodeling) prior constriction. This is because the luminal area decreases as the ASM shortens, but the area of the material inner the ASM does not; assuming that this material is uncompressible. With a greater fraction of the total area occupied by uncompressible material to begin with, a thicker airway wall would exaggerate the changes in the luminal area during ASM shortening. In other words, for a given change in total area (i.e., area inner the ASM layer), if more area is occupied by uncompressible material, more changes in the compressible area (i.e., lumen) would have to occur. Importantly, filling the airway lumen by inflammatory cells, mucus accumulation or

*In vivo*, even if the material inner the ASM would be somewhat compressible, an increased amount of material inner the ASM layer would still contribute to AHR, unless the compressibility of this material is severely affected in asthma. Computation models of the human bronchial tree confirmed that thicker airway wall inner the ASM increases airflow resistance caused by any given degree of ASM activation (126). This effect may not be small. Wagers and coworkers have developed a computational model based on morphometric and functional data derived from a mouse model of allergic airway inflammation to address this question. They predicted that thickening of the airway wall combined with an increased propensity for airway closure (discussed later in subsection 4.4.2) in the 'asthmatic' mice were sufficient to explain entirely AHR, without the need of increasing ASM shortening (237). Taken together, any lung defect that increase the volume of material inner the ASM layer leads to more luminal narrowing for any given degree of ASM shortening. This is a

good example showing that AHR may be present despite normal ASM contractility.

The load impeding muscle shortening is just as important as the force-generating capacity of the ASM. A weaker load will allow the ASM to shorten more but also to shorter faster for the same reasons as a stronger ASM would shorten more and faster against a given load

The loads impeding ASM shortening originate from different airway wall and lung elements. One of the most important load is the parenchymal recoil; i.e., the radial tethering force offered by the parenchymal attachments on the outer edge (adventitia) of the airway wall. In fact, this load seems to be crucial for the maintenance of intraparenchymal airway patency. In condition where the ASM would be fully relaxed, this load is totally counterbalanced by the circumferential tension of the airway wall; an inwardly-directed force equal to the force of recoil but acting in opposite direction. To overcome a greater recoil, more strain within the airway wall would be required to reach a circumferential tension equal but opposite to the recoil, which would mean more airway dilatation. Therefore, greater

**4.1.4 Reduced ASM-load due to remodeling** 

(discussed in subsection 2.1).

**4.1.3 Increased amount of material inner the ASM layer** 

by plasma extravasation would have the same detrimental consequence.

parenchymal recoil leads to greater airway caliber. The extent of the recoil depends on lung volume, the elasticity of the parenchymal tissue and the strength of the connection between the airway wall and the parenchyma (the later is sometimes called the force of interdependence). Any lung defect affecting these parameters may influence the strength of the recoil at a given lung volume and, concomitantly, change airway caliber. The parenchymal recoil also offers an additional load during airway narrowing because the lung parenchyma is progressively distorted as the ASM shortens. This load can be calculated from the shear modulus () of the lung parenchyma, which was estimated to be 0.7 time the transpulmonary pressure (124), and the changes in adventitial diameter during airway narrowing, which is geometrically related to the changes in ASM length during shortening (126). So in addition to keep the intraparenchymal airways patent prior constriction, the lung recoil imposes an afterload that further limits airway narrowing once the ASM begins to shorten. Lung defects previously observed in asthma, such as alveolar breaks (149) and adventitial thickening (190), were shown to decouple the airways from the parenchyma and affect the extent of both the pre- and the after-load offered by the recoil. These lung defects may contribute to AHR.

The ECM within and surrounding the ASM bundle also affects the after-load impeding muscle shortening. In conditions where the muscle is fully relaxed, the circumferential tension (which is carrying the load of the recoil) would be born from the passive elements of the airway wall, including the resting tension of the ASM. However, during narrowing induced by ASM shortening, the load carried by the parallel elastic elements is progressively transferred to the ASM. This transfer proceeds until these parallel elastic elements are no longer in tension but in compression. At that time, they become a direct load impeding muscle shortening. So whether these parallel elastic elements are in tension and progressively transfer the load to the ASM during airway narrowing or whether they pass the transition from being in tension to become in compression, they still limit ASM shortening by increasing the after-load. If this material is stiffer for example, it would be carrying more load for less strain (i.e., for a given lung recoil pressure there will be less airway dilatation). In those conditions, while the ASM begins to shorten upon activation, a greater load would be transferred to the ASM for a given amount of airway narrowing. It will also be harder to compress this material if airway narrowing proceeds until the parallel elastic elements are no longer in tension but in compression. In addition, when a cell or a bundle of ASM shortens, its center tempts to bulge. Anything that would prevent the modification of cell shape required for shortening may limit airway narrowing. This load has been called radial constraint (190). Together, this suggests that the quantity and the mechanical properties of the ECM, but also any other wall constituents, within and surrounding the ASM cells or bundles may affect the degree of airway narrowing. In support of this contention, pre-treatment of airway wall strips with collagenase was shown to increase ASM shortening in response to a given contractile stimulus (29). This result suggested that degradation of the collagenous matrix in the airway wall reduces the load, allowing the ASM to shorten further in response to a given level of activation.

Epithelial buckling pressure and airway wall corrugation that occur during airway narrowing are also believed to provide an after-load hampering ASM shortening. The number of mucosal folds developing during narrowing seems to be of major importance. The number of folds, in turn, may be determined by the flexibility, stiffness and thickness of the airway wall, by the stiffness and thickness ratios of the different layers composing the airway wall, or by structural features such as longitudinal elastic bundles (37) or blood and lymphatic vessels (239), which could dictate the location of the folds. More studies are warranted to establish a consensus concerning the factors influencing the pattern of mucosal folding and how it affects ASM-load. Current evidences suggested that the number of folds is not different between asthmatic and non-asthmatic airways (37).

The load impeding ASM shortening may either decrease or increase due to airway remodeling. Thickening and/or fibrosis of the airway wall, for instance, may protect against airway narrowing. Accordingly, airway reactivity was shown to correlate negatively with airway wall thickness in humans with asthma (155, 175). Similar observations were made in a mouse model of asthma (2). Therefore, even if thickening due to fibrosis can theoretically increase airway narrowing in response to spasmogens because of geometrical effects, airway wall stiffening due to thicker or more fibrotic wall may well protect against AHR. Clearly, it is not just the geometric effects of wall thickening that influence airway narrowing (189). The mechanical properties of the material that causes the thickening are also important since ASM has to deform this material to narrow the lumen. The composition of the ECM may also affect the ASM phenotype (discussed in subsection 4.1.6).

Taken together, we have seen that the caliber of the airways embedded in the parenchyma is determined by the dilating force of the lung elastic recoil and the stiffness of the airway wall. Anything affecting the parenchymal recoil, the airway-parenchymal interdependence, the parallel elastic elements, the radial constraint and the pattern of mucosal folding may reduce (or augment) the load and, consequently, causes more (or less) ASM shortening for any given degree of ASM activation.

#### **4.1.5 Length adaptation**

A decrease in airway caliber caused by any lung defect attenuating the pre-load would cause a reduction in ASM length. Because the force-generating capacity of the ASM is proportional to its length, decreasing ASM length simultaneously decreases its forcegenerating capacity. However, the ASM is endowed with an intrinsic ability to adapt to length changes (reviewed in (27)). This phenomenon was called length adaptation and is defined as the recovery of ASM force-generating capacity that was loss following length perturbations. Adaptation of ASM to a shorter length is particularly relevant to the understanding of AHR. This is because a muscle adapted to shorten length is able to generate more force at any given length during shortening comparatively to a muscle adapted to a longer length. In contradistinction, adaptation to longer length would have a protective effect in terms of airway responsiveness. This is a good example to show how a normal ASM behavior (i.e., length adaptation), may contribute to AHR by increasing a contractile property of the ASM (herein force).

Length adaptation is likely to occur in different situations. Emphysema, for example, decreases airway caliber by reducing lung recoil, which will allow the ASM to adapt to shorter length. Other factors reducing lung volume such as recumbent posture (247), high spinal cord injury (201) and obesity could have a similar effect. However, a recent review on obesity and AHR (206) rather concludes that obesity has little direct effect on airway caliber despite reducing functional residual capacity (FRC) and expiratory reserve volume. Based on the authors, the expiratory flow limitation observed in individuals with low lung volume (like the obese) is due to an increased propensity for airway closure (discussed in subsection 4.4.2). That may also apply to other conditions affecting lung volume.

Another contractile property that is affected by length adaptation is the ASM-passive stiffness. It was shown that the passive stiffness initially declines following a length reduction but slowly re-develops over time (28). However, since the contribution of ASM-

passive stiffness to the overall stiffness of the airway wall is neglectable, it is insignificant to the understanding of AHR in asthma.

#### **4.1.6 Alterations in the composition to the ECM**

It was suggested that the phenotype of ASM cells can be switched around from a contractile phenotype to a synthetic/proliferative phenotype (95). ECM components are thought to play an important role in determining ASM phenotypes. Laminin, for example, was shown to be required for the transformation of ASM cells into a more contractile phenotype (233). The existing interplay between ECM and ASM phenotypes and functions has been the subject of other reviews (232). It is known that the composition of the ECM is altered in asthma due to either a change in the rate of ECM component synthesis or a change in the rate of their degradation, the later being controlled by a balance between proteases and their inhibitors. Taken together, this suggests that a fine balance of ECM components is required to keep the ASM phenotype in check. Any alteration in this balance may affect ASM contractility and airway responsiveness.

#### **4.1.7 Enlargement of ASM mass**

20 Lung Diseases – Selected State of the Art Reviews

warranted to establish a consensus concerning the factors influencing the pattern of mucosal folding and how it affects ASM-load. Current evidences suggested that the number of folds

The load impeding ASM shortening may either decrease or increase due to airway remodeling. Thickening and/or fibrosis of the airway wall, for instance, may protect against airway narrowing. Accordingly, airway reactivity was shown to correlate negatively with airway wall thickness in humans with asthma (155, 175). Similar observations were made in a mouse model of asthma (2). Therefore, even if thickening due to fibrosis can theoretically increase airway narrowing in response to spasmogens because of geometrical effects, airway wall stiffening due to thicker or more fibrotic wall may well protect against AHR. Clearly, it is not just the geometric effects of wall thickening that influence airway narrowing (189). The mechanical properties of the material that causes the thickening are also important since ASM has to deform this material to narrow the lumen. The composition of the ECM may

Taken together, we have seen that the caliber of the airways embedded in the parenchyma is determined by the dilating force of the lung elastic recoil and the stiffness of the airway wall. Anything affecting the parenchymal recoil, the airway-parenchymal interdependence, the parallel elastic elements, the radial constraint and the pattern of mucosal folding may reduce (or augment) the load and, consequently, causes more (or less) ASM shortening for

A decrease in airway caliber caused by any lung defect attenuating the pre-load would cause a reduction in ASM length. Because the force-generating capacity of the ASM is proportional to its length, decreasing ASM length simultaneously decreases its forcegenerating capacity. However, the ASM is endowed with an intrinsic ability to adapt to length changes (reviewed in (27)). This phenomenon was called length adaptation and is defined as the recovery of ASM force-generating capacity that was loss following length perturbations. Adaptation of ASM to a shorter length is particularly relevant to the understanding of AHR. This is because a muscle adapted to shorten length is able to generate more force at any given length during shortening comparatively to a muscle adapted to a longer length. In contradistinction, adaptation to longer length would have a protective effect in terms of airway responsiveness. This is a good example to show how a normal ASM behavior (i.e., length adaptation), may contribute to AHR by increasing a

Length adaptation is likely to occur in different situations. Emphysema, for example, decreases airway caliber by reducing lung recoil, which will allow the ASM to adapt to shorter length. Other factors reducing lung volume such as recumbent posture (247), high spinal cord injury (201) and obesity could have a similar effect. However, a recent review on obesity and AHR (206) rather concludes that obesity has little direct effect on airway caliber despite reducing functional residual capacity (FRC) and expiratory reserve volume. Based on the authors, the expiratory flow limitation observed in individuals with low lung volume (like the obese) is due to an increased propensity for airway closure (discussed in subsection 4.4.2).

Another contractile property that is affected by length adaptation is the ASM-passive stiffness. It was shown that the passive stiffness initially declines following a length reduction but slowly re-develops over time (28). However, since the contribution of ASM-

is not different between asthmatic and non-asthmatic airways (37).

also affect the ASM phenotype (discussed in subsection 4.1.6).

any given degree of ASM activation.

contractile property of the ASM (herein force).

That may also apply to other conditions affecting lung volume.

**4.1.5 Length adaptation** 

We came to realize that the mass of ASM is increased in asthmatic airways almost a century ago (106). This structural change was one of the first hypothesis advanced to explain AHR in asthma and has not been refuted yet. Although a recent study suggested that the increased ASM mass is related predominantly to cellular hyperplasia rather than hypertrophy (199), the exact origin of ASM enlargement is still a matter of debate. In addition to the possibilities of cellular hyperplasia and hypertrophy, alternative hypotheses have been proposed, such as myositis (i.e., inflammation of the ASM) and increased ECM deposition. The origin of hyperplasia is also unclear but could stem from increased proliferation, decreased rate of apoptosis and/or migration of ASM progenitors into the ASM bundle with their subsequent differentiation into ASM cells (reviewed in (25)). The role of proliferation in ASM hyperplasia has gained credibility recently when Hassan and coworkers (100) provided data suggestive of an increased rate of ASM cell proliferation in humans with severe asthma. Irrespective of the mechanism of ASM thickening, the increased mass could lead to a greater total force generation for any given level of ASM activation; assuming that the ASM from asthmatics produce the same stress (force per cross-sectional area) as nonasthmatic ASM. *In vivo*, that would be translated into increased airway narrowing for any given dose of inhaled spasmogen and, therefore, AHR.

To reiterate, asthmatic remodeling is characterized by relatively permanent changes in the architecture of the airways, the lung parenchyma and their interconnection. Many of these changes can affect, positively or negatively, the degree of airway responsiveness. The integrity of the epithelium and vascular remodeling may influence the delivery and the clearance of spasmogens toward and away from the ASM, respectively. Increased amount of material inner the ASM layer can increase airway narrowing by a geometrical effect. Decremented load impeding ASM shortening can cause more ASM shortening for any given degree of ASM activation. Remodeling can also promote ASM hypercontractility by fostering normal ASM behaviors such as length adaptation or phenotype switching. Finally, factors fostering the growing of the ASM mass encircling the airways may increase ASMforce per unit of airway length. Those are all different ways by which a normal ASM may account for AHR in a remodeled environment.

#### **4.2 Airway inflammation**

Airway inflammation is a hallmark of asthma. The use of the term 'airway inflammation' is vague though. Asthma can be characterized by different types of inflammation, which may be useful to distinguish for diagnosis purposes and to offer patients the optimal therapeutic strategy (248). There is clearly a link between airway inflammation and AHR. Interventions altering airway inflammation are effective in changing the degree of airway responsiveness. For example, AHR can be increased in asthmatics following the induction of acute inflammation by inhalation of allergens (47, 143) or IL-5 (218). On the other hand, reducing inflammation by the use of glucocorticoids attenuates AHR (reviewed in (30)).

Airway inflammation can affect airway responsiveness by many means. It is sometime hard to dissociate the effect of inflammatory changes from remodeling changes, since both can affect features of the airway wall, such as its thickness, that participate to AHR. It is also hard to dissociate because of the interplay that exists between inflammation and remodeling. Many believe that inflammation is the cause of remodeling, as the damage caused by inflammation may lead to impaired repair, scaring and loss of function. For the sake of this chapter, we considered inflammation as the transient and potentially reversible changes occurring in the lungs of asthmatics following exposure to the triggering agent. We also consider the direct influence of asthma triggers and inflammation-derived molecules on ASM contractile properties.

#### **4.2.1 Mucus accumulation**

Any encroachment of the airway lumen, whether it is provoked by narrowing or by filling the lumen, causes airway obstruction, increases airway resistance and contributes to AHR. The mucus layer is required for normal lung homeostasis. It is recognized as a physical and immunological barrier protecting the host against inhaled pathogens, particulates and pollutants. However, its hyper-secretion can encroach the lumen and causes airway obstruction. Goblet cell hyperplasia (1), as well as enlargement and hyperplasia of the submucosal glands (16), can contribute to mucus hyper-secretion. These are remodeling changes but their contribution to lung dysfunction is only manifested upon exposure to airway stresses. In the extreme cases, mucus can also cause complete occlusion by forming mucus plugs, which contain, in addition to mucus, proteinaceous exudate, inflammatory cells and isolated epithelial cells and creola bodies (123). Complete occlusion of some airways severely compromises airflow into the lungs, and may thus be an important player in the manifestation of AHR. It also has severe repercussions on ventilation.

The accumulation of mucus can also originate from improper clearance. Mucociliary clearance was shown to be slower in asthmatics (108). The cephalad transport of mucus into the lungs is motored by a synchronized rotational beating of the epithelial cils and by cough when necessary. It also relies on the integrity of a low-viscosity solution lying atop the epithelial cells called the periciliary liquid layer (PCL), which prevents the shear friction between the epithelial surface glycocalyx and the mucus layer (120). Any alteration in mucus clearance, such as cil diskinesia, changes in mucus consistency, overly thin PCL and problems associated with cough reflex can all lead to mucus accumulation and airway obstruction. In addition, stagnant mucus can increase the risk of infection (120).

Mucus accumulation also increases the surface tension. It was estimated that replacing the normal fluid lining the epithelium of peripheral airways by mucus would increase surface tension by 5 folds (141). Surface tension is sometime overlook but can play an important role in determining the degree of airway responsiveness. This was well demonstrated when we came to realize that the pressure required to achieve a given lung volume is significantly higher when the lung is inflated with a gas compare to when it is filled with a liquid. In addition to its great impact on airway resistance, surface tension also increases the propensity for airway closure. In fact, a 5-fold increase in surface tension was predicted to be sufficient to cause airway instability and collapse (141). The influence of airway closure in airway responsiveness is discussed further in subsection 4.4.2. The role of surfactant in controlling surface tension is thus of major importance. Any alteration in surfactant caused by allergen exposure (51) or plasma exudation (238) is likely to affect surface tension and, concomitantly, lung functions (257). Finally, combined with ASM constriction, the baseline airway obstruction induced by mucus accumulation can synergistically increase airway responsiveness for geometric reasons mentioned in section 4.1.3.

#### **4.2.2 Edema**

22 Lung Diseases – Selected State of the Art Reviews

Airway inflammation is a hallmark of asthma. The use of the term 'airway inflammation' is vague though. Asthma can be characterized by different types of inflammation, which may be useful to distinguish for diagnosis purposes and to offer patients the optimal therapeutic strategy (248). There is clearly a link between airway inflammation and AHR. Interventions altering airway inflammation are effective in changing the degree of airway responsiveness. For example, AHR can be increased in asthmatics following the induction of acute inflammation by inhalation of allergens (47, 143) or IL-5 (218). On the other hand, reducing

Airway inflammation can affect airway responsiveness by many means. It is sometime hard to dissociate the effect of inflammatory changes from remodeling changes, since both can affect features of the airway wall, such as its thickness, that participate to AHR. It is also hard to dissociate because of the interplay that exists between inflammation and remodeling. Many believe that inflammation is the cause of remodeling, as the damage caused by inflammation may lead to impaired repair, scaring and loss of function. For the sake of this chapter, we considered inflammation as the transient and potentially reversible changes occurring in the lungs of asthmatics following exposure to the triggering agent. We also consider the direct influence of asthma triggers and inflammation-derived molecules on

Any encroachment of the airway lumen, whether it is provoked by narrowing or by filling the lumen, causes airway obstruction, increases airway resistance and contributes to AHR. The mucus layer is required for normal lung homeostasis. It is recognized as a physical and immunological barrier protecting the host against inhaled pathogens, particulates and pollutants. However, its hyper-secretion can encroach the lumen and causes airway obstruction. Goblet cell hyperplasia (1), as well as enlargement and hyperplasia of the submucosal glands (16), can contribute to mucus hyper-secretion. These are remodeling changes but their contribution to lung dysfunction is only manifested upon exposure to airway stresses. In the extreme cases, mucus can also cause complete occlusion by forming mucus plugs, which contain, in addition to mucus, proteinaceous exudate, inflammatory cells and isolated epithelial cells and creola bodies (123). Complete occlusion of some airways severely compromises airflow into the lungs, and may thus be an important player

The accumulation of mucus can also originate from improper clearance. Mucociliary clearance was shown to be slower in asthmatics (108). The cephalad transport of mucus into the lungs is motored by a synchronized rotational beating of the epithelial cils and by cough when necessary. It also relies on the integrity of a low-viscosity solution lying atop the epithelial cells called the periciliary liquid layer (PCL), which prevents the shear friction between the epithelial surface glycocalyx and the mucus layer (120). Any alteration in mucus clearance, such as cil diskinesia, changes in mucus consistency, overly thin PCL and problems associated with cough reflex can all lead to mucus accumulation and airway

Mucus accumulation also increases the surface tension. It was estimated that replacing the normal fluid lining the epithelium of peripheral airways by mucus would increase surface tension by 5 folds (141). Surface tension is sometime overlook but can play an important role

in the manifestation of AHR. It also has severe repercussions on ventilation.

obstruction. In addition, stagnant mucus can increase the risk of infection (120).

inflammation by the use of glucocorticoids attenuates AHR (reviewed in (30)).

**4.2 Airway inflammation** 

ASM contractile properties.

**4.2.1 Mucus accumulation** 

Edema is a characteristic feature of inflammation. It is due to an increased permeability of the vessels. This increased leakiness of the vascular endothelium swells the tissue by fostering extravasation of inflammatory cells, as well as plasmatic fluid and proteins, into the interstitial tissue. It may also encroach the airway lumen. Plasma exudation onto the epithelial lining is increased in asthma and the extent of it is associated with the level of airway responsiveness (235). Many inflammatory molecules that are overexpressed in asthma were shown to increase vascular permeability, such as substance P, histamine and many others. Newly formed blood vessels are also more permeable than the existing vessels (10). Angiogenic remodeling seen in asthma may thus render the airways more prone to tumefaction.

Edema can alter the degree of airway responsiveness by many means. Plasma exudation induced experimentally by infusion of saline in dogs was shown to increase airway wall thickness and to decrease the caliber of the airway lumen over the level achieved simply by increasing the vascular pressure (35). This model of airway edema suggested that both the airway wall thickness and the size of the lumen are affected at baseline by airway turgidity. These changes in lumen and airway wall geometry can severely increase the degree of airway responsiveness as discussed in section 4.1.3. In fact, using this dog model of airway engorgement by saline infusion, it was shown that airway responsiveness to histamine increases for the same level of ASM shortening (34). Leakage of plasma protein in the epithelial lining fluid can also be detrimental for airway narrowing and closure. Fibrin, for example, inactivates the surfactant and can increase airway responsiveness by increasing the surface tension (238).

#### **4.2.3 Inflammatory cells and molecules**

In addition to mucus accumulation and edema, which increase airway responsiveness mainly by changing the luminal and airway wall geometry, other inflammatory changes may increase directly ASM contractility.

At the cellular level, mast cells have been linked to increased ASM contractility. The number of mast cells has been shown to be elevated in ASM tissues of asthmatics compared to nonasthmatic subjects with or without other inflammatory disorders (31). The participation of mast cells in AHR has been suggested based on correlations observed between the percentage of mast cells recovered in the bronchoalveolar lavage fluid (BALF) (64) or interspersed in the ASM tissue (113) and the degree of airway responsiveness. Interestingly, the later correlation was even stronger when the mast cells with a fibroblastoid phenotype, which spontaneously produce more histamine, were counted (113). It was also demonstrated using immunohistological sections of human airways that ASM cells that localized in vicinity of mast cells express higher levels of -smooth muscle actin (-SMA), suggesting a paracrine influence of mast cells that increases ASM contractility (255). Taken together, these results suggest that the release of mast cell-derived mediators or direct mast cell-ASM contact could contribute to AHR by making the ASM stronger.

An earlier study also suggested that a short period of mast cell activation alone is sufficient to induce AHR in mice. In that study, it was shown that acute activation of mast cells by an anti-IgE antibody 20 min prior to methacholine (MCh) challenge increases airway responsiveness in wild-type naïve (i.e., non-sensitized and challenged) animals but not in mast cell-deficient naïve animals (146). Apart from histamine, mast cells can release a plethora of inflammatory mediators upon activation. As it is the case for histamine, some of these mediators increase ASM-tone directly by triggering ASM contraction, such as prostaglandin D2 and leukotrienes. It would be a hard task to distinguish the effect of inflammation from the effect of increased ASM-tone on airway responsiveness. This is because a lot of molecules that are part of the asthmatic inflammatory processes are spasmogens. The increase ASM-tone caused by inflammation-derived spasmogens will be discussed separately in a following subsection (4.3). In the present subsection, the focus is on the inflammatory mediators that are not spasmogenic but have been shown to increase ASM contractility.

At the molecular level, several asthma triggers have been shown to increase ASM contractility *ex vivo*. For example, ASM contractility has been shown to be enhanced following prolong (at least 16 h) incubation with atopic serum (15, 19, 78, 89, 91, 161, 211, 245, 246), IgE immune complex (80, 89, 250) and exogenous asthma triggers such as the house dust mite allergen *Der p 1* (82), the bacterial endotoxin lipopolysaccharide (LPS) (8, 166, 216, 220) and the rhinovirus (serotype 16) (78, 81) or the virus mimetic toll-like receptor (TLR)3 ligand polyinosinic polycytidylic acid (poly-IC) (8). Most of these studies assessed ASM contractility by measuring its force-generating capacity. However, some studies also reported that some of these inflammatory insults change the velocity and amount of shortening of the ASM in response to a spasmogen (161), as well as its ability to relax in response to bronchodilators (8, 15, 78, 81, 89, 216). The half-time of relaxation after short EFS-induced tetani has also been shown to increase (double) in ASM strips derived from dogs sensitized to ragweed (109).

The effect of asthma triggers on *ex vivo* ASM contractility can be indirect; i.e., due to an autocrine loop of mediators that are produced by the ASM in response to asthma triggers (79, 80, 91, 166). *In vivo*, the paracrine influence of other cells that are responsive to asthma triggers can also impact ASM contractility. Ultimately, all the inflammatory mediators overexpressed in asthma, whether they originate from other cells or from the ASM itself, can contribute individually or in combination to ASM hypercontractility and AHR. Supports for this contention are accumulating. Cytokines such as interleukin (IL)-1 (11, 92, 200, 250), tumor necrosis factor (TNF) (4, 7, 167, 168, 172, 191, 193, 200, 227), the combination of TNF and IL-1 (90, 92), IL-13 (41, 62, 80, 228), IL-5 (93, 250), IL-10 (79), granulocytemacrophage colony-stimulating factor (GM-CSF) (94), interferon (IFN) (5), leukemia inhibitory factor (LIF) (63, 119) and transforming growth factor (TGF) (75, 255), as well as

interspersed in the ASM tissue (113) and the degree of airway responsiveness. Interestingly, the later correlation was even stronger when the mast cells with a fibroblastoid phenotype, which spontaneously produce more histamine, were counted (113). It was also demonstrated using immunohistological sections of human airways that ASM cells that localized in vicinity of mast cells express higher levels of -smooth muscle actin (-SMA), suggesting a paracrine influence of mast cells that increases ASM contractility (255). Taken together, these results suggest that the release of mast cell-derived mediators or direct mast

An earlier study also suggested that a short period of mast cell activation alone is sufficient to induce AHR in mice. In that study, it was shown that acute activation of mast cells by an anti-IgE antibody 20 min prior to methacholine (MCh) challenge increases airway responsiveness in wild-type naïve (i.e., non-sensitized and challenged) animals but not in mast cell-deficient naïve animals (146). Apart from histamine, mast cells can release a plethora of inflammatory mediators upon activation. As it is the case for histamine, some of these mediators increase ASM-tone directly by triggering ASM contraction, such as prostaglandin D2 and leukotrienes. It would be a hard task to distinguish the effect of inflammation from the effect of increased ASM-tone on airway responsiveness. This is because a lot of molecules that are part of the asthmatic inflammatory processes are spasmogens. The increase ASM-tone caused by inflammation-derived spasmogens will be discussed separately in a following subsection (4.3). In the present subsection, the focus is on the inflammatory mediators that are not spasmogenic but have been shown to increase ASM

At the molecular level, several asthma triggers have been shown to increase ASM contractility *ex vivo*. For example, ASM contractility has been shown to be enhanced following prolong (at least 16 h) incubation with atopic serum (15, 19, 78, 89, 91, 161, 211, 245, 246), IgE immune complex (80, 89, 250) and exogenous asthma triggers such as the house dust mite allergen *Der p 1* (82), the bacterial endotoxin lipopolysaccharide (LPS) (8, 166, 216, 220) and the rhinovirus (serotype 16) (78, 81) or the virus mimetic toll-like receptor (TLR)3 ligand polyinosinic polycytidylic acid (poly-IC) (8). Most of these studies assessed ASM contractility by measuring its force-generating capacity. However, some studies also reported that some of these inflammatory insults change the velocity and amount of shortening of the ASM in response to a spasmogen (161), as well as its ability to relax in response to bronchodilators (8, 15, 78, 81, 89, 216). The half-time of relaxation after short EFS-induced tetani has also been shown to increase (double) in ASM strips derived from

The effect of asthma triggers on *ex vivo* ASM contractility can be indirect; i.e., due to an autocrine loop of mediators that are produced by the ASM in response to asthma triggers (79, 80, 91, 166). *In vivo*, the paracrine influence of other cells that are responsive to asthma triggers can also impact ASM contractility. Ultimately, all the inflammatory mediators overexpressed in asthma, whether they originate from other cells or from the ASM itself, can contribute individually or in combination to ASM hypercontractility and AHR. Supports for this contention are accumulating. Cytokines such as interleukin (IL)-1 (11, 92, 200, 250), tumor necrosis factor (TNF) (4, 7, 167, 168, 172, 191, 193, 200, 227), the combination of TNF and IL-1 (90, 92), IL-13 (41, 62, 80, 228), IL-5 (93, 250), IL-10 (79), granulocytemacrophage colony-stimulating factor (GM-CSF) (94), interferon (IFN) (5), leukemia inhibitory factor (LIF) (63, 119) and transforming growth factor (TGF) (75, 255), as well as

cell-ASM contact could contribute to AHR by making the ASM stronger.

contractility.

dogs sensitized to ragweed (109).

the protease -tryptase (212, 255) were shown to increase ASM contractility. In turn, the effect of these mediators is not always direct. For example, the potentiating effect of IL-1 on bradykinin-induced contraction is due to a greater release of thromboxane (Tx)A2 in response to bradykinin (166). As for the asthma triggers, most of these studies assessed ASM contractility by measuring its force-generating capacity. However, inflammatory mediators found in asthmatic airways can also impair ASM relaxation. For example TNF (90, 92), IL-1 (90, 92), IL-13 (127), IL-10 (79), IL-5 (79) and lysophosphatidic acid (LPA) (230) have all been shown to attenuate ASM relaxation elicited by 2-adrenoceptor agonists. In addition, TNF (9, 92) and IL-1 (92) were shown to attenuate the relaxant effect of prostaglandin (PG)E2.

The molecular mechanisms governing the transformation of ASM into a hypercontractile phenotype may differ from one inflammatory mediator to another. In the case of TGF, increased expression of the contractile protein -SMA and actin filamentogenesis have been proposed (75, 76). On the other hand, many inflammatory mediators increase ASM contractility through shared mechanisms related to alterations in calcium handling; either by Ca2+ sensitization via the Rho-ROCK pathway or by increasing the intracellular mobilization of Ca2+ via the CD38/cADPR/RyR pathway (both briefly described below and illustrated in Figure 4).

Ca2+ sensitization is a phenomenon that allows the ASM to produce more force in response to a given mobilization of intracellular calcium concentration ([Ca2+]i). Ca2+ sensitization seems to rely mainly on a signaling pathway running in parallel to canonical Ca2+ signaling pathways (inositol 3-phosphate receptor (IP3R)-dependent and entry from the extracellular compartment). The pathway is referred to as the Rho-Rho-associated, coiled coil-containing kinase (ROCK) pathway and is activated by certain G protein-coupled receptors (GPCRs) following ligation with their cognate spasmogen (see Figure 4). Rho is a small G (GTPase) protein activated by the exchange of GDP for GTP. The identity of the guanine exchange factor (GEF) that is activated by the GPCR and involved in Rho activation is unclear, and is probably receptor specific. However, one of the downstream signals mediated by Rho that leads to Ca2+ sensitization is well characterized. GTP-bound (active) Rho initially activates ROCK, which then inhibits myosin light chain (MLC) phosphatase (MLCP) both directly, via phosphorylation of the myosin phosphatase-targeting subunit 1 (MYPT1) of MLCP, and indirectly, via CPI-17 (17-kDa PKC-potentiated inhibitory protein of PP1; PP1 stands for protein phosphatase 1, which is the catalytic subunit of the heterotrimeric MLCP). Since the level of MLC phosphorylation depends on a balance between its phosphorylation by MLCK and its dephosphorylation by MLCP, direct or indirect inhibition of MLCP by ROCK causes more MLC phosphorylation for the same degree of MLCK activation. Therefore, when the Rho-ROCK pathway is activated, the same degree of MLCK activation induced by a given sarcoplasmic Ca2+ mobilization leads to more MLC phosphorylation and sequentially more activated cross-bridges and more force.

AHR in animal models of airway inflammation has been attributed to an increased ASMsensitivity to Ca2+ (40, 43). Ca2+ sensitization is more typically triggered by inflammationderived spasmogens (discussed in section 4.3.1), which bind to GPCRs capable of activating the Rho-ROCK pathway. However, non-spasmogenic inflammatory mediators such as TNF have also been shown to potentiate ASM-force by fostering Ca2+ sensitization (107, 172, 191). In addition, the extent of Ca2+ sensitization can be regulated by mediators affecting the levels of expression or activation of key regulatory proteins involved in the Rho-ROCK pathway. For example, IL-13 (41) increases the level of RhoA expression in ASM, which consequently increases the proficiency of Rho-ROCK signaling pathway to induce Ca2+

Fig. 4. Pathways transducing the extracellular signal of the spasmogens from their cognate cell-surface receptor to the contractile apparatus. See text for descriptions and abbreviations. The green and red dots represent Ca2+ and Na+, respectively. The reverse mode of the Na+/Ca2+ exchanger (NCX) is shown. Reproduced with permission from: Bossé and Paré. Airway smooth muscle responsiveness: The origin of airway hyperresponsiveness in asthma? Current Respiratory Medicine Reviews. 7(4): 289-301, 2011.

Fig. 4. Pathways transducing the extracellular signal of the spasmogens from their cognate cell-surface receptor to the contractile apparatus. See text for descriptions and abbreviations. The green and red dots represent Ca2+ and Na+, respectively. The reverse mode of the Na+/Ca2+ exchanger (NCX) is shown. Reproduced with permission from: Bossé and Paré. Airway smooth muscle responsiveness: The origin of airway hyperresponsiveness in

asthma? Current Respiratory Medicine Reviews. 7(4): 289-301, 2011.

sensitization. On the other hand, mediators capable of reducing the expression/activity of intermediate proteins involved in this pathway may have therapeutic potential for diminishing Ca2+ sensitization and AHR. One example is the effect of glucocorticoids in reducing the expression of RhoA, which concomitantly attenuates antigen-induced ASM hyperresponsiveness in a rat model of allergic airway inflammation (40).

In addition to canonical Ca2+ signaling pathways, at least one other Ca2+ signaling pathway contributes to the release of Ca2+ into the sarcoplasm (see Figure 4). This pathway is referred to as the CD38/cyclic adenosine-5'-diphosphate (ADP) ribose (cADPR) signaling pathway and has received significant attention recently in regard to its potential role in AHR. CD38 is a type II transmembrane protein of 45-kDa with dual ecto-enzymatic activity. It possesses an ADP-ribosyl cyclase activity, converting -NAD into cADPR and a cADPR hydrolase activity, converting the cADPR into ADPR. The product of the ADP-ribosyl cyclase activity, cADPR, sensitizes the ryanodine receptor (RyR) for Ca2+ release and, more specifically, potentiates Ca2+-induced Ca2+ release (CICR) (70). Whether cADPR binds directly to RyR or requires other intermediates to open the RyR channel is still a matter of debate (54). What is sure is that cADPR increases the open probability of the RyR, liberating more Ca2+ from the sarcoplasmic reticulum (SR) (196). As is the case for IP3R-dependent Ca2+ release, the CD38 pathway is also activated by binding of specific spasmogens to their cognate receptors (252). Thus, this pathway acts in parallel with the canonical Ca2+ signaling pathways to amplify the mobilization of Ca2+ into the sarcoplasm following spasmogenic stimulation.

The amplitude of Ca2+ release by the CD38/cADPR/RyR pathway, as well as its contribution in ASM-force generation, is influenced by CD38 expression. CD38 expression and/or activity were shown to be upregulated by many of the pro-inflammatory cytokines present in asthmatic airways, such as IFN (229), IFN (53), TNF (53, 229), IL-1 (53) and IL-13 (52). Therefore, in the presence of inflammation, the proficiency of this signaling pathway to liberate the Ca2+ from the internal stores may be enhanced. Consequently, higher force generation would be attained for a given contractile stimulus since more Ca2+ would be released. The influence of inflammatory mediators on the CD38/cADPR/RyR pathway may thus play a role in AHR seen in asthma.

Taken together, these results suggested that the contractile properties of ASM are not fixed but rather plastic; i.e., they can change rapidly in the face of external (inflammatory) cues. So in an inflammatory lung disorder such as asthma, the ASM may be stronger or faster, for example, because of the 'bad' environment in which it is embedded. This increased contractility may only be present *in vivo* when the muscle is exposed to inflammationderived mediators, but might be lost once the muscle is removed from the airways and washed repetitively before being studied *ex vivo.* These results may offer an explanation for the failure of *ex vivo* studies to show an increased strength of ASM from asthmatics (reviewed in (153)). The ability of ASM to rapidly change its contractile capacity in response to inflammatory molecules can also contribute to changes in airway responsiveness observed in response to interventions altering airway inflammation (30, 47, 143, 218). Those are all different ways by which a normal ASM may account for AHR in an inflamed environment. Another way by which inflammation can increase airway responsiveness is by producing spasmogens, which increases ASM-tone.

#### **4.3 Increased ASM-tone**

The responsiveness of asthmatics to bronchodilators, together with more direct evidence (165), indicate that ASM-tone is increased in asthmatic airways. The origin of this augmented ASM-tone is not clear and likely varies between asthmatic individuals. Nonetheless, asthmatic lungs are characterized by the overexpression of inflammationderived spasmogens. These spasmogens trigger sustained ASM contraction and, acting individually or collectively, are a likely cause of increased ASM-tone in asthma. In the present subsection, we discuss the mechanisms by which inflammation-derived spasmogens, together with the attendant increase in ASM-tone, can contribute to AHR.

The rationale is that the amount of airway narrowing in response to a given concentration of a chosen spasmogen (e.g., MCh) is due to the combined effect of this extrinsically delivered spasmogen plus the baseline tone, which is caused by the endogenous spasmogens already present. In other words, it is the cooperative effect of all the spasmogens present at the time of the challenge that determines the total level of ASM activation and, ultimately, the amount of airway narrowing achieved. In addition, one of the causes of AHR can be the contractile synergistic interactions that are often observed when different spasmogens are used in combination. In this regard, a sizable literature exists documenting contractile synergisms between different spasmogens (Table 1). The mechanisms involved in these synergistic interactions have been debated in the last few decades and it is now clear that all the mechanisms that may be involved are not mutually exclusive. The nature of these interplays can give us important clues regarding the role of baseline airway tone in determining the degree of airway responsiveness.

#### **4.3.1 Interactive synergisms between spasmogens**

Initial studies in that area begin in the late 1970s and have started by looking at the combined effect of spasmogens on different measures of lung function in animals (45, 46, 57, 99, 102, 112, 131, 152, 180, 181, 192, 208, 209, 224, 244), as well as in humans (49, 68, 97, 101, 110, 156, 158, 194, 202, 240). Apart from an interactive synergism at the level of ASM contraction, many other factors can account for these *in vivo* interactive synergisms. In fact, although these synergistic interactions can be extremely relevant to the understanding of AHR, the mechanisms involved are sometime impossible to delineate.

First, the interactions between spasmogens were assessed *in vivo* by measuring airway resistance (or conductance) and/or flow parameters (flow rate at different lung volumes, forced expiratory volume in 1 sec (FEV1), etc...). All of these measurements are geometrydependent. As mentioned earlier, airway resistance is inversely proportional to the luminal radius at the 4th power. The potentiation of the effect of one spasmogen by another spasmogen (or an increased baseline tone) might be related to a geometrical effect more than a true synergistic interaction of spasmogens on ASM-force. In other words, an additive effect at the level of muscle activation that may result in an additive effect in terms of ASMforce generation and shortening can be perceived as a synergistic effect when looking at airway resistance.

To circumvent these limitations, others have used *in situ* preparations allowing the assessment of tracheal smooth muscle-isometric force (12, 33, 131) or have measured airway caliber using tantalum bronchography (17). Some of these studies confirmed the synergistic effect of spasmogens on airway narrowing and ASM-force generation. However, these results are also confounded by the acute effect of some spasmogens on vessels and nerves, or by the effect of some spasmogens on airway inflammation and the epithelium. In regard to the vessels, many airway spasmogens also have vasoactive properties. Both vasoconstrictors and vasodilators have significant impact on the degree of airway

augmented ASM-tone is not clear and likely varies between asthmatic individuals. Nonetheless, asthmatic lungs are characterized by the overexpression of inflammationderived spasmogens. These spasmogens trigger sustained ASM contraction and, acting individually or collectively, are a likely cause of increased ASM-tone in asthma. In the present subsection, we discuss the mechanisms by which inflammation-derived spasmogens, together with the attendant increase in ASM-tone, can contribute to AHR. The rationale is that the amount of airway narrowing in response to a given concentration of a chosen spasmogen (e.g., MCh) is due to the combined effect of this extrinsically delivered spasmogen plus the baseline tone, which is caused by the endogenous spasmogens already present. In other words, it is the cooperative effect of all the spasmogens present at the time of the challenge that determines the total level of ASM activation and, ultimately, the amount of airway narrowing achieved. In addition, one of the causes of AHR can be the contractile synergistic interactions that are often observed when different spasmogens are used in combination. In this regard, a sizable literature exists documenting contractile synergisms between different spasmogens (Table 1). The mechanisms involved in these synergistic interactions have been debated in the last few decades and it is now clear that all the mechanisms that may be involved are not mutually exclusive. The nature of these interplays can give us important clues regarding the role of baseline airway tone in

Initial studies in that area begin in the late 1970s and have started by looking at the combined effect of spasmogens on different measures of lung function in animals (45, 46, 57, 99, 102, 112, 131, 152, 180, 181, 192, 208, 209, 224, 244), as well as in humans (49, 68, 97, 101, 110, 156, 158, 194, 202, 240). Apart from an interactive synergism at the level of ASM contraction, many other factors can account for these *in vivo* interactive synergisms. In fact, although these synergistic interactions can be extremely relevant to the understanding of

First, the interactions between spasmogens were assessed *in vivo* by measuring airway resistance (or conductance) and/or flow parameters (flow rate at different lung volumes, forced expiratory volume in 1 sec (FEV1), etc...). All of these measurements are geometrydependent. As mentioned earlier, airway resistance is inversely proportional to the luminal radius at the 4th power. The potentiation of the effect of one spasmogen by another spasmogen (or an increased baseline tone) might be related to a geometrical effect more than a true synergistic interaction of spasmogens on ASM-force. In other words, an additive effect at the level of muscle activation that may result in an additive effect in terms of ASMforce generation and shortening can be perceived as a synergistic effect when looking at

To circumvent these limitations, others have used *in situ* preparations allowing the assessment of tracheal smooth muscle-isometric force (12, 33, 131) or have measured airway caliber using tantalum bronchography (17). Some of these studies confirmed the synergistic effect of spasmogens on airway narrowing and ASM-force generation. However, these results are also confounded by the acute effect of some spasmogens on vessels and nerves, or by the effect of some spasmogens on airway inflammation and the epithelium. In regard to the vessels, many airway spasmogens also have vasoactive properties. Both vasoconstrictors and vasodilators have significant impact on the degree of airway

determining the degree of airway responsiveness.

airway resistance.

**4.3.1 Interactive synergisms between spasmogens** 

AHR, the mechanisms involved are sometime impossible to delineate.



Abbreviations in the table: AII, angiotensin II; ACh, acetylcholine; ATP, adenosine triphosphate; BK, bradykinin; CCh, carbachol; Cys-LTs, cysteinyl-leukotrienes; ET-1, endothelin-1; HIST, histamine; 5-HT, 5-hydroxytryptamine (also called serotonine); K+, potassium; LPA, lysophosphatidic acid; LTB4, leukotriene B4; MCh, methacholine; NaF, sodium fluoride; NE, norepinephrine; PA, phosphatidic acid; PAF, platelet activating factor; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2, prostaglandin F2; SM, smooth muscle; SP, substance P; S1P, sphingosine-1-phosphate; TXA2, thromboxane A2.

Table 1. Interactions between spasmogens increasing ASM contractility

responsiveness (151). The potentiating effect of histamine on the responsiveness to muscarinic agonists (33, 131), for example, can likely be due to its vasodilating effect, especially when the drugs are administered intravenously or via the tracheal vasculature. This is because vasodilatation facilitates the delivery of the bronchoactive substance (ACh) to the ASM. Histamine also increases airway epithelium permeability by altering Ecadherin-based adhesions (262). The effect of a second inhaled spasmogen may thus be potentiated by histamine pre-treatment because the second spasmogen will have easier access to the ASM.

In regard to the nerves, many studies have shown synergistic interactions between spasmogens and electrical stimulation of the vagal nerves (17, 33, 57, 88, 114, 137, 217, 258). Similarly, a decrease in responsiveness to spasmogens (other than ACh (88)) was observed following vagotomy (59, 137, 157, 258), cooling of the vagal nerves (58, 88), or treatments with either atropine (59, 219), tetrodotoxin (219) or hexamethonium (59). These results suggest that the basal cholinergic tone synergistically interacts with the exogenously delivered spasmogen. These studies are not included in Table 1 because it is not clear whether the potentiating effect is due to a true synergistic effect at the level of ASM-force generation or due to the ability of the spasmogens to either trigger a vagal reflex (36, 58, 74, 157) or to increase cholinergic neurotransmission and/or synaptic transmission at the

**Mediators** *in vivo ex vivo in vitro* 

rings;

strips;

(42)

rings

S1P MCh (122) in guinea pig tracheal

LPA MCh (230), 5-HT (230), SP (230) in

PA MCh (230) in rabbit tracheal rings

Table 1. Interactions between spasmogens increasing ASM contractility

Abbreviations in the table: AII, angiotensin II; ACh, acetylcholine; ATP, adenosine triphosphate; BK, bradykinin; CCh, carbachol; Cys-LTs, cysteinyl-leukotrienes; ET-1, endothelin-1; HIST, histamine; 5-HT, 5-hydroxytryptamine (also called serotonine); K+, potassium; LPA, lysophosphatidic acid; LTB4, leukotriene B4; MCh, methacholine; NaF, sodium fluoride; NE, norepinephrine; PA, phosphatidic acid; PAF, platelet activating factor; PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2, prostaglandin F2; SM, smooth muscle; SP, substance P; S1P, sphingosine-1-phosphate; TXA2, thromboxane A2.

responsiveness (151). The potentiating effect of histamine on the responsiveness to muscarinic agonists (33, 131), for example, can likely be due to its vasodilating effect, especially when the drugs are administered intravenously or via the tracheal vasculature. This is because vasodilatation facilitates the delivery of the bronchoactive substance (ACh) to the ASM. Histamine also increases airway epithelium permeability by altering Ecadherin-based adhesions (262). The effect of a second inhaled spasmogen may thus be potentiated by histamine pre-treatment because the second spasmogen will have easier

In regard to the nerves, many studies have shown synergistic interactions between spasmogens and electrical stimulation of the vagal nerves (17, 33, 57, 88, 114, 137, 217, 258). Similarly, a decrease in responsiveness to spasmogens (other than ACh (88)) was observed following vagotomy (59, 137, 157, 258), cooling of the vagal nerves (58, 88), or treatments with either atropine (59, 219), tetrodotoxin (219) or hexamethonium (59). These results suggest that the basal cholinergic tone synergistically interacts with the exogenously delivered spasmogen. These studies are not included in Table 1 because it is not clear whether the potentiating effect is due to a true synergistic effect at the level of ASM-force generation or due to the ability of the spasmogens to either trigger a vagal reflex (36, 58, 74, 157) or to increase cholinergic neurotransmission and/or synaptic transmission at the

tracheal strips;

pig tracheal rings

rabbit tracheal rings;

K+ (215) and CCh (215) in porcine

ACh (48) and HIST (48) in guinea

K+ in bronchial rings from normal but not antigen-challenged mice

> ATP (205) and K+ (205) in bovine tracheal SM cells (205)

MCh (230) in cat tracheal rings; ACh (98) in guinea pig tracheal

ET-1 (195) in bovine bronchial

LTB4 ACh (181) in dogs Cys-LTs HIST (194) and PGD2

access to the ASM.

(194) in humans; PAF (192) in rhesus

monkeys

ganglia (217). These latter phenomena would lead to more ACh release and concomitantly higher ASM activation for the same neural input. On the other hand, the bronchoconstricting effect of spasmogens is known to decrease the vagal tone (13, 210). This is because of their indirect effect on the slowly adapting stretch receptors (SARs), which, upon an enhanced discharge, relax the ASM by reducing parasympathetic tone to the airways. However, other spasmogens, such as bradykinin, would synergize with the effect of these mechanoreceptors via C-fibers activation to initiate the bronconconstricting reflex (150). Together, the influence of the nervous system is intricate and certainly a major confounding factor *in vivo* when one tries to elucidate the synergistic interaction between spasmogens.

Finally, thickening of the airway wall by edema and inflammatory infiltrates, encroachment of the airway lumen by mucus hypersecretion and cellular debris, and reduction in the forces of interdependence between the airway wall and the lung parenchyma by the accumulation of inflammatory exudates in the adventitial layer are all means by which airway inflammation can increase airway responsiveness without affecting ASM-force (reviewed in (26)). Therefore, the hyperresponsiveness observed in response to the second spasmogen, when the first spasmogen is administered hours before the second spasmogen (180), may be due to the inflammation provoked by the initial spasmogen.

To avoid these confounding factors and to investigate whether the synergistic action of two spasmogens on airway responsiveness relies on mechanisms operating at the level of ASM, the only solution is to study the ASM tissue in isolation. Many investigators have focused on this strategy. Most studies were performed using freshly dissected ASM strips or bronchial rings from which isometric force was measured *ex vivo* (12, 32, 38, 48, 71, 87, 98, 122, 135, 156, 173, 174, 184, 185, 195, 203, 204, 215, 230). *In vitro* preparations (i.e., cell culture) have also been used on occasions (205). These reported synergisms are also enumerated inTable 1. The molecular mechanisms involved in contractile synergisms observed between different pairs of spasmogens have been extensively studied. The synergisms involve the co-activation of ASM by two spasmogens acting on two distinct GPCRs. The activation of two distinct GPCRs can lead to intracellular signaling synergisms that ultimately potentiate ASM-force. The more commonly discussed mechanism is Ca2+ sensitization, defined as an increased ASMforce generation in response to a given amount of intracellular Ca2+ mobilization (as discussed in subsection 4.2.3). Many spasmogenic mediators, such as leukotriene C4 (215), isoprostanes (135), ATP (184), endothelin-1 (260), LPA (205), TxA2 (167) and S1P (122), have been shown to increase the responsiveness of the contractile apparatus to Ca2+.

However, these synergisms in terms of force generation can also be related to an additive effect in terms of ASM activation, especially when the concentrations of spasmogens used are low. This is because the dose-response curve to any spasmogen is sigmoidal, regardless of whether the response is measured in terms of ASM-force or shortening. That means that initially, a threshold concentration is needed before any response can be measured, but then there is a progressive increase toward a steeper, relatively linear slope followed by a progressive decrease in slope to reach a plateau. When two different spasmogens are used in combination, their additive effect on ASM-activation could be erroneously interpreted as being synergistic because sub-threshold concentrations of two spasmogens could have a measurable effect on ASM-force or shortening. Similarly, if one uses a concentration just above the response threshold, the subsequent addition of a different or the same spasmogen will have more effect simply because it is now acting on the steeper part of the doseresponse curve. In fact, many of the previously documented examples of 'synergism' performed either *in vivo* (110, 112, 131, 158, 244) or *ex vivo* (38, 71, 87, 135) were involving low concentrations of spasmogens, and were unable to demonstrate a synergism at higher concentrations for at least some of the reported spasmogenic interactions (71, 87, 131). So the level of basal tone, even if low, can seemingly synergize with the extrinsically delivered spamogen to cause AHR simply by an additive effect at the level of ASM activation

Collectively, the force potentiation of one spasmogen (or baseline tone) on another spasmogen can be due to many factors such as: 1-use of sub-threshold or barely active concentrations of spasmogens, which simply act additively in terms of ASM activation but will be perceived as being synergistic in terms of ASM-force, ASM shortening, airway narrowing or airway resistance; 2-use of geometry-dependent measurements such as airway resistance; so that the effect of the second spasmogen is augmented by the reduced airway caliber caused by the first spasmogen; 3-a synergistic effect due to the contribution of vascular, neural or inflammatory effects when tested *in vivo* or *in situ*, which can be observed when the spasmogens used have vasoactive and inflammatory activities or when they can trigger either a vagal reflex and/or an increase in the efferent cholinergic traffic; and 4-a true synergistic effect on myosin cross-bridge cycling due to the convergence of intracellular signaling pathways that are activated by the ligation of two distinct GPCRs (such as Ca2+ sensitization).

#### **4.3.2 Force adaptation**

There is an additional, more subtle way by which the tone can contribute to AHR. Recent studies from our laboratory have demonstrated that a short period (~30 min) of increased ASM-tone augments ASM strength over time (20, 21). Specifically, ASM was exposed to ACh and ASM-force generation in response to EFS was monitored both before and after the induction of the ACh-induced tone. The first EFS in the presence of tone was given 1 min post ACh administration and then at 5-min intervals thereafter. Addition of ACh immediately increased the total force (ACh-induced tone + EFS-induced force), suggesting that ASM generates more force in response to two sub-maximal stimuli (ACh + EFS) compared with EFS alone. More surprisingly, while the EFS-induced force decreased immediately following the administration of ACh, it recovered significantly over time. Since the tone produced by the exogenous ACh remained relatively constant, the total force increased over time. We termed this phenomenon 'force adaptation' (20). The potential significance of force adaptation on airway narrowing based on a computational model has been demonstrated recently (Bossé *et al*., in press in the journal of Respiratory Physiology & Neurobiology).

Force adaptation is distinct from the synergisms in force that were attributed to the convergence of intracellular signaling pathways described above, which occur as a result of activation of two distinct GPCRs. This form of synergism cannot occur when ACh is combined with activation of the ASM by EFS, which was the experimental setting that was used to describe force adaptation. This is because EFS triggers ASM contraction by releasing ACh from the nerve endings (145), which, in turn, binds on the same M3 receptor as the exogenously administered ACh. In fact, all studies investigating the acute effect of a muscarinic agonist on EFS-induced force have failed to show a synergistic effect (57, 114). Therefore, despite abundant literature reporting synergistic effects between spasmogens on ASM strength, force adaptation is a phenomenon with no precedent.

The molecular mechanisms involved in force adaptation are also likely to be different from those described above because it occurs over minutes. This time-course would be too fast for

performed either *in vivo* (110, 112, 131, 158, 244) or *ex vivo* (38, 71, 87, 135) were involving low concentrations of spasmogens, and were unable to demonstrate a synergism at higher concentrations for at least some of the reported spasmogenic interactions (71, 87, 131). So the level of basal tone, even if low, can seemingly synergize with the extrinsically delivered

Collectively, the force potentiation of one spasmogen (or baseline tone) on another spasmogen can be due to many factors such as: 1-use of sub-threshold or barely active concentrations of spasmogens, which simply act additively in terms of ASM activation but will be perceived as being synergistic in terms of ASM-force, ASM shortening, airway narrowing or airway resistance; 2-use of geometry-dependent measurements such as airway resistance; so that the effect of the second spasmogen is augmented by the reduced airway caliber caused by the first spasmogen; 3-a synergistic effect due to the contribution of vascular, neural or inflammatory effects when tested *in vivo* or *in situ*, which can be observed when the spasmogens used have vasoactive and inflammatory activities or when they can trigger either a vagal reflex and/or an increase in the efferent cholinergic traffic; and 4-a true synergistic effect on myosin cross-bridge cycling due to the convergence of intracellular signaling pathways that are activated by the ligation of two distinct GPCRs

There is an additional, more subtle way by which the tone can contribute to AHR. Recent studies from our laboratory have demonstrated that a short period (~30 min) of increased ASM-tone augments ASM strength over time (20, 21). Specifically, ASM was exposed to ACh and ASM-force generation in response to EFS was monitored both before and after the induction of the ACh-induced tone. The first EFS in the presence of tone was given 1 min post ACh administration and then at 5-min intervals thereafter. Addition of ACh immediately increased the total force (ACh-induced tone + EFS-induced force), suggesting that ASM generates more force in response to two sub-maximal stimuli (ACh + EFS) compared with EFS alone. More surprisingly, while the EFS-induced force decreased immediately following the administration of ACh, it recovered significantly over time. Since the tone produced by the exogenous ACh remained relatively constant, the total force increased over time. We termed this phenomenon 'force adaptation' (20). The potential significance of force adaptation on airway narrowing based on a computational model has been demonstrated recently (Bossé *et al*., in press in the journal of Respiratory Physiology &

Force adaptation is distinct from the synergisms in force that were attributed to the convergence of intracellular signaling pathways described above, which occur as a result of activation of two distinct GPCRs. This form of synergism cannot occur when ACh is combined with activation of the ASM by EFS, which was the experimental setting that was used to describe force adaptation. This is because EFS triggers ASM contraction by releasing ACh from the nerve endings (145), which, in turn, binds on the same M3 receptor as the exogenously administered ACh. In fact, all studies investigating the acute effect of a muscarinic agonist on EFS-induced force have failed to show a synergistic effect (57, 114). Therefore, despite abundant literature reporting synergistic effects between spasmogens on

The molecular mechanisms involved in force adaptation are also likely to be different from those described above because it occurs over minutes. This time-course would be too fast for

ASM strength, force adaptation is a phenomenon with no precedent.

spamogen to cause AHR simply by an additive effect at the level of ASM activation

(such as Ca2+ sensitization).

**4.3.2 Force adaptation** 

Neurobiology).

*de novo* gene expression; so it is not likely due to transcriptional activation of genes involved in ASM contraction induced by the initial tone. On the other hand, if it was due to biochemical events such as increased phosphorylation of MLC due MLCP deactivation leading to Ca2+ sensitization (as described in subsection 4.2.3), the potentiating effect would be very fast and seen at the first EFS following ACh administration (1 min). Taken together, the kinetic of force adaptation is too slow to be explained by fast biochemical reactions, such as protein phosphorylation, but too fast for *de novo* gene expression. We thus suggest that this time-dependent increase in ASM-force is due to post-translational mechanisms. All the necessary machinery responsible for this increased ASM-force might be present within the cell prior to activation, but it is only upon stimulation (i.e., induction of tone) that this protein machinery re-organizes to optimize generation of force. Processes such as actin (264) or myosin (213) filamentogenesis and/or re-arrangement of the cytoskeleton and its connectivity with the contractile apparatus, the plasmalemma and the ECM (264) are likely possibilities. However, these remain pure speculations as the mechanisms underlying force adaptation have never been investigated.

#### **4.3.3 Increased tone on airway wall stiffness**

Apart from ASM-force, increased ASM-tone can also impact other ASM contractile properties. For instance, stiffness is greatly enhanced upon ASM activation. By increasing ASM stiffness, an increased tone would reduce the strains experienced by the airway wall due to the cyclical stresses of breathing. Interestingly, Noble and coworkers (178) have demonstrated that the amount of airway wall strain induced by a DI is proportional to its bronchodilating effect (178). They also showed that the main factor limiting the strain was the magnitude of ASMtone, which was controlled by delivering different concentrations of ACh. These observations suggested that the ASM needs not only to be stressed but to be stretched for a DI to be effective in reducing ASM contractility. They concluded by saying that a stiffer airway caused by an increased ASM-tone reduces the strain induced by the dynamic load associated with DI and, for this reason, reduces the bronchodilating effect of a DI.

Taken all together, these observations suggest that baseline tone is of crucial importance in determining the degree of airway responsiveness to an inhalational challenge with a spasmogen. The increased tone observed in asthma can be from inflammatory and/or vagal origin. In both cases, this increased tone acts additively with the extrinsically delivered spamogen in terms of ASM activation, force generation, ASM shortening and airway diameter narrowing. This can also be translated into a synergistic effect when geometrydependent measurements are taken, such as airway cross-sectional area of the lumen or airflow resistance (or other measurements relying on it, such as peak expiratory flow (PEF), FEV1, …). In cases where the increased tone is mediated by inflammation-derived spasmogens, the potentiating effect of increased tone on airway responsiveness can also be due to a vascular effect, a neural effect, an inflammatory effect and/or an increase in ASMforce caused by either Ca2+ sensitization or force adaptation. Finally, augmented tone also increases ASM stiffness and, concomitantly, airway wall stiffness, which can further enhance airway responsiveness by many ways as discussed in subsections 2.2 and 2.3.

#### **4.4 Heterogeneity**

To assess airway responsiveness airway resistance is measured at the mouth using the forced oscillation technique or, alternatively, flow parameters are measured using standard spirometry. These measurements take into account airway resistance but also lung tissue and chest wall resistance. Since chest wall resistance does not change during bronchoprovocation, and lung tissue resistance does not change by much, the progressive increase of resistance occurring in response to increasing doses of a spasmogen is thought to reflect changes of airway caliber. In other words, the change of resistance along the course of a challenge is viewed as being due to airway narrowing caused by ASM shortening. However, even this change of resistance is intricate. This is because this resistance represents represents the combined resistance of all generations of airways arranged in series and in parallel. Therefore, simply looking at the factors affecting resistance in a single airway, as we have done since the beginning of this chapter, may not be sufficient to understand the full nature of airway responsiveness.

#### **4.4.1 Mathematical link between airway narrowing heterogeneity and airway responsiveness**

The link between heterogeneity and AHR was initially though as being simply due to geometric considerations. As aforementioned, the relationship between airway luminal radius and resistance to airflow is not linear. A decrease in radius causes an exponential increase in airway resistance. For this reason, even if the total cross sectional area within a given airway generation is the same, inhomogeneous airway caliber increases airway resistance. In other words, the increase in resistance caused by narrowing of some airways is not compensated for by the decrease in resistance caused by dilation of other airways. Airway narrowing heterogeneity upon a bronchoprovocative challenge with a spasmogen will have the same consequence. So that the increases in resistance caused by augmented narrowing in some airways are not compensated for by the attenuated increases in resistance caused by reduced narrowing happening in other airways.

The consequences of baseline heterogeneity on baseline resistance and of non-homogeneous airway narrowing on airway responsiveness were predicted based on computational models. Bates (14) was one of the first to address the issue of heterogeneity. He developed a stochastic model of the airway tree in which the values of airway radii within every airway generation were chosen randomly according to a probability distribution function. The mean values were based on realistic values (morphometric data of human lungs). He found that, while the mean values were kept the same, increasing the standard deviations progressively leads to greater airway resistance. This suggested that the simple presence of heterogeneity increases airway resistance. The model further predicted that, upon ASM activation, both baseline airway caliber heterogeneity and ASM shortening heterogeneity increase airway responsiveness. Obviously, this is only true if one assumes that the flow is not redistributed into more patent airways.

More sophisticated computational models have then been developed and brought additional insights about the origin of this increased resistance evoked by baseline or airway narrowing heterogeneity. The main contributor to this increased resistance comes from nearly closed peripheral airways, especially when resistance is measured at frequency near the breathing frequency (138). These airway closures (or near closed) ultimately lead to ventilation defects and attendant flow limitation. They might be the link between ventilation heterogeneity and AHR.

#### **4.4.2 Non-uniform ventilation with air trapping and AHR**

Using the forced oscillation technique, several groups noticed that the frequencydependence of resistance and elastance was increased at baseline in asthmatics (139) and

and chest wall resistance. Since chest wall resistance does not change during bronchoprovocation, and lung tissue resistance does not change by much, the progressive increase of resistance occurring in response to increasing doses of a spasmogen is thought to reflect changes of airway caliber. In other words, the change of resistance along the course of a challenge is viewed as being due to airway narrowing caused by ASM shortening. However, even this change of resistance is intricate. This is because this resistance represents represents the combined resistance of all generations of airways arranged in series and in parallel. Therefore, simply looking at the factors affecting resistance in a single airway, as we have done since the beginning of this chapter, may not be sufficient to

**4.4.1 Mathematical link between airway narrowing heterogeneity and airway** 

resistance caused by reduced narrowing happening in other airways.

The link between heterogeneity and AHR was initially though as being simply due to geometric considerations. As aforementioned, the relationship between airway luminal radius and resistance to airflow is not linear. A decrease in radius causes an exponential increase in airway resistance. For this reason, even if the total cross sectional area within a given airway generation is the same, inhomogeneous airway caliber increases airway resistance. In other words, the increase in resistance caused by narrowing of some airways is not compensated for by the decrease in resistance caused by dilation of other airways. Airway narrowing heterogeneity upon a bronchoprovocative challenge with a spasmogen will have the same consequence. So that the increases in resistance caused by augmented narrowing in some airways are not compensated for by the attenuated increases in

The consequences of baseline heterogeneity on baseline resistance and of non-homogeneous airway narrowing on airway responsiveness were predicted based on computational models. Bates (14) was one of the first to address the issue of heterogeneity. He developed a stochastic model of the airway tree in which the values of airway radii within every airway generation were chosen randomly according to a probability distribution function. The mean values were based on realistic values (morphometric data of human lungs). He found that, while the mean values were kept the same, increasing the standard deviations progressively leads to greater airway resistance. This suggested that the simple presence of heterogeneity increases airway resistance. The model further predicted that, upon ASM activation, both baseline airway caliber heterogeneity and ASM shortening heterogeneity increase airway responsiveness. Obviously, this is only true if one assumes that the flow is

More sophisticated computational models have then been developed and brought additional insights about the origin of this increased resistance evoked by baseline or airway narrowing heterogeneity. The main contributor to this increased resistance comes from nearly closed peripheral airways, especially when resistance is measured at frequency near the breathing frequency (138). These airway closures (or near closed) ultimately lead to ventilation defects and attendant flow limitation. They might be the link between ventilation

Using the forced oscillation technique, several groups noticed that the frequencydependence of resistance and elastance was increased at baseline in asthmatics (139) and

understand the full nature of airway responsiveness.

not redistributed into more patent airways.

**4.4.2 Non-uniform ventilation with air trapping and AHR** 

heterogeneity and AHR.

**responsiveness** 

upon MCh challenge in both asthmatics and non-asthmatics (111, 139). In accordance to the computational models discussed above, this is indicative of a heterogenous pattern of constriction that includes randomly distributed airway closures or near closures (138). These observations are supported by ventilation imaging studies such as hyperpolarized 3He MRI (39, 207, 234), single-photon emission computed tomography (116) and positron emission tomography (169). These studies showed that asthmatics have increased ventilation heterogeneity at baseline and, even if heterogeneous airway response to a spamogen is also observed in non-asthmatics, the level of ventilation heterogeneity achieved is greater in asthmatics (39, 207, 234). This heterogeneous pattern of ventilation is characterized by relatively large zone of non-ventilated area, which supports the presence of airway closure. These ventilations defects represent zones of air trapping, which are characteristic of obstructive diseases and known to contribute to airflow limitation. Ventilation heterogeneity can thus be one of the sources of AHR seen in asthmatics. This is consistent with recent reports showing that baseline ventilation heterogeneity measured by nitrogen washouts correlates with the severity of AHR in both younger (60) and older (96) asthmatic subjects. The increase in ventilation heterogeneity in response to a spasmogenic challenge may simply be due to an amplifying effect over the baseline heterogeneity. However, upon

induced bronchoconstriction, airway narrowing heterogeneity can also contribute to ventilation heterogenetity. King and coworkers (115) have used high resolution computed tomography (HRCT) to measure the heterogeneity of airway narrowing in response to MCh challenge in human subjects. The heterogeneity was measured by the standard deviations of the changes in luminal airway caliber caused by MCh. They showed that in airways of 2 mm of diameter that the variability of airway narrowing was greater than the measure of repeatability (the variability in the changes in luminal airway caliber when two pre-MCh scans were compared), suggesting that airways narrow heterogeneously. They also shown that for the same level of bronchoconstriction achieved, asthmatic airways narrowed more heterogeneously than non-asthmatics. Together, these observations suggest that the larger and more numerous ventilation defects observed in asthmatics during a spasmogenic challenge are due to both greater baseline heterogeneity and greater airway narrowing heterogeneity.

#### **4.4.3 Mechanistic link between airway narrowing heterogeneity and airway responsiveness**

As aforementioned, the emergence of ventilation defects during bronchoconstriction is essentially the link between ventilation heterogeneity and AHR. In other words, simple heterogeneity without airway closure (or near closure) does not seem to cause AHR. It is true that for the same mean level of bronchoconstriction, heterogeneity would increase resistance but only if the flow is still evenly distributed among the different airways. However, during heterogeneous constriction, the flow is more likely to be redirected into more conductant (patent) airways and correspondingly less flow would be redirected into more resistant (constricted) airways. Consequently, the overall resistance would rather decrease with heterogeneity. On the other hand, when the airways are closed (or nearly), the volume of air subtended by these airways is trapped and will not be able to be expelled (at least not as fast) upon maximal expiratory flow maneuvers and that would be the cause of AHR.

It is also unlikely that the ventilation defects are due to random closure of single peripheral airways. Based on the size of these ventilation defects, they are more likely due to clustering of constricted peripheral airways. In a computational model developed by Venegas and coworkers (236, 253), it was shown that airway closure occurs in cluster. In their model, the bronchial tree branches dichotomously, where every bifurcation is composed of one parent airway and two daughter airways with slight heterogeneity between them. During bronchoconstriction, the flow is redistributed according to divergence of patency among daughter airways. The model predicted that all the airways shorten uniformly when the level of ASM activation is low. However, passed a certain threshold of ASM activation, daughter airways develop a dichotomic response. Whereas one constricts excessively the other one dilates. This is because the initial heterogeneity between the two daughter airways fosters the redistribution of flow into the more conductant airway. The insufflation pressure thus rises in the hyperventilated areas subtended by this airway, which increases the load impeding ASM shortening. In fact, the model predicted that this airway dilates despite the rise in ASM activation. On contrary, diminution of flow in the other daughter airway causes regional lung deflation and loss of parenchymal tethering recoil. The load impeding muscle shortening thus decreases and, for the same level of ASM activation, more narrowing occurs. Because of axial interdependence of pathways in series, all the smaller airways downstream of the excessively closed airways are also affected. The size of the ventilation defects depends on the first airway generation afflicted by this dichotomic response. Since more ASM activation is required to close larger airways, this dichotomic response begins in more peripheral airways. But as the ASM activation progressively rises, the model predicted that larger and larger airways are affected and larger and larger patches of lung become non-ventilated.

Taken together, Venegas and colleagues suggested that only a slight degree of heterogeneity at baseline can trigger a self-perpetuating feedback loop when a certain level of ASM activation is achieved; such that the redistribution of flow in slightly more patent airways, makes the other airways and their downstream pathways unstable because of the loss of elastic recoil. This ultimately leads to clustering of peripheral airway constriction and the emergence of large ventilation defects, which, in turn, cause flow limitation. The chase for the factors determining this baseline heterogeneity, which was heretofore ignored, is now on.

#### **4.4.4 Factors potentially involved in determining baseline heterogeneity**

The amplifying effect described by Venegas and coworkers (236), which can lead to large ventilation defects and attendant AHR, relies on subtle structural and/or functional changes that were already present prior the inhalation of the spasmogen. Therefore, the identification of factors responsible for this baseline airway wall heterogeneity is of major interest.

Remodeling can certainly impact heterogeneity and can potentially discriminate the different levels of heterogeneity observed between asthmatics and non-asthmatics. Remodeling occurs non-uniformly throughout the tracheobronchial tree. The occurrence of epithelium desquamation or inflammatory infiltrates for example is patchy in nature. The extent of subepithelial fibrosis, ASM enlargement, and other structural changes present in asthmatic lungs could also vary at different locations in the lung. Together, these disparities alter the mechanical properties of the airway wall non-uniformly along the bronchial tree. It can also modify airway geometry and sometimes lead to the formation of bottlenecks, a characteristic feature found in terminal bronchioles of chronic obstructive pulmonary disease (COPD) patients (McDonough *et al*., provisionally accepted in the New England Journal of Medicine). The effect of a bottleneck is treacherous because not only it increases the resistance to airflow in an airway that otherwise may look normal but also because it reduces ventilation in the parenchymal tissue subtended by this airway. This ultimately fosters the development of a ventilation defect due to a decrease in the force of airway-parenchymal interdependence. Greater ventilation defects at baseline seen in asthmatics can thus be attributed to remodeling.

However, ventilation heterogeneity is mainly attributable to the fact that airways narrow heterogeneously. So, it is upon bronchoconstriction that the functional consequences of these disparities (localized remodeling changes) along the length of a pathway are exacerbated. For example, remodeling heterogeneity can affect locally the initial geometry, which has a huge influence on airway responsiveness (as discussed in subsection 4.1.3). It can also modify locally the after-load impeding ASM shortening. Another example would be the patchiness of ASM mass enlargement, which can potentially amplify regional differences in airway narrowing upon ASM activation.

Gain in contractile properties (e.g., force-generating capacity) due to ASM behaviors could also be patchy within the lungs. For example, acquisition of supplemental force by length adaptation can occur in an area where the airway caliber is smaller because of a localized decrease in the force of airway-parenchymal interdependence. The baseline ASM-tone also likely differs in different areas of the lung. Its magnitude may be spatially correlated with zones of inflammation, where inflammation-derived spasmogens are produced/released. That would also lead to force adaptation and the attendant gain in force-generating capacity in those areas. All these factors can affect ASM contractile properties in a localized manner and contribute to narrowing heterogeneity and AHR.

Hence, remodeling and inflammatory changes seen in asthmatic lungs can give rise to a greater baseline level of airway wall heterogeneity. It can also cause inhomogeneity of narrowing upon a bronchoprovocative challenge with a spasmogen by changing the load impeding ASM shortening or by fostering the development of increased ASM contractility. Together with the strong interplay between the parenchymal tethering and airway patency, this increased level of heterogeneity can be sufficient to trigger a self-perpetuating loop leading to patches of hypo- or non-ventilated area and the appearance of AHR in asthmatics.

## **5. Conclusion**

36 Lung Diseases – Selected State of the Art Reviews

of constricted peripheral airways. In a computational model developed by Venegas and coworkers (236, 253), it was shown that airway closure occurs in cluster. In their model, the bronchial tree branches dichotomously, where every bifurcation is composed of one parent airway and two daughter airways with slight heterogeneity between them. During bronchoconstriction, the flow is redistributed according to divergence of patency among daughter airways. The model predicted that all the airways shorten uniformly when the level of ASM activation is low. However, passed a certain threshold of ASM activation, daughter airways develop a dichotomic response. Whereas one constricts excessively the other one dilates. This is because the initial heterogeneity between the two daughter airways fosters the redistribution of flow into the more conductant airway. The insufflation pressure thus rises in the hyperventilated areas subtended by this airway, which increases the load impeding ASM shortening. In fact, the model predicted that this airway dilates despite the rise in ASM activation. On contrary, diminution of flow in the other daughter airway causes regional lung deflation and loss of parenchymal tethering recoil. The load impeding muscle shortening thus decreases and, for the same level of ASM activation, more narrowing occurs. Because of axial interdependence of pathways in series, all the smaller airways downstream of the excessively closed airways are also affected. The size of the ventilation defects depends on the first airway generation afflicted by this dichotomic response. Since more ASM activation is required to close larger airways, this dichotomic response begins in more peripheral airways. But as the ASM activation progressively rises, the model predicted that larger and larger airways are affected and larger and larger patches of lung become

Taken together, Venegas and colleagues suggested that only a slight degree of heterogeneity at baseline can trigger a self-perpetuating feedback loop when a certain level of ASM activation is achieved; such that the redistribution of flow in slightly more patent airways, makes the other airways and their downstream pathways unstable because of the loss of elastic recoil. This ultimately leads to clustering of peripheral airway constriction and the emergence of large ventilation defects, which, in turn, cause flow limitation. The chase for the factors determining

The amplifying effect described by Venegas and coworkers (236), which can lead to large ventilation defects and attendant AHR, relies on subtle structural and/or functional changes that were already present prior the inhalation of the spasmogen. Therefore, the identification

Remodeling can certainly impact heterogeneity and can potentially discriminate the different levels of heterogeneity observed between asthmatics and non-asthmatics. Remodeling occurs non-uniformly throughout the tracheobronchial tree. The occurrence of epithelium desquamation or inflammatory infiltrates for example is patchy in nature. The extent of subepithelial fibrosis, ASM enlargement, and other structural changes present in asthmatic lungs could also vary at different locations in the lung. Together, these disparities alter the mechanical properties of the airway wall non-uniformly along the bronchial tree. It can also modify airway geometry and sometimes lead to the formation of bottlenecks, a characteristic feature found in terminal bronchioles of chronic obstructive pulmonary disease (COPD) patients (McDonough *et al*., provisionally accepted in the New England Journal of Medicine). The effect of a bottleneck is treacherous because not only it increases the resistance

this baseline heterogeneity, which was heretofore ignored, is now on.

**4.4.4 Factors potentially involved in determining baseline heterogeneity** 

of factors responsible for this baseline airway wall heterogeneity is of major interest.

non-ventilated.

Despite abundant evidence implicating the ASM as the culprit of AHR in asthma, the latest genetic studies are relatively weak to support the role of asthma susceptibility gene risk variants in causing ASM dysfunction. The more reliable genetic analyses performed to date have instead suggested that the well-recognized genetic predisposition to suffer from asthma may be related to polymorphisms in genes that are predominately involved in immunology and/or epithelial integrity and functions. Even if we cannot rule out the contribution of genetic alterations affecting the ASM in AHR at the present time, the bulk of evidence rather suggests that if alterations in ASM mechanics contribute to AHR, these alterations are acquired as a result of inflammatory mediators and/or extracellular matrix remodeling that are present in asthmatic lungs. For example, lung defects can foster the appearance of normal ASM behaviors that render the muscle hypercontractile, such as length adaptation, force adaptation and changes in ASM cell's phenotype. These ASM behaviors are the testimony that he contractile properties of the ASM are not fixed but rather plastic (adaptable).

On the other hand, alterations in ASM contractile properties are not necessarily a prerequisite to suffer from AHR. Many lung defects are sufficient alone (i.e., without the need of increased ASM contractility) to cause AHR. These include but are not restricted to: 1-alterations in epithelial integrity, which increase the bioavailability of the inhaled spasmogen onto the ASM; 2-increased ASM-tone (due to the preponderance of inflammation-derived spasmogens, an augmented vagal input and/or decreased expression of relaxant factors), which acts additively with the extrinsically-delivered spasmogen in terms of ASM activation and ASM shortening, and synergistically in terms of narrowing of the cross-sectional area of the lumen and airway resistance; 3-obstruction of the vessels irrigating the ASM, which prevents clearance of the spasmogens into the circulation and concomitantly sequesters the spasmogens in the vicinity of ASM; 4-increased mass of the material inner the ASM layer, which increases airway narrowing for any given degree of ASM shortening; 5-decreased ASM-load (due to adventitial thickening, detachment of the parenchymal tethers from the outer edge of the airway wall, decrease in lung recoil, reduction in lung volume, diminution of the radial constraint limiting ASM bulging during shortening, other changes in ECM constitution which may change the mechanical properties of the airway wall to make it easier to compress by affecting, or not, its pattern of folding), which increases ASM shortening for any given degree of ASM activation; and 6-ventilation heterogeneity with airway closure, which affects flow measurements (such as FEV1, PEF and others) because of air trapping. For all of these reasons, some asthmatics can be hypperresponsive even when their ASM operates as normal. In conclusion, we think that the ASM is often 'blamed' for the AHR seen in asthma simply because of its unequivocal role in airway responsiveness. We would like to propose that without further or more convincing proofs to incriminate ASM in AHR, this obeisant tissue should still be considered 'innocent'.

#### **6. References**


need of increased ASM contractility) to cause AHR. These include but are not restricted to: 1-alterations in epithelial integrity, which increase the bioavailability of the inhaled spasmogen onto the ASM; 2-increased ASM-tone (due to the preponderance of inflammation-derived spasmogens, an augmented vagal input and/or decreased expression of relaxant factors), which acts additively with the extrinsically-delivered spasmogen in terms of ASM activation and ASM shortening, and synergistically in terms of narrowing of the cross-sectional area of the lumen and airway resistance; 3-obstruction of the vessels irrigating the ASM, which prevents clearance of the spasmogens into the circulation and concomitantly sequesters the spasmogens in the vicinity of ASM; 4-increased mass of the material inner the ASM layer, which increases airway narrowing for any given degree of ASM shortening; 5-decreased ASM-load (due to adventitial thickening, detachment of the parenchymal tethers from the outer edge of the airway wall, decrease in lung recoil, reduction in lung volume, diminution of the radial constraint limiting ASM bulging during shortening, other changes in ECM constitution which may change the mechanical properties of the airway wall to make it easier to compress by affecting, or not, its pattern of folding), which increases ASM shortening for any given degree of ASM activation; and 6-ventilation heterogeneity with airway closure, which affects flow measurements (such as FEV1, PEF and others) because of air trapping. For all of these reasons, some asthmatics can be hypperresponsive even when their ASM operates as normal. In conclusion, we think that the ASM is often 'blamed' for the AHR seen in asthma simply because of its unequivocal role in airway responsiveness. We would like to propose that without further or more convincing proofs to incriminate ASM in AHR, this obeisant tissue should still be

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Xie ZF. Effect of inhaled interleukin-5 on airway hyperreactivity and eosinophilia

vitro studies on alpha-receptors in human airways; potentiation with bacterial

and their relationship to bronchial hyperresponsiveness. *Clin Rev Allergy Immunol* 

Rafaels NM, Michel S, Bonnelykke K, Zhang H, Kim CE, Frackelton EC, Glessner JT, Hou C, Otieno FG, Santa E, Thomas K, Smith RM, Glaberson WR, Garris M, Chiavacci RM, Beaty TH, Ruczinski I, Orange JM, Allen J, Spergel JM, Grundmeier R, Mathias RA, Christie JD, von Mutius E, Cookson WO, Kabesch M, Moffatt MF, Grunstein MM, Barnes KC, Devoto M, Magnusson M, Li H, Grant SF, Bisgaard H, and Hakonarson H. Variants of DENND1B associated with asthma in children. *N* 

adenosine monophosphate inhalation of sensitized guinea-pigs. *Clin Exp Allergy* 38:

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## **Polymerization and Oxidation of Alpha-1-Antitrypsin in Pathogenesis of Emphysema**

Aleksandra Topic1 and Dragica Radojkovic2

*1University of Belgrade, Faculty of Pharmacy, Department of Medical Biochemistry, 2University of Belgrade, Institute of Molecular Genetics and Genetic Enginieering, Belgrade Serbia* 

#### **1. Introduction**

54 Lung Diseases – Selected State of the Art Reviews

[250] Whelan R, Kim C, Chen M, Leiter J, Grunstein MM, and Hakonarson H. Role and

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[254] Winter MC, Shasby SS, Ries DR, and Shasby DM. PAR2 activation interrupts E-

of beta-agonists. *Am J Physiol Lung Cell Mol Physiol* 291: L628-635, 2006. [255] Woodman L, Siddiqui S, Cruse G, Sutcliffe A, Saunders R, Kaur D, Bradding P, and

autocrine up-regulation of TGF-beta1. *J Immunol* 181: 5001-5007, 2008. [256] Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter

[257] Wright SM, Hockey PM, Enhorning G, Strong P, Reid KB, Holgate ST, Djukanovic R,

[258] Yanta MA, Loring SH, Ingram RH, Jr., and Drazen JM. Direct and reflex

[259] Yoo J, Ellis R, Morgan KG, and Hai CM. Mechanosensitive modulation of myosin

[260] Yoshimura H, Jones KA, Perkins WJ, Kai T, and Warner DO. Calcium sensitization

[261] Youn T, Kim SA, and Hai CM. Length-dependent modulation of smooth muscle

[262] Zabner J, Winter MC, Shasby S, Ries D, and Shasby DM. Histamine decreases E-

[263] Zhang W, and Gunst SJ. Dynamic association between alpha-actinin and beta-integrin

[264] Zhang W, and Gunst SJ. Interactions of airway smooth muscle cells with their tissue matrix: implications for contraction. *Proc Am Thorac Soc* 5: 32-39, 2008. [265] Zhang W, Wu Y, Wu C, and Gunst SJ. Integrin-linked kinase regulates N-WASp-

lung function in asthma. *J Appl Physiol* 89: 1283-1292, 2000.

muscle. *Eur Respir J* 24: 559-567, 2004.

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muscle. *J Biol Chem* 282: 34568-34580, 2007.

482-484, 2003.

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Brightling C. Mast cells promote airway smooth muscle cell differentiation via

R, Wong HH, Cadbury PS, and Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. *Am J Respir Crit* 

and Postle AD. Altered airway surfactant phospholipid composition and reduced

bronchoconstriction induced by histamine aerosol inhalation in dogs. *J Appl Physiol* 

phosphorylation and phosphatidylinositol turnover in smooth muscle. *Am J Physiol* 

produced by G protein activation in airway smooth muscle. *Am J Physiol Lung Cell* 

activation: effects of agonist, cytochalasin, and temperature. *Am J Physiol* 274:

cadherin-based adhesion to increase permeability of human airway epithelium.

regulates contraction of canine tracheal smooth muscle. *J Physiol* 572: 659-676, 2006.

mediated actin polymerization and tension development in tracheal smooth

The last two decades efforts have been made in investigation of genes that encode proteins involved in pathogenesis of emphysema and chronic obstructive pulmonary disease (COPD). So far, SERPINA1 gene which encodes protein alpha-1-antitrypsin (A1AT) is the only defined genetic risk factor associated with early development of emphysema.

The A1AT is dominant protein of 1 electrophoretic fraction of serum proteins, whose main physiological role is to inhibit neutrophil elastase (NE) in the lower respiratory tract, and protect pulmonary connective tissue from NE released from triggered neutrophiles. Neutrophil elastase is serine protease that degrades elastin of the alveolar walls as well other structural proteins of a variety of tissues.

Hereditary alpha-1-antitrypsin deficiency (A1ATD) is associated with retention of mutant A1AT polymers in hepatocytes which leads to decrease of circulating A1AT with less than 15% of normal level in A1ATD homozygotes. Since the integrity of lung alveoli is maintained by proper circulating level of A1AT, severe deficiency of this protein was identified as genetic risk factor for emphysema and COPD. Clinical manifestation of emphysema in patients with A1ATD occurs in 3th decade in smokers and in the 5th decade in non-smokers (Larsson, 1978; Janus et al., 1985) and requires replacement therapy with purified A1AT pooled from donor plasma.

Genetic epidemiologic studies show that A1ATD may affect 1 in about 1,500 individuals in Europe (De Serres, 2002). Approximately 3.4 million individuals of all racial subgroups are affected by A1ATD worldwide (De Serres, 2002).

Liver disease in early childhood is second clinically significant consequence resulting from retention of mutant A1AT polymers in hepatocytes (Eriksson, 1986; Sveger, 1976). Clinically it is presents as neonatal cholestasis which may progress to juvenile chirosis or slowly progress to the liver disease in adults (Mahadeva and Lomas, 1998).

In the early sixties of the last century, Laurell and Eriksson discovered that the absence of the electrophoretic 1-globulin pattern of serum is associated with A1AT deficiency (Laurell and Eriksson, 1963). At the same time was discovered an association between A1ATD and emphysema in relatively young patients in fourth decade of life (Eriksson, 1964; Lieberman, 1969). These observations suggested a significant role of A1ATD in pathogenesis of emphysema. The proteinase-antiproteinase hypothesis, established by Janoff (Janoff, 1985) still remains central in our understanding of the pathogenesis of lung disease. According to this hypothesis, emphysema in A1ATD arises from an imbalance of neutrophil elastase and A1AT as antielastase, which leads to inappropriate antielastase defense and the relatively excessive activity of neutrophil elastase and consequent degradation of elastin and other extracellular matrix components of the lower respiratory tract.

However, only 1% of patients with COPD are A1ATD (Lieberman, et al., 1986), indicating that A1ATD alone is not sufficient to induce emphysema (Silverman, et al., 1989). The additional factor which may induce emphysema in A1ATD is inflammation, when elastin repair mechanisms are overwhelmed by a massive attack of elastase from triggered neutrophils and cigarette smoke. Studies of the genetic and environmental factors have shown a difference in the reduction of pulmonary function in A1ATD, indicating that additional genetic factors (modifier genes) may influence the pulmonary function in A1ATD subjects (Silverman et al., 1990). Also, the single-nucleotide polymorphisms (SNPs) were identified in the six haplotypes of the SERPINA1 gene, which controls synthesis of A1AT (Chappell, et al., 2006). Several environmental factors that accelerate the onset of symptoms in A1ATD patients, such as personal and second hand exposure to tobacco smoke in childhood, respiratory infections (Mayer et al., 2006), and higher exposures to ozone (Wood et al., 2009) have been also identified.

In addition to the low circulating levels of A1AT in hereditary A1ATD, the risk of emphysema includes reduced antielastase activity. Functional inactivation of A1AT by oxidants present in cigarette smoke could impair antielastase defence in lower respiratory tract, and represent acquired A1AT inactivation. Furthermore, in hereditary A1ATD smoking could impair the function of A1AT both quantitatively and qualitatively.

## **2. Alpha-1-antitrypsin**

Alpha-1-antitrypsine (A1AT) is the archetype of the serpin family of proteins. SERPINs (SER*ine* P*roteinase* IN*hibitors*) are the superfamily of structurally related proteins that control many physiological processes. A1AT is a highly polymorphic, acute-phase glycoprotein, synthesised in hepatocytes (Koj et al., 1978) and subsequently secreted into the plasma. Hepatic synthesis of this acute phase protein by SERPINA1 gene is under control of different cytokines, such as interleukin-1 (IL-1), tumour necrosis factors (TNF) and most effectively the interleukin-6 family of cytokines (interleukin-6, leukaemia inhibitory factor, oncostatin M) (Richards and Gauldie, 1991). Besides liver, the small quantities of A1AT are produced by alveolar macrophages, circulating monocytes and intestinal, renal and lung-derived epithelial cells (Mornex et al., 1986; Carlson et al., 1988; Molmenti et al., 1993; Cichy et al., 1997; Mulgrew et al., 2004). Extra hepatic synthesis of A1AT is important in preventing tissue damage in the site of inflammation or injury. For instance, synthesis of A1AT in monocytes is up-regulated by inflammatory mediators such as IL-1 and TNF in lung tissue (Knoell et al., 1998). Serum level of A1AT is elevated in inflammation, trauma, and pregnancy.

Healthy individuals produce 34 mg of A1AT per kilogram of body weight per day (Jones, 1978). Normal reference interval for antigenic concentration of serum A1AT measured by nephelometry is 15-40 M (0.83-2.20 g/L) (ATS/ERS Statement, 2003). The threshold level of 11 M (0.59 g/L) provides relevant antielastase protection of lower respiratory tract (WHO Meeting, 1996.). As a relatively small protein (52 kD), the mature A1AT is capable to diffuse into many organs. The concentration of A1AT in organs is lower than in plasma. Thus, Olsen et al. (Olsen et al., 1975) reported that A1AT level in a bronchoalveolar lavage fluid of non-smokers is 7% of serum level, with a higher value (11%) in smokers. Also, total amount of A1AT in the lavage fluid of smokers was significantly greater than in non-smokers. The same authors reported about five times higher A1AT concentration in pulmonary alveolar macrophages in smokers in comparison to non-smokers. All these results suggest an increased A1AT concentration in the air spaces of the cigarette smokers.

#### **2.1 Structure and function of alpha-1-antitrypsin**

56 Lung Diseases – Selected State of the Art Reviews

1969). These observations suggested a significant role of A1ATD in pathogenesis of emphysema. The proteinase-antiproteinase hypothesis, established by Janoff (Janoff, 1985) still remains central in our understanding of the pathogenesis of lung disease. According to this hypothesis, emphysema in A1ATD arises from an imbalance of neutrophil elastase and A1AT as antielastase, which leads to inappropriate antielastase defense and the relatively excessive activity of neutrophil elastase and consequent degradation of elastin and other

However, only 1% of patients with COPD are A1ATD (Lieberman, et al., 1986), indicating that A1ATD alone is not sufficient to induce emphysema (Silverman, et al., 1989). The additional factor which may induce emphysema in A1ATD is inflammation, when elastin repair mechanisms are overwhelmed by a massive attack of elastase from triggered neutrophils and cigarette smoke. Studies of the genetic and environmental factors have shown a difference in the reduction of pulmonary function in A1ATD, indicating that additional genetic factors (modifier genes) may influence the pulmonary function in A1ATD subjects (Silverman et al., 1990). Also, the single-nucleotide polymorphisms (SNPs) were identified in the six haplotypes of the SERPINA1 gene, which controls synthesis of A1AT (Chappell, et al., 2006). Several environmental factors that accelerate the onset of symptoms in A1ATD patients, such as personal and second hand exposure to tobacco smoke in childhood, respiratory infections (Mayer et al., 2006), and higher exposures to ozone (Wood

In addition to the low circulating levels of A1AT in hereditary A1ATD, the risk of emphysema includes reduced antielastase activity. Functional inactivation of A1AT by oxidants present in cigarette smoke could impair antielastase defence in lower respiratory tract, and represent acquired A1AT inactivation. Furthermore, in hereditary A1ATD

Alpha-1-antitrypsine (A1AT) is the archetype of the serpin family of proteins. SERPINs (SER*ine* P*roteinase* IN*hibitors*) are the superfamily of structurally related proteins that control many physiological processes. A1AT is a highly polymorphic, acute-phase glycoprotein, synthesised in hepatocytes (Koj et al., 1978) and subsequently secreted into the plasma. Hepatic synthesis of this acute phase protein by SERPINA1 gene is under control of different cytokines, such as interleukin-1 (IL-1), tumour necrosis factors (TNF) and most effectively the interleukin-6 family of cytokines (interleukin-6, leukaemia inhibitory factor, oncostatin M) (Richards and Gauldie, 1991). Besides liver, the small quantities of A1AT are produced by alveolar macrophages, circulating monocytes and intestinal, renal and lung-derived epithelial cells (Mornex et al., 1986; Carlson et al., 1988; Molmenti et al., 1993; Cichy et al., 1997; Mulgrew et al., 2004). Extra hepatic synthesis of A1AT is important in preventing tissue damage in the site of inflammation or injury. For instance, synthesis of A1AT in monocytes is up-regulated by inflammatory mediators such as IL-1 and TNF in lung tissue (Knoell et al., 1998). Serum

Healthy individuals produce 34 mg of A1AT per kilogram of body weight per day (Jones, 1978). Normal reference interval for antigenic concentration of serum A1AT measured by nephelometry is 15-40 M (0.83-2.20 g/L) (ATS/ERS Statement, 2003). The threshold level of 11 M (0.59 g/L) provides relevant antielastase protection of lower respiratory tract (WHO Meeting, 1996.). As a relatively small protein (52 kD), the mature A1AT is capable to diffuse into many organs. The concentration of A1AT in organs is lower than in plasma. Thus,

smoking could impair the function of A1AT both quantitatively and qualitatively.

level of A1AT is elevated in inflammation, trauma, and pregnancy.

extracellular matrix components of the lower respiratory tract.

et al., 2009) have been also identified.

**2. Alpha-1-antitrypsin** 

The mature A1AT protein is a single chain composed of 394 amino acids. The main characteristics of the protein are: Met358 residue at the active site, isoelectric point ranging from 4.4 to 4.6, and a total molecular weight of 52 kDa. Crystalographic analysis of the mature protein reveals that A1AT is a globular protein with tree N asparigynil-linked carbohydrate side chains on the external surface of the one end of the molecule (Loebermann et al., 1984). The side chains are composed of N-acetylglukosamine, mannose, galactose and sialic acid and they are N-linked to amino acids Asn46, Asn83 and Asn247. These carbohidrate side chains are on the outside surface of one-half of the elongated structure. The difference in carbohydrate side chains at position of Asn83 is responsible for the two major bands of A1AT when serum focused at pH 4-4.9 on thin-layer polyacrilamide gel. The internal structure of A1AT is highly ordered with 30 percent -helices and 40 percent -pleated sheets. There are nine -helices (AI) and three -sheets (AI).

Similar to other inhibitory serpins, A1AT is "suicide" or "single use" inhibitor that employs a unique and extensive conformational change in the process of inhibition of target proteases (Figure 1.). The hallmark of serpins is the reactive centre loop (RCL) that presents the key P1-P'1 methionine–serine bond as a pseudosubstrate for the cognate proteinase, neutrophil elastase (Johnson and Travis, 1978). The reactive centre loop of A1AT is highly stressed external loop protruding from the molecule with Met358-Ser359 in the active center. Inhibitory process begins by docking of the serpin and the protease, and formation of Michaelis complex. Like the other inhibitory serpins, the structure of the RCL is crucial for the ability of the inhibitor to undergo a "stressed to relaxed" (S→R) conformational change. The active A1AT is in metastable or "stressed form", which is essential for inhibition of proteases. During the process of inhibition, A1AT is like mousetrap with spring-like shift from a metastable to a hyperstable state (Hunington et al., 2000; Carrell and Lomas, 2002). After the formation of Michaelis complex there are two possible different ending of the reaction. One is inactivation of protease, where serpin has undergone the S → R transition, and the protease hangs distorted at the base of the molecule. The other possibility is A1AT substrate-like behavior, where RCL forms the fourth -sheet, providing the opportunity for the protease to escape the conformational trap, leaving active protease and inactive cleaved serpin. Thus, *in vivo*, A1AT can exist in: native inhibitory conformation with an exposed RCL, latent conformation with a partially inserted RCL and non-inhibitory conformation.

Non-inhibitory conformation of A1AT occurs in certain circumstances: when A1AT is in complex with neutrophil elastase, when the reactive center loop of A1AT is cleaved by nontarget proteinases, when reactive oxygen species oxidized A1AT, and when A1ATD variants form polymers.

Moreover, A1AT non-inhibitory conformations show other biological effects. For instance, oxidized A1AT and the cleaved peptide fragment of A1AT stimulate monocyte activation, and A1AT-elastase complexes and polymeric A1AT are chemotactic for neutrophils (Banda et al., 1988; Dabbagh et al., 2001; Moraga and Janciauskiene, 2000; Moraga et al., 2001).

Fig. 1. Structure of alpha-1antitrypsin and reaction with target protease (modified from Law, et al., 2006)

#### **2.2 Physiological roles of alpha-1-antitrypsin**

The main physiological role of A1AT is protection of lower respiratory tract by inhibiting proteases released from triggered neutrophiles including neutrophil elastase, cathepsin G, and proteinase-3 (Carrell, 1986). The target proteases of A1AT derive from azurophilic granules of polymorphonuclear neutrophils which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at the sites of inflammation. This protective role of A1AT occurs primarily extracellular. A1AT enters the lung from the circulation by passive diffusion (Stockley, 1984). Besides direct inhibition of NE, there are evidences that A1AT exhibits anti-inflammatory properties to suppress cigarette smoke induced production of tumor necrosis factor (TNF) and matrix metalloproteinase 12 (MMP12) by alveolar macrophages, and subsequent inflammatory cell infiltration (Churg et al., 2007). Furthermore, studies have shown that native A1AT modulates function of immune cells, such as neutrophils (Bergin et al., 2010), monocytes (Janciauskiene et al., 2007), and T cells (Lu et al., 2006). *Ex vivo* and *in vitro* experiments have shown that endogenous A1AT in blood contributes to the suppression of proinflammatory cytokine synthesis (Pott et al., 2009). Thus, A1AT is an endogenous inhibitor of proinflammatory cytokine production in whole blood, and may participate in innate immune response to an inflammation-inducing stimulus. The recently discovered role of A1AT in prevention of emphysema is the inhibition of lung endothelial cell apoptosis due to inactivation of intracellular caspase-3 (Petrache et al., 2006a; Petrache et al., 2006b). Antiapoptotic role of A1AT in the lung *in vivo* and *in vitro* in micro vascular endothelial cells is associated with intracellular presence of A1AT. Lung endothelial cells don't produce A1AT and they take it. Sohrab et al. (Sohrab et al., 2009) showed that clathrin-mediated endocytosis predominantly regulates A1AT intracellular function in the lung endothelium, and might represent an important determinant of the serpin's protection against development of cigarette smoke-induced emphysema. The uptake is severely affected by exposure to cigarette smoke extract *in vitro* and *in vivo*, probably directly influencing clathrin-mediated endocytosis. Furthermore, polymers of A1AT exhibit a marked decrease in lung endothelial cell uptake. Inhibition of A1AT uptake by cigarette smoke may further weaken the A1AT protective role in the lung.

In the last decade other physiological roles of A1AT have been discovered, such as roles in atherogenesis (Talmud et al., 2003), angiogenesis (Huang et al., 2004), fibroblast proliferation, and procollagen synthesis (Dabbagh et al., 2001).

#### **2.3 Genetics of alpha-1-antitrypsin**

58 Lung Diseases – Selected State of the Art Reviews

Fig. 1. Structure of alpha-1antitrypsin and reaction with target protease (modified from Law,

The main physiological role of A1AT is protection of lower respiratory tract by inhibiting proteases released from triggered neutrophiles including neutrophil elastase, cathepsin G, and proteinase-3 (Carrell, 1986). The target proteases of A1AT derive from azurophilic granules of polymorphonuclear neutrophils which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at the sites of inflammation. This protective role of A1AT occurs primarily extracellular. A1AT enters the lung from the circulation by passive diffusion (Stockley, 1984). Besides direct inhibition of NE, there are evidences that A1AT exhibits anti-inflammatory properties to suppress cigarette smoke induced production of tumor necrosis factor (TNF) and matrix metalloproteinase 12 (MMP12) by alveolar macrophages, and subsequent inflammatory cell infiltration (Churg et al., 2007). Furthermore, studies have shown that native A1AT modulates function of immune cells, such as neutrophils (Bergin et al., 2010), monocytes (Janciauskiene et al., 2007), and T cells (Lu et al., 2006). *Ex vivo* and *in vitro* experiments have shown that endogenous A1AT in blood contributes to the suppression of proinflammatory cytokine synthesis (Pott et al., 2009). Thus, A1AT is an endogenous inhibitor of proinflammatory cytokine production in whole blood, and may participate in innate immune response to an inflammation-inducing stimulus. The recently discovered role of A1AT in prevention of emphysema is the inhibition of lung endothelial cell apoptosis due to inactivation of intracellular caspase-3 (Petrache et al., 2006a; Petrache et al., 2006b). Antiapoptotic role of A1AT in the lung *in vivo* and *in vitro* in micro vascular endothelial cells is associated with intracellular presence of A1AT. Lung endothelial cells don't produce

et al., 2006)

**2.2 Physiological roles of alpha-1-antitrypsin** 

The alpha-1-antitrypsin is encoded by SERPINA1 gene (serpin peptidase inhibitor, clade A) located in proteinase inhibitor (Pi) locus on the long arm of chromosome 14q32.1 (Schroeder et al., 1985; Billingsley et al., 1993). The Pi locus is 12.2 kb long and consists of 4 coding exons, 3 non-coding exons and 6 introns (Figure 2.). At the 5 region of the SERPINA1 gene there are three non-protein coding exons (IA, IB, IC) which control gene transcription. Exons referred as exons II-V are coding and containing the sequence information that defines the protein itself. The start codon (ATG) for translational of the mRNA and the signal peptide are in exon II, and the stop codon (TAA) is in exon V, followed by the polyadenilatyon signal (ATTAA). The carbohydrate attachmnent site (Asn46, Asn83, Asn247) are coded for in exon II and III. The region coding for the reactive loop with the active inhibitory centre Met358 is within exon V.

Following transcription, A1AT mRNA is translated on ribosome bound to the the rough endoplasmatic reticulum, producing a preprotein of 418 amino acids. The signal peptide of 24 residues is removed during secretion into the cisterne of the rough endoplasmatic reticulum where the protein is glycosylated with high-mannose type carbohydrates, and folds into appropriate globular tree-dimensional configuration. Complete protein maturation is accomplished within the Golgi apparatus and protein is secreted.

Fig. 2. Structure of the SERPINA1 gene (Crystal et al., 1989)

Hepatocytes and monocytes have two different promotors (Perlino et al., 1987) that operate via different mechanisms. The SERPINA1 gene in macrophages is transcribed from a macrophags-specific promoter located about 2,000 bp upstream of the hepatocyte-specific promoter. Transcription from the two SERPINA1 promoters is mutually exclusive; the macrophage promoter is silent in hepatocytes, and the hepatocyte promoter is silent in macrophages. In macrophages, two distinct mRNAs are generated by alternative splicing. In addition, Hafeez et al. (Hafeez et al., 1992) demonstrated that the SERPINA1 gene has 3 monocyte-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression, but also switch to transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator interleukin-6 (IL-6).

#### **2.4 Polymorphism of alpha-1-antitrypsin**

The A1AT coding gene SERPINA1 is a highly polimorphic, with more than 125 SNPs reported in public SNP databases (Entrez SNP). Protein variants of A1AT are classified by the Pi (P*rotese* i*nhibitor*) system and each variant is identified by migration on agarose gel electrophoresis. These differences in migration relate to variations in protein charge resulting from amino acid alterations (Fagerhol and Laurell, 1970; Cox, 1978). Isoelectric focusing in the narrow range of pH (4.2-4.9) has enabled identification of more A1AT variants then in agarose gel electrophoresis. The alleles were given symbols according to the relative electrophoretic mobility of the allele product, so anodal variants are marked with the first letters, and cathodal with last letters. All A1AT variants are categorized according to the serum level and functional activity as normal, deficient, null and dysfunctional.

#### **2.4.1 Normal A1AT variants**

Normal A1AT variants have normal serum level and functional activity to inhibit neutrophil elastase. More than 95% of normal variants are the "common" M1 (Ala213), M1 (Val213), M2 and M3. Among Caucasians, M1 (Val213) is the most common, and M1 (Ala213), M2 and M3 are less frequent. The "rare" normal variants with frequencies less than 1% are: M4, Balhambra, F, PStAlbans and XChirstchurch. Usually, rare variants are named by the birthplace of the oldest individual tested in pedegree. PiM homozygotes and heterozygotes are characterized by normal serum level of A1AT (20-50 M) and normal functional activity (ATS/ERS Statement, 2003).

#### **2.4.2 Deficient A1AT variants**

Deficient variants are associated with lower serum level of A1AT than normal variants. Several mutations associated with A1ATD have been identified, and the most common are Z and S alleles. Rare A1ATD variants are MMalton, MMineralSprings, MNichinan, MProcida, PLowell, SIiyama and others.

Gene-mapping studies have shown that the PiZ allele probably arose in Northern Europe (Cox et al., 1985). Age estimates of A1AT variants based on microsatellite variation, suggest that the Z deficiency allele appeared 107 to 135 generations ago and could have been spread in neolithic times. Frequency of the Z allele shows a large variation in Caucasians, but is rare or absent in Asians and Africans (De Croo et al., 1991; Hutchison, 1998.).

The PiS deficiency allele has an older 279-generation to 470-generation age and from its high incidence on the Iberia peninsula it has been suggested that it could have originated in this region (Seixas et al., 2001).

#### **2.4.2.1 Z variant**

60 Lung Diseases – Selected State of the Art Reviews

Hepatocytes and monocytes have two different promotors (Perlino et al., 1987) that operate via different mechanisms. The SERPINA1 gene in macrophages is transcribed from a macrophags-specific promoter located about 2,000 bp upstream of the hepatocyte-specific promoter. Transcription from the two SERPINA1 promoters is mutually exclusive; the macrophage promoter is silent in hepatocytes, and the hepatocyte promoter is silent in macrophages. In macrophages, two distinct mRNAs are generated by alternative splicing. In addition, Hafeez et al. (Hafeez et al., 1992) demonstrated that the SERPINA1 gene has 3 monocyte-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression, but also switch to transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator

The A1AT coding gene SERPINA1 is a highly polimorphic, with more than 125 SNPs reported in public SNP databases (Entrez SNP). Protein variants of A1AT are classified by the Pi (P*rotese* i*nhibitor*) system and each variant is identified by migration on agarose gel electrophoresis. These differences in migration relate to variations in protein charge resulting from amino acid alterations (Fagerhol and Laurell, 1970; Cox, 1978). Isoelectric focusing in the narrow range of pH (4.2-4.9) has enabled identification of more A1AT variants then in agarose gel electrophoresis. The alleles were given symbols according to the relative electrophoretic mobility of the allele product, so anodal variants are marked with the first letters, and cathodal with last letters. All A1AT variants are categorized according to the serum level and functional activity as normal, deficient, null and dysfunctional.

Normal A1AT variants have normal serum level and functional activity to inhibit neutrophil elastase. More than 95% of normal variants are the "common" M1 (Ala213), M1 (Val213), M2 and M3. Among Caucasians, M1 (Val213) is the most common, and M1 (Ala213), M2 and M3 are less frequent. The "rare" normal variants with frequencies less than 1% are: M4, Balhambra, F, PStAlbans and XChirstchurch. Usually, rare variants are named by the birthplace of the oldest individual tested in pedegree. PiM homozygotes and heterozygotes are characterized by normal serum level of A1AT (20-50 M) and normal functional activity (ATS/ERS

Deficient variants are associated with lower serum level of A1AT than normal variants. Several mutations associated with A1ATD have been identified, and the most common are Z and S alleles. Rare A1ATD variants are MMalton, MMineralSprings, MNichinan, MProcida, PLowell, SIiyama

Gene-mapping studies have shown that the PiZ allele probably arose in Northern Europe (Cox et al., 1985). Age estimates of A1AT variants based on microsatellite variation, suggest that the Z deficiency allele appeared 107 to 135 generations ago and could have been spread in neolithic times. Frequency of the Z allele shows a large variation in Caucasians, but is rare

or absent in Asians and Africans (De Croo et al., 1991; Hutchison, 1998.).

interleukin-6 (IL-6).

**2.4.1 Normal A1AT variants** 

**2.4.2 Deficient A1AT variants** 

Statement, 2003).

and others.

**2.4 Polymorphism of alpha-1-antitrypsin** 

The Z variant represents a "classic"A1ATD variant and derives from M1 (Ala213) following a point mutation which is the same for the all Z individuals who are probably descendants of a single ancestral progenitor. The result of mutation is the substitution of a *G*AG that codes for Glu342 with an *A*AG that code for Lys342 (Nukiwa et al., 1987). The Z mutation perturbs the folding (Yu et al., 1995.) and structure of the protein (Lomas et al., 1993a). It distorts the relationship between the reactive centre loop and -sheet A, and the consequent perturbation in structure allows opening of -sheet A to favor partial loop insertion and formation of an unstable intermediate (M\*) (Figure 3.). Then the patent -sheet A accepts the loop of another A1AT molecule to form a dimer (D) which then extends to form chains of loop-sheet polymer (P) (Lomas, et al., 1992; Elliott et al., 1996a; Lomas, 2000).

Fig. 3. Formation of Z polymers (Lomas, 2005)

Process of polymerization depends on concentration and temperature. Abnormality in posttranslational modification of protein causes accumulation of the A1AT polymers in the cisterna of the rough endoplsmatic reticulum with a drastic reduction in secretion rates. The abnormal protein accumulates in hepatocytes and forms inclusion bodies (aggregates) that are positive to diastase-resistant periodic acid Schiff (PAS-D) staining and visible on microscopy. The retained A1AT polymers are cytotoxic for hepatocytes and can cause a diverse liver damages, ranging from neonatal hepatitis to juvenile cirrhosis, and hepatocellular carcinoma in adults (Eriksson, et al., 1986). As a consequence of polymer accumulation, hepatocytes of PiZZ homozygote secrete only 10–15 % of normal quantity. PiMZ heterozygotes have about 50 % of normal A1AT circulating level.

Besides low circulation level, the Z protein also less efficiently inhibits elastase (Ogushi, et al., 1987). Consequently, in PiZZ individuals, quantitative and qualitative defects of A1AT lead to early-onset COPD including emphysema and chronic bronchitis.

A similar form of loop-sheet polymers *in vivo* with hepatic inclusions and plasma deficiency was found in two other variants, MMalton (Phe52 deleted) (Lomas et al., 1995) and SIiyama (Ser53→Phe) (Lomas et al., 1993b.), that are common in Sardinia and Japan, respectively.

A1AT polymers were also detected in bronchoalveolar lavage fluid (BALF) from PiZ homozygotes with emphysema. This conformational transition may further reduce the levels of functional proteinase inhibitor in the lungs, and consequently exacerbate lung tissue damage (Elliott et al., 1998).

Studies of Parmar et al. (Parmar et al., 2002) and Mulgrew et al. (Mulgrew et al., 2004) showed that Z A1AT locally produced on the epithelial surface of the lung polymerizes and A1AT polymers demonstrate proapoptotic and proinflammatory effects. These studies also revealed that unlike M A1AT protein, Z A1AT protein polymerized at body temperature, and in addition of being an ineffective antiprotease inhibitor, might become a strong neutrophil chemoattractant, thus representing an ongoing source of inflammation in the lungs of individuals with A1ATD. Thus, polymerization of locally produced ZA1AT is a contributory factor to the lung inflammation experienced by those with A1AT deficiency and that standard antiprotease therapies may not address this problem. Other studies reported that even PiZZ patients with near-normal lung function had high concentrations of neutrophils on respiratory epithelial surfaces (Rouhani et al., 2000). Neutrophil burden in PiZZ and in PiMZ (on a lesser extent) is attributed to leukotriene B4 or IL-8 released from neutrophils or epithelial cells (Woolhouse et al., 2002; Malerba et al., 2006). Neutrophil accumulation in the lung of PiZZ deficient individuals is multifactorial and chemoattraction due to polymerized Z protein represents another potential cause of neutrophil dominated inflammation. These findings suggest a novel mechanism in pathogenesis of emphysema associated with Z antitrypsin deficiency.

Lomas (Lomas, 2006) highlighted a possible role of Z mutant in systemic response to infection. In the case of invasion of pathogens, organism initiates a systemic inflammatory response that results in increased secretion of Z A1AT as acute phase protein, by hepatocytes. Factors such as: elevation of body temperature, increased concentration of mutant Z A1AT, and lower pH at the site of bacterial invasion of lung (Stockley and Burnett, 1979) favor polymerization of mutant Z A1AT. Polymers possess chemotactic properties, which in turn amplify inflammatory response and enhance the recruitment of neutrophils. Excessive burden of neutrophils may cause increase of proinflamatory and proxidative factors.

Therefore, a rational approach in therapy of A1ATD would be to inhibit the polymerization of the Z protein (intracellularly and extracellularly), accompanied by standard augmentation therapy. It is clear that in addition to increased level of A1AT above a putative therapeutic threshold, it is necessary to increase the secretion of active nonpolymerized form of Z protein. This approach could potentially ameliorate the liver disease, and defend respiratory epithelial surface, providing antielastase protection and avoiding the proinflammatory effects of polymerized Z A1AT.

Currently, there is some progress in development of synthetic peptide designed to selectively inhibit Z polymerisation (Mahadeva et al., 2002; Parfrey et al., 2004; Chang et al., 2006; Mallya et al., 2007; Chang et al., 2009).

#### **2.4.2.2 S variant**

In contrast to the Z allele, S causes only mild plasma deficiency. The genetic sequence of the S variant derives from M1 (Val213) as a result of a mutation which cause substitution of G*A*A that codes for Glu264 with an G*T*A that codes for Val264 (Owen et al., 1976; Yoshida et al., 1977).

The single mutation of the S variant leads to spontaneous polymer formation, but slower than the Z variant, without affecting the ability to inhibit neutrophil elastase (Elliott, et al*.,*

1996b; Mahadeva et al., 1999; Dafforn et al., 1999). The slower polymerization causes less retention of S variant in the liver, and hence the plasma levels are 60% of the normal M allele. Thus, carriers of S allele (PiSS, PiSZ and PiMS) have levels of 52%, 32% and 75% of normal level, respectively. Furthermore, Z A1AT forms heteropolymers with S A1AT (Mahadeva et al., 1999), which explains cases of hepatic cirrhosis in PiSZ patients (Cruz et al., 1975; Campra et al., 1973; Craig et al., 1975.). Also, PiSZ smokers are at significant risk of the development of COPD, while in nonsmoking individuals the PiSZ phenotype may confer little or no risk to develop COPD (Turino et al., 1996).

#### **2.4.2.3 Null A1AT variants**

62 Lung Diseases – Selected State of the Art Reviews

A1AT polymers were also detected in bronchoalveolar lavage fluid (BALF) from PiZ homozygotes with emphysema. This conformational transition may further reduce the levels of functional proteinase inhibitor in the lungs, and consequently exacerbate lung

Studies of Parmar et al. (Parmar et al., 2002) and Mulgrew et al. (Mulgrew et al., 2004) showed that Z A1AT locally produced on the epithelial surface of the lung polymerizes and A1AT polymers demonstrate proapoptotic and proinflammatory effects. These studies also revealed that unlike M A1AT protein, Z A1AT protein polymerized at body temperature, and in addition of being an ineffective antiprotease inhibitor, might become a strong neutrophil chemoattractant, thus representing an ongoing source of inflammation in the lungs of individuals with A1ATD. Thus, polymerization of locally produced ZA1AT is a contributory factor to the lung inflammation experienced by those with A1AT deficiency and that standard antiprotease therapies may not address this problem. Other studies reported that even PiZZ patients with near-normal lung function had high concentrations of neutrophils on respiratory epithelial surfaces (Rouhani et al., 2000). Neutrophil burden in PiZZ and in PiMZ (on a lesser extent) is attributed to leukotriene B4 or IL-8 released from neutrophils or epithelial cells (Woolhouse et al., 2002; Malerba et al., 2006). Neutrophil accumulation in the lung of PiZZ deficient individuals is multifactorial and chemoattraction due to polymerized Z protein represents another potential cause of neutrophil dominated inflammation. These findings suggest a novel mechanism in pathogenesis of emphysema

Lomas (Lomas, 2006) highlighted a possible role of Z mutant in systemic response to infection. In the case of invasion of pathogens, organism initiates a systemic inflammatory response that results in increased secretion of Z A1AT as acute phase protein, by hepatocytes. Factors such as: elevation of body temperature, increased concentration of mutant Z A1AT, and lower pH at the site of bacterial invasion of lung (Stockley and Burnett, 1979) favor polymerization of mutant Z A1AT. Polymers possess chemotactic properties, which in turn amplify inflammatory response and enhance the recruitment of neutrophils. Excessive burden of neutrophils may cause increase of proinflamatory and proxidative

Therefore, a rational approach in therapy of A1ATD would be to inhibit the polymerization of the Z protein (intracellularly and extracellularly), accompanied by standard augmentation therapy. It is clear that in addition to increased level of A1AT above a putative therapeutic threshold, it is necessary to increase the secretion of active nonpolymerized form of Z protein. This approach could potentially ameliorate the liver disease, and defend respiratory epithelial surface, providing antielastase protection and avoiding the

Currently, there is some progress in development of synthetic peptide designed to selectively inhibit Z polymerisation (Mahadeva et al., 2002; Parfrey et al., 2004; Chang et al.,

In contrast to the Z allele, S causes only mild plasma deficiency. The genetic sequence of the S variant derives from M1 (Val213) as a result of a mutation which cause substitution of G*A*A that codes for Glu264 with an G*T*A that codes for Val264 (Owen et al., 1976; Yoshida et al., 1977). The single mutation of the S variant leads to spontaneous polymer formation, but slower than the Z variant, without affecting the ability to inhibit neutrophil elastase (Elliott, et al*.,*

tissue damage (Elliott et al., 1998).

associated with Z antitrypsin deficiency.

proinflammatory effects of polymerized Z A1AT.

2006; Mallya et al., 2007; Chang et al., 2009).

factors.

**2.4.2.2 S variant** 

Null variants are characterized by the modification of an important part of the gene with no detectable mRNA. Although extremely rare, they have been found in all populations. Frequencies of null variants among Caucasians are estimated to be less than 0.1%. Nullallelic variants are denoted as Q0 rather than Pi. The Null mutations do not result in secreted protein or the formation of polymers. Subjects with Null mutations show significantly lower lung function values than PiSZ and PiZZ individuals, and they are at particularly high risk to develop emphysema (Cox and Levison, 1988; Fregonese et al., 2008). Early detection of Null carriers is important for preventive and therapeutic interventions.

The PiNullBellingham differs from the normal M1 (Val213) gene by the mutation in exon II, where the codon for Lys217 (*A*AG) is altered to Stop codon (*T*AG) (Satoh et al., 1988). Homozygotes for PiNullBellingham have complete absence of A1AT, and develop premature emphysema much earlier than more common PiZZ individuals (Cook et al., 1994). The Nullisola di procida is caused by complete deletion of exons II-V of SERPINA1 gene (Takahashi and Crystal, 1990). The Nullgranite falls allele derives from the M1 (Ala213) by the deletion of a single base in exon II in the codon for Tyr160 (TA*C*) with deletion of the *C*. Consequently, there is 5' frame shift of the downstream nucleotides, moving the G form the next codon, Val161 G*T*C in place of the normal Tyr160 (Holmes et al., 1989).

The Nullmattawa allele is a consequence of the insertion of a single nucleotide within the coding region of exon V, causing a 3' frameshift with generation of a premature stop signal (Curiel et al., 1989).

Prins et al. (Prins et al., 2008) performed genotyping by direct sequencing of the SERPINA1 gene coding region in patients with A1AT concentrations ≤1.0 g/L, and this approach allowed them to discover Q0soest and Q0amersfoort null alleles.

### **2.4.2.4 Dysfunctional variants**

Dysfunctional A1AT variants are synthesized in normal quantities, but have altered protein function. The PiPittsburgh allele is a mutation which occurs at the A1AT active site, and represents an example of a mutation responsible for altered function of the gene product. A1AT becomes a potent inhibitor of thrombin and factor XI rather than of elastase, which results in a bleeding disorder (Lewis et al., 1978; Owen et al., 1983).

### **3. Hereditary alpha-1-antitrypsin deficiency and emphysema**

The risk of developing early-onset emphysema caused by hereditary A1ATD is inversely correlated with the serum A1AT level (ATS/ERS Statement, 2003). Only PiZ homozygotes with severe decreased A1AT serum level, or carriers of M-like or Null alleles are at significant risk to develop panlobular emphysema with typical dilatation or destruction of all lower lobules. However, the risk of COPD in PiMZ individuals is still controversial. Heterozygous, PiMZ individuals have moderately reduced serum levels A1AT, but whether they have increased risk of COPD is uncertain. Tarjan et al. (Tarjan et al., 1994) in longitudinal lung function study in heterozygous PiMZ subjects observed a decrease in elasticity and deterioration lung function parameters in comparison to those without A1ATD, which supports the concept of PiMZ phenotype being a risk factor for pulmonary emphysema development at a younger age. Dahl et al. (Dahl et al., 2002) found that PiMZ heterozygotes had a slightly greater rate of decrease in FEV1. Meta analysis by Hersh et al. (Hersh et al., 2004) has shown an increased odd of COPD in PiMZ individuals with suggestion that variability in study design and quality limits interpretation. Recent study (Sørheim et al., 2010) suggests that PiMZ individuals may be slightly more susceptible to the development of the airflow obstruction than PiMM individuals.

#### **3.1 Epidemiology of A1ATD**

The majority of the data regarding frequency and geographical distribution of severe A1ATD genotypes refer to the most frequent deficient variants PiZZ and PiSZ. Considering that severe A1ATD predisposes the development of emphysema that requires expensive diagnostic methods and treatment, it would be very useful to determine prevalence of severe A1ATD in every population.

The most comprehensive study that has been performed on 200,000 Swedish newborns revealed the prevalence rate of PiZZ phenotype of approximately 1 in 1,600 newborns (Sveger, 1976). Despite the lack of reliable epidemiological studies and marked differences between countries, Blanco et al. (Blanco et al., 2006) estimated numbers of individuals carrying two most common deficiency alleles, Z and S in Europe. Highest prevalence of the PiZZ phenotype is in the Scandinavian Peninsula, Latvia and Denmark and progressively decreases towards the South and the East of Europe. While the highest prevalence of the PiSZ is in the Iberian Peninsula and it gradually decreases towards the North, South and East of the continent. Prevalence of the moderate PiMZ is highest in the South of the Scandinavian Peninsula, Baltic Republics, Denmark and the UK, and progressively decreases towards the East, South and North of the continent. The estimated prevalence of PiZZ, PiSZ and PiMZ in European adults was 1/4727, 1/1051 and 1/36 respectively, with large variation in different countries. In this regard, it was estimated that there are 124,594 PiZZ, 560,515 PiSZ, and even 16 million PiMZ individuals in all Europe. Globally, A1ATD affects all major racial subgroups, and there are at least 116 million carriers (PiMZ and PiMS) and 3.4 million deficiency allele's combinations (PiSS, PiSZ and PiZZ) worldwide (De Serres, 2002). According to these data, frequency of the Z allele is lowest in Far East Asia (0.04%), and highest in Northern Europe (1.53%), while the S allele is lowest in Far East Asia (0.07%), and highest in Southern Europe (5.64%).

Although the epidemiological data indicate a large of number of A1ATD individuals worldwide, this condition is largely undiagnosed and exact prevalence of A1ATD in most population remains unknown. Owing to data from international registry of A1ATD, established in several countries, it was estimated that only 0.35% of severe A1ATD (PiZZ and PiSZ) are actually recognized (Luisetti and Seersholm, 2004). One of the reasons may be significantly delayed onset of symptoms. In 1994 Stoller at al. (Stoller et al., 1994) reported a mean interval of 7.2 years between initial symptom and first diagnosis. A decade later, a decrement in the overall diagnostics of 5.6 years was noted (Stoller et al., 2005a), which was attributed to the better education of physicians about recognising A1ATD. Authors concluded that despite this decrement in the overall diagnostics, underrecognition of individuals with A1ATD persisted. Under-recognition of A1ATD may be a part of a larger phenomenon of under-recognition of individuals with COPD. The second reason could be a low penetrance of the PiZ gene, so that the relationship between genotype and clinical phenotype is not strong.

There are many benefits of early detection of A1ATD, such as avoidance of exposures to cigarette smoke and air pollution in prevention of pulmonary emphysema. Also, measure of prevention is protection from pneumonia, which is frequently reported in medical history of A1ATD patients with emphysema (McElvaney et al., 1997). In A1ATD patients, pulmonary infection further increases the risk of developing emphysema. Pulmonary infection favors increasing of elastase activity with subsequent destruction of lung due to compromised antiprotease defenses, and promotion of A1AT polymers due to elevated body temperature in inflammation. In this regard, it is very important to protect lung function of A1ATD individuals trough aggressive treatment of pulmonary infections and by vaccination with pneumococcal and influenza A vaccines.

Data concerning genetic epidemiology of the rare A1ATD variants are incomplete, and therefore raise a suspicion that the prevalence of these variants might be higher than 2-4%, as previously considered, due to misclassification as Z variant (Luisetti and Seersholm, 2004). Phenotyping by isoelectric focusing is often used to characterize 1AT deficiency, but this method may lead to misdiagnosis (e.g., by missing null alleles). Zorzetto et al. (Zorzetto et al., 2008) sequenced exons II, III, IV, and V of subjects whose are negative for Z and S alleles, and detected even 7% rare A1ATD alleles. Moreover, Prins et al. (Prins et al., 2008) have analyzed patients with A1ATD by sequencing of exons II, III, and V of the SERPINE1 gene and reported that up to 22% of deficiency variants were missed by conventional diagnostic methods.

#### **3.2 Emphysema caused by A1ATD**

64 Lung Diseases – Selected State of the Art Reviews

all lower lobules. However, the risk of COPD in PiMZ individuals is still controversial. Heterozygous, PiMZ individuals have moderately reduced serum levels A1AT, but whether they have increased risk of COPD is uncertain. Tarjan et al. (Tarjan et al., 1994) in longitudinal lung function study in heterozygous PiMZ subjects observed a decrease in elasticity and deterioration lung function parameters in comparison to those without A1ATD, which supports the concept of PiMZ phenotype being a risk factor for pulmonary emphysema development at a younger age. Dahl et al. (Dahl et al., 2002) found that PiMZ heterozygotes had a slightly greater rate of decrease in FEV1. Meta analysis by Hersh et al. (Hersh et al., 2004) has shown an increased odd of COPD in PiMZ individuals with suggestion that variability in study design and quality limits interpretation. Recent study (Sørheim et al., 2010) suggests that PiMZ individuals may be slightly more susceptible to the

The majority of the data regarding frequency and geographical distribution of severe A1ATD genotypes refer to the most frequent deficient variants PiZZ and PiSZ. Considering that severe A1ATD predisposes the development of emphysema that requires expensive diagnostic methods and treatment, it would be very useful to determine prevalence of

The most comprehensive study that has been performed on 200,000 Swedish newborns revealed the prevalence rate of PiZZ phenotype of approximately 1 in 1,600 newborns (Sveger, 1976). Despite the lack of reliable epidemiological studies and marked differences between countries, Blanco et al. (Blanco et al., 2006) estimated numbers of individuals carrying two most common deficiency alleles, Z and S in Europe. Highest prevalence of the PiZZ phenotype is in the Scandinavian Peninsula, Latvia and Denmark and progressively decreases towards the South and the East of Europe. While the highest prevalence of the PiSZ is in the Iberian Peninsula and it gradually decreases towards the North, South and East of the continent. Prevalence of the moderate PiMZ is highest in the South of the Scandinavian Peninsula, Baltic Republics, Denmark and the UK, and progressively decreases towards the East, South and North of the continent. The estimated prevalence of PiZZ, PiSZ and PiMZ in European adults was 1/4727, 1/1051 and 1/36 respectively, with large variation in different countries. In this regard, it was estimated that there are 124,594 PiZZ, 560,515 PiSZ, and even 16 million PiMZ individuals in all Europe. Globally, A1ATD affects all major racial subgroups, and there are at least 116 million carriers (PiMZ and PiMS) and 3.4 million deficiency allele's combinations (PiSS, PiSZ and PiZZ) worldwide (De Serres, 2002). According to these data, frequency of the Z allele is lowest in Far East Asia (0.04%), and highest in Northern Europe (1.53%), while the S allele is lowest in Far East Asia

Although the epidemiological data indicate a large of number of A1ATD individuals worldwide, this condition is largely undiagnosed and exact prevalence of A1ATD in most population remains unknown. Owing to data from international registry of A1ATD, established in several countries, it was estimated that only 0.35% of severe A1ATD (PiZZ and PiSZ) are actually recognized (Luisetti and Seersholm, 2004). One of the reasons may be significantly delayed onset of symptoms. In 1994 Stoller at al. (Stoller et al., 1994) reported a mean interval of 7.2 years between initial symptom and first diagnosis. A decade later, a decrement in the overall diagnostics of 5.6 years was noted (Stoller et al., 2005a), which was

development of the airflow obstruction than PiMM individuals.

**3.1 Epidemiology of A1ATD** 

severe A1ATD in every population.

(0.07%), and highest in Southern Europe (5.64%).

The main lung manifestations of severe A1ATD are emphysema and COPD (ATS/ERS Statement, 2003). In A1ATD-smokers, the first symptoms usually occur between 32 and 41 years, with considerable variability in the time of onset of symptoms (Larsson, 1978; Tobin et al., 1983).

Panlobular emphysema is dominant clinical manifestation in A1ATD patients, and affects the lower half of the lungs. Pulmonary vessels of the emphysematous lung appear fewer and smaller than normal (Stein et al., 1971). In severe A1ATD changes at the level of bronchioli such as bronchiolitis obliterans, bronchiolectasia, acute and chronic bronchiolitis and bronchiolitis with organizing pneumonia are more frequent than in emphysema without A1ATD (Theegarten et al., 1998).

First representative study that included 124 patients with A1ATD and symptomatic emphysema (Brantly et al., 1988) showed predominance of male gender, ex-smoke status, levels of A1AT ≤ 5.5 M (0.3 g/L), and abnormalities in a lower zone distribution. About one third of patients had pulmonary hypertension. The lung function tests were typical for emphysema: the FEV1 and DLCO were dramatically reduced, and their annual rate of decline was greater than in general population. The cumulative probability of survival of the patients indicated a significantly shortened lifespan with a mean survival of 16% at 60 yr of age compared with 85% for normal persons.

The largest study ever conducted has been included 1,129 patients who participated in the National Heart, Lung, and Blood Institute (NHLBI) Registry of Individuals with Severe Deficiency of A1AT (McElvaney et al., 1997). Most frequent were PiZZ (97%), and very few PiSZ (1%), and rare variants (2%). Pulmonary function test results were consistent with emphysema. The pulmonary function impairment was moderate to severe, frequently associated with a bronchodilator response, but generally with preservation of the PaCO2 until the development of severe airway obstruction. Medical history of lung function revealed that initial diagnoses included asthma (in 35% of participants), respiratory tract allergies (28%), pneumonia (43%), and chronic bronchitis (36%). The most frequent symptoms in A1ATD patients were dyspnea on exertion (in 84% of participants), selfreported wheezing during respiratory tract infections (76%), and wheezing independent of infections (65%), usual cough (45%), and "annual" cough in phlegm episodes (52%). Significant number of patients who initially diagnosed as asthma had symptoms that suggest airway hyper-responsiveness such as cough and wheezing, responded to aerosol bronchodilator moderately (Eden et al., 1997). It is interesting that a subgroup of individuals in the Registry with relatively normal lung function was younger, more likely to have never smoked and more likely to have come to medical attention owing to a family history of A1ATD.

Cigarette smoking is associated with more accelerated decline of lung function and early development of emphysema in PiZZ individuals leading to a considerably reduced life expectancy (Larsson, 1978). A mortality study showed that emphysema was a major determinant of mortality in population of severe A1ATD patients (Stoller et al., 2005b*).*  Negative impact of smoking on survival of A1ATD patients was demonstrated in two recently published studies. Tanassh et al. (Tanash et al., 2008) reported that PiZZ individuals who have never smoked and have been identified trough screening do not have an increased mortality risk in comparison to general Swedish population. Larger study which included 1,349 PiZZ individuals selected from the Swedish National AATD Registry showed that smokers with severe A1ATD had a significantly higher mortality risk than the general Swedish population (Tanash et al., 2010). The pulmonary emphysema has been more common in PiZZ smokers (78%) than in PiZZ never smokers (47%), and respiratory diseases have been main cause of death among PiZZ smokers (58%).

#### **3.3 Laboratory diagnosis of hereditary A1ATD**

Although being one of the most prevalent and potentially severe hereditary disorders, A1ATD still remains under-recognized. Affected individuals often visit several physicians before obtaining the correct diagnosis. The main reason is generally low knowledge about A1ATD among internists and respiratory therapists (Taliercio et al., 2010). Clinically relevant A1ATD is often caused by homozygous inheritance of the Z allele, but A1ATD can also be due to the combination of other rare deficient or null alleles at the Pi locus. Even moderate A1ATD in PiMZ heterozygote is associated with reduced pulmonary functions in individuals with clinically established COPD (Dahl et al., 2001).

The guidelines of the American Thoracic Society and the European Respiratory Society (ATS/ERS Statement, 2003) recommend quantitative and qualitative laboratory testing for A1ATD for all patients with COPD, asthma, unexplained liver disease, and necrotizing panniculitis, as well as for asymptomatic subjects with persistent airflow limitation and siblings of A1ATD individuals. Laboratory testing of suspected A1ATD individuals involve

The largest study ever conducted has been included 1,129 patients who participated in the National Heart, Lung, and Blood Institute (NHLBI) Registry of Individuals with Severe Deficiency of A1AT (McElvaney et al., 1997). Most frequent were PiZZ (97%), and very few PiSZ (1%), and rare variants (2%). Pulmonary function test results were consistent with emphysema. The pulmonary function impairment was moderate to severe, frequently associated with a bronchodilator response, but generally with preservation of the PaCO2 until the development of severe airway obstruction. Medical history of lung function revealed that initial diagnoses included asthma (in 35% of participants), respiratory tract allergies (28%), pneumonia (43%), and chronic bronchitis (36%). The most frequent symptoms in A1ATD patients were dyspnea on exertion (in 84% of participants), selfreported wheezing during respiratory tract infections (76%), and wheezing independent of infections (65%), usual cough (45%), and "annual" cough in phlegm episodes (52%). Significant number of patients who initially diagnosed as asthma had symptoms that suggest airway hyper-responsiveness such as cough and wheezing, responded to aerosol bronchodilator moderately (Eden et al., 1997). It is interesting that a subgroup of individuals in the Registry with relatively normal lung function was younger, more likely to have never smoked and more likely to have come to medical attention owing to a family

Cigarette smoking is associated with more accelerated decline of lung function and early development of emphysema in PiZZ individuals leading to a considerably reduced life expectancy (Larsson, 1978). A mortality study showed that emphysema was a major determinant of mortality in population of severe A1ATD patients (Stoller et al., 2005b*).*  Negative impact of smoking on survival of A1ATD patients was demonstrated in two recently published studies. Tanassh et al. (Tanash et al., 2008) reported that PiZZ individuals who have never smoked and have been identified trough screening do not have an increased mortality risk in comparison to general Swedish population. Larger study which included 1,349 PiZZ individuals selected from the Swedish National AATD Registry showed that smokers with severe A1ATD had a significantly higher mortality risk than the general Swedish population (Tanash et al., 2010). The pulmonary emphysema has been more common in PiZZ smokers (78%) than in PiZZ never smokers (47%), and respiratory

Although being one of the most prevalent and potentially severe hereditary disorders, A1ATD still remains under-recognized. Affected individuals often visit several physicians before obtaining the correct diagnosis. The main reason is generally low knowledge about A1ATD among internists and respiratory therapists (Taliercio et al., 2010). Clinically relevant A1ATD is often caused by homozygous inheritance of the Z allele, but A1ATD can also be due to the combination of other rare deficient or null alleles at the Pi locus. Even moderate A1ATD in PiMZ heterozygote is associated with reduced pulmonary functions in

The guidelines of the American Thoracic Society and the European Respiratory Society (ATS/ERS Statement, 2003) recommend quantitative and qualitative laboratory testing for A1ATD for all patients with COPD, asthma, unexplained liver disease, and necrotizing panniculitis, as well as for asymptomatic subjects with persistent airflow limitation and siblings of A1ATD individuals. Laboratory testing of suspected A1ATD individuals involve

diseases have been main cause of death among PiZZ smokers (58%).

individuals with clinically established COPD (Dahl et al., 2001).

**3.3 Laboratory diagnosis of hereditary A1ATD** 

history of A1ATD.

analysis of A1AT concentrations in serum and identification of specific alleles by genotyping or phenotyping. Therefore it is important to identify appropriate cutoff that balances costs of testing identification of deficiency alleles in the general population.

Diagnostic algorithms for laboratory testing of the A1AT deficiency that were proposed (Snyder et al., 2006; Bornhorst et al., 2007; Miravitlles et al., 2010) should lead to improve diagnostics of A1ATD (Figure 4.). Initial testing involves quantification of A1AT concentrations and genotyping. Quantification of A1AT alone is not sufficient to diagnose genetic causes of A1ATD due to secondary causes of reduced concentration of A1AT in severe liver diseases, protein-losing enteropathies or nephrotic syndrome which may cause a general decrease of serum proteins. If serum level of A1AT lay in the expected range for the certain genotype, than the results and interpretation should be reported to physician. Qualitative analyses of A1ATD include two complementary methods, genotyping and phenotyping, each with advantages and disadvantages. Using conventional phenotyping of the A1AT by isoelectic focusing (IEF) necessarily leads to misdiagnoses of the null alleles (Klaassen et al., 2001). Therefore, the replacement of IEF with direct sequencing of the relevant parts of the SERPINA1 gene enables an efficient and reliable approach to reveal A1ATD patient. Direct sequencing of exons II, III and V of the SERPINA1 gene is the preferred method in initial phase of diagnostic algorithm for laboratory testing of A1ATD (Prins et al., 2008), as it allows detection of disease-associated A1AT allele combinations, including null alleles.

However if quantitative result are in discrepancy with obtained genotype, laboratory should perform phenotype assay. Determination of phenotypes serve as complementary to the genetic assay, in order to clarify cases that cannot be detected by genotyping. Besides phenotyping, other techniques that can be considered as complementary include wholegene sequencing and the addition of other alleles to the melting curve genotype assay (Rodriguez et al., 2002).

There are two approaches to assess the complementarity between serum level of A1AT and genotype result. Previously, the estimate was based on established threshold of A1AT serum levels for the most frequent A1AT phenotypes/genotypes in the general population. The various ranges for A1AT serum level of the most common phenotypes, which can be found in the literature, are results of different methods of quantification, different commercially available standards of A1AT, and samples size. (Brantly et al., 1991; Lee et al., 2002; ATS/ERS Statement, 2003). Additional difficulty is the estimation of the ranges for rare phenotypes because it is difficult to collect a representative number of samples in a given population.

In the context of these concerns, particular problem is the presence of inflammation in examined population. The A1AT is an acute phase protein and its production and secretion increases with inflammation. Thus, serum levels might be "falsely elevated" and are not reflecting the genotype, especially in moderate A1ATD. This was confirmed in a recent study which found that PiMZ individuals with a higher level of C-reactive protein (CRP, a sensitive marker of inflammation) had higher level of A1AT than those with lower level of CRP (Zorzetto et al., 2008). Also, we should bear in mind that in healthy blood donors only 26% of the variance of circulating A1AT level is explained by known SERPINA1 gene variants (Oakeshott et al., 1985). Recently, large population-based study on the Swiss SAPALDIA cohort (Senn et al., 2008) revealed that female gender, hormone intake, systolic blood pressure, age in men and in postmenopausal women, as well active and passive smoking were positively, whereas alcohol intake and body mass index (BMI) were inversely correlated with serum A1AT levels, independent of CRP adjustment. The results of this study reflect a complexity of relationship between tobacco exposures, gender, circulating A1AT, systemic inflammatory status and lung function.

Nowadays, the efforts are directed towards determination of the cut-off values with high specificity and sensitivity, in order to separate normal from deficient phenotypes. The value of A1AT serum level of 22 M (1, 2 g/L) has been determined as a reliable cut-off able to identify A1ATD with a specificity of 73% and a sensitivity of 97% (Corda et al., 2006). The lower value of 18.5 M (1.00 g/L) was able to detect heterozygous A1ATD (Simsek et al., 2011), while cut-off value of 14.7 M (0.8 g/L) was proposed for detection of all patients who are at risk of A1ATD (Prins et al., 2008).

Fig. 4. Alpha-1-antitrypsin deficiency−testing algorithm (Snyder et al., 2006)

#### **4. Oxidation of alpha-1-antitrypsin and emphysema**

Cigarette smoke and lung inflammation leads to proteolytic destruction of the lung parenchyma with characteristic loss of alveolar integrity and an enlargement of alveolar space (reviewed in: Sharafkhaneh et al., 2008). Oxidative stress, as a result of an imbalance between oxidants and antioxidants, plays a critical role in the pathogenesis of emphysema (Rahman, 2005; Janoff et al., 1983). There are two main significant sources of oxidants:

inversely correlated with serum A1AT levels, independent of CRP adjustment. The results of this study reflect a complexity of relationship between tobacco exposures, gender,

Nowadays, the efforts are directed towards determination of the cut-off values with high specificity and sensitivity, in order to separate normal from deficient phenotypes. The value of A1AT serum level of 22 M (1, 2 g/L) has been determined as a reliable cut-off able to identify A1ATD with a specificity of 73% and a sensitivity of 97% (Corda et al., 2006). The lower value of 18.5 M (1.00 g/L) was able to detect heterozygous A1ATD (Simsek et al., 2011), while cut-off value of 14.7 M (0.8 g/L) was proposed for detection of all patients

circulating A1AT, systemic inflammatory status and lung function.

Fig. 4. Alpha-1-antitrypsin deficiency−testing algorithm (Snyder et al., 2006)

Cigarette smoke and lung inflammation leads to proteolytic destruction of the lung parenchyma with characteristic loss of alveolar integrity and an enlargement of alveolar space (reviewed in: Sharafkhaneh et al., 2008). Oxidative stress, as a result of an imbalance between oxidants and antioxidants, plays a critical role in the pathogenesis of emphysema (Rahman, 2005; Janoff et al., 1983). There are two main significant sources of oxidants:

**4. Oxidation of alpha-1-antitrypsin and emphysema** 

who are at risk of A1ATD (Prins et al., 2008).

exogenous from cigarette smoke or air pollutants and endogenous from activated neutrophils and macrophages.

Oxidative stress caused by cigarette smoke and airway inflammation together form a vicious circle. It has been shown that cigarette smoke-mediated oxidative stress induces the release of proinflammatory cytokine by activation of NF-kappaB and posttranslational modifications of histone deacetylase in macrophages (Yang et al., 2006). Chronic inflammation that persists in emphysema leads to the activation of macrophages and neutrophils (Finkelstein et al., 1995), which are a significant source of reactive oxygen species (ROS). When ROS overwhelm lung antioxidant defenses, the oxidative stress arises. Thus, cigarette smoke and activated macrophages/neutrophils represent considerable source of reactive oxygen species in emphysema.

Cigarette smoke contains free radicals with tremendous oxidative power which serve as direct damaging agents and/or precursor of the other damaging substances. The main free radicals in cigarette smoke are superoxide (O2-•), hydroxyl radical (•OH), and hydrogen peroxide (H2O2) (Pryor, 1997).

Harmful effects of oxidative stress are numerous: inactivation of antiproteases, disregulation of cell proliferation, induction of apoptosis, modulation the immune system, direct damages of proteins, lipids, and nucleic acids. The products of lipid peroxidation, protein oxidation, and nucleic acid oxidation have been shown in emphysema (Mohsenin, 1991; Sahin et al., 2001; Hackett et al., 2010; Torres-Ramos et al., 2009; Deslee et al., 2009; Deslee et al., 2010).

#### **4.1 Oxidation of A1AT - structural and functional consequences**

A1AT is a protein susceptible to oxidation, and its exposure to pro-oxidative enzymes and chemicals results in their oxidation. Exposure of one A1AT molecule to oxidants results in oxidation of Met358 and Met351 residues to methionine sulfoxide (Johnson and Travis, 1979). Oxidation of both Met358 and Met351 significantly reduces the ability of A1AT to inhibit neutrophil elastase (Beatty et al., 1980; Taggart et al., 2000). Oxidation of Cys232 is far more likely to occur in oxidizing environments comparing with oxidation of exposed and reactive methionine residues (Griffiths et al., 2002). The structural and biological aspects of Cys232 oxidation are still unknown.

Methionine residues can be oxidized by cigarette smoke-derived oxidants (Pryor et al., 1984), produced *in vivo* such as peroxide, hydroxyl radicals, chloramines, hypochloride, inducible nitric oxide, and peroxynitrite (Vogt, 1995), or with mineral dust (coal, amosite asbestos, silica, or titanium dioxide) (Li et al., 1997). Thus the oxidation of A1AT by cigarette smoke or free radicals *in vivo* could lead to a functional deficiency of A1AT and has been suggested as a mechanism contributing to the development of emphysema in non-deficient PiM individuals. Cigarette smoke-mediated oxidation of the Z mutant accelerates process of polymerization, which further reduces defense of lung, increases neutrophil influx into the lungs (Alam et al., 2011), and contributes to premature emphysema in PiZZ homozygotes who smoke.

It has been reported that cigarette smoke oxidize A1AT (oxyA1AT), and reduces inhibitory activity of A1AT against elastase (Janoff et al., 1979) and caspase-3 (Petrache et al., 2006a). Substantial constituents of cigarette smoke that oxidise and inactivate A1AT include hydrogen peroxyde, nitrogen dioxide, transition metals, and products of lipid peroxidation initiated by cigarette smoke (reviewed in: Evans and Pryor, 1994). Inactivation of antielastase activity of A1AT is reversible and phenolic antioxidants prevented the suppression of serum elastase-inhibition by cigarette smoke (Carp and Janoff, 1978).

Another mechanism of A1AT oxidation is its oxidative inactivation in the microenvironment of inflammatory cells, at sites of acute or chronic inflammation. The lower airways of smokers are infiltrated with phagocitic cells. Induced neutrophils and alveolar macrophage release a spectrum of oxidants and pro-oxidative enzymes that may inactivate A1AT in their local environment. Increased release of proteases from triggered phagocytes and reduced antiprotease defense leads to damage of lung tissue (Carp and Janoff, 1980). Myeloperoxidase-dependent production of oxidants from neutrophils is increased in inflammation, and can cause significant damage of A1AT. Myeloperoxidase (MPO) is located in the azurophilic granules of the neutrophils, and plays an important role in the human immune system by killing bacteria and invading pathogen. Under certain circumstances, a MPO can be released into the extracellular space. In the presence of hydrogen peroxide and chloride ions, MPO produces hypochlorous acid (HOCl), the major strong oxidant which reacts readily with free amino groups to form N-chloramines. *In vitro* studies show that MPO inactivates purified A1AT trough oxidation of two methionine residues (Matheson et al., 1979; Summers et al., 2008). Hydrogen peroxide released from macrophages in the small airways of smokers synergistically with hydrogen peroxide from tobacco may contribute to the oxidative inhibition of A1AT (Cohen and James, 1982). Moreover, A1AT oxidized by the myeloperoxidase-hydrogen peroxide system (MPO-H2O2), in inflammation, promotes the formation of IgA-A1AT complexes, and consequently the elastase inhibitory activity of A1AT is reduced (Scott et al., 1999).

Recent studies have shown that although oxyA1AT loses antielastase activity it gets new biological properties that may be important in the pathogenesis of certain diseases. Several studies have shown that oxyA1AT behaves as a proinflammatory stimulus. Moraga et al. (Moraga and Janciauskiene, 2000) revealed that oxyA1AT activates monocytes, which is reflected in significant elevation in monocyte chemoattractant protein-1, cytokine IL-6, and TNF expression, as well in increased activity of NADPH oxidase. Furthermore, oxyA1AT by activation of pro-oxidative NADPH oxidase may promote its own formation and thereby contributes to inflammation. OxyA1AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways (Li et al., 2009). The release of oxidants by these inflammatory cells could oxidize A1AT, perpetuating the cycle and potentially contributing to the pathogenesis of COPD.

#### **4.2 Oxidation of A1AT - clinical aspects**

Centrolobular emphysema in smokers as a clinical manifestation is strongly associated with functional deficiency caused by oxidation of A1AT. Several studies supported the mechanism by which tobacco smoke increased the risk of developing emphysema. According to this mechanism, cigarette smoke reduces protease inhibitory capacity, causing the increase of the lung vulnerability to elastolytic destruction and thereby increasing the risk for the development of emphysema (Carp et al., 1982; Janoff et al., 1983; Ogushi et al., 1991). By losing the antiprotease ability and becoming pro-inflammatory stimulus, the oxyA1AT favors the development of clinical emphysema.

OxyA1AT has a potential clinical significance in atherogenesis. Mashiba et al. (Mashiba et al., 2001) have revealed that A1AT produced and oxidized by macrophages, attaches to low density lipoprotein (LDL) in the intima of the arterial wall and contributes to the lipid accumulation in arterial wall cells in the early stage of atherogenesis.

In addition, reduction of antielastase activity was observed in hemodialysis patients (Hashemi et al., 2009). Furthermore, Honda et al. (Honda, et al., 2009) reported that serum levels of oxyA1AT positively correlated with myeloperoxidase in patients on hemodialysis, and could represent a useful marker for the estimation of the increasing carotid intimamedia thickness. They also found that oxyA1AT might be an independent predictor of protein-energy wasting in patients on hemodialysis. Oxidized A1AT was also detected in patients with Alzheimer's disease, heart failure, and in premature rupture of the fetal membrane (Choi et al., 2002; Banfi et al., 2008; Izumi-Yoneda et al., 2009).

## **4.3 Perspective of the determination of oxidized A1AT**

Determination of oxyA1AT as biomarker is not used in routine practice, although it may be useful in assessment of pulmonary emphysema risk, and other pathological conditions associated with oxidative stress and inflammation. Our knowledge of the clinical significance of oxyA1AT is still insufficient, as well the optimal quantification of oxyA1AT.

Previously used method for quantification of oxyA1AT was based on determination of elastase- (EIC) and trypsin inhibitory capacity (TIC) (Beatty et al., 1982). As oxyA1AT loses its ability to inhibit porcine elastase but retains antitrypsin activity, the increased TIC/EIC ratio correlates with degree of A1AT oxidation. Progress in oxyA1AT methodology includes development of immunochemical method (Ueda et al., 2002).

Advanced methodology of quantification of oxyA1AT should be developed as a sensitive, specific method which would be suitable for routine practice.

## **5. Conclusion**

70 Lung Diseases – Selected State of the Art Reviews

Another mechanism of A1AT oxidation is its oxidative inactivation in the microenvironment of inflammatory cells, at sites of acute or chronic inflammation. The lower airways of smokers are infiltrated with phagocitic cells. Induced neutrophils and alveolar macrophage release a spectrum of oxidants and pro-oxidative enzymes that may inactivate A1AT in their local environment. Increased release of proteases from triggered phagocytes and reduced antiprotease defense leads to damage of lung tissue (Carp and Janoff, 1980). Myeloperoxidase-dependent production of oxidants from neutrophils is increased in inflammation, and can cause significant damage of A1AT. Myeloperoxidase (MPO) is located in the azurophilic granules of the neutrophils, and plays an important role in the human immune system by killing bacteria and invading pathogen. Under certain circumstances, a MPO can be released into the extracellular space. In the presence of hydrogen peroxide and chloride ions, MPO produces hypochlorous acid (HOCl), the major strong oxidant which reacts readily with free amino groups to form N-chloramines. *In vitro* studies show that MPO inactivates purified A1AT trough oxidation of two methionine residues (Matheson et al., 1979; Summers et al., 2008). Hydrogen peroxide released from macrophages in the small airways of smokers synergistically with hydrogen peroxide from tobacco may contribute to the oxidative inhibition of A1AT (Cohen and James, 1982). Moreover, A1AT oxidized by the myeloperoxidase-hydrogen peroxide system (MPO-H2O2), in inflammation, promotes the formation of IgA-A1AT complexes, and consequently the

Recent studies have shown that although oxyA1AT loses antielastase activity it gets new biological properties that may be important in the pathogenesis of certain diseases. Several studies have shown that oxyA1AT behaves as a proinflammatory stimulus. Moraga et al. (Moraga and Janciauskiene, 2000) revealed that oxyA1AT activates monocytes, which is reflected in significant elevation in monocyte chemoattractant protein-1, cytokine IL-6, and TNF expression, as well in increased activity of NADPH oxidase. Furthermore, oxyA1AT by activation of pro-oxidative NADPH oxidase may promote its own formation and thereby contributes to inflammation. OxyA1AT generated in the airway interacts directly with epithelial cells to release chemokines IL-8 and MCP-1, which in turn attracts macrophages and neutrophils into the airways (Li et al., 2009). The release of oxidants by these inflammatory cells could oxidize A1AT, perpetuating the cycle and potentially contributing

Centrolobular emphysema in smokers as a clinical manifestation is strongly associated with functional deficiency caused by oxidation of A1AT. Several studies supported the mechanism by which tobacco smoke increased the risk of developing emphysema. According to this mechanism, cigarette smoke reduces protease inhibitory capacity, causing the increase of the lung vulnerability to elastolytic destruction and thereby increasing the risk for the development of emphysema (Carp et al., 1982; Janoff et al., 1983; Ogushi et al., 1991). By losing the antiprotease ability and becoming pro-inflammatory stimulus, the

OxyA1AT has a potential clinical significance in atherogenesis. Mashiba et al. (Mashiba et al., 2001) have revealed that A1AT produced and oxidized by macrophages, attaches to low density lipoprotein (LDL) in the intima of the arterial wall and contributes to the lipid

elastase inhibitory activity of A1AT is reduced (Scott et al., 1999).

to the pathogenesis of COPD.

**4.2 Oxidation of A1AT - clinical aspects** 

oxyA1AT favors the development of clinical emphysema.

accumulation in arterial wall cells in the early stage of atherogenesis.

The only proven genetic risk factor in pathogenesis of emphysema is severe alpha-1 antitrypsin deficiency. However, several known and unknown genetic and environmental factors contribute to the differences in the susceptibility of A1ATD individuals to develop lung disease. Among the most important are cigarette smoke and air pollutants that could provoke oxidative stress and inflammatory response.

So far, a lot of attention and efforts in A1ATD research was given to the deficient A1AT variants and A1AT polymerization, while the oxidation of A1AT protein has been generally overlooked. Non-deficient, heavy smokers may have normal serum level of A1AT, but with reduced functional activity due to functional deficiency caused by oxidation. The physiological role of oxyA1AT could be particularly important regarding growing evidence of different biological functions of A1AT that go beyond those usually linked to its antiprotease activities. Future studies will elucidate the role of A1AT oxidation in modulation of inflammation and tissue destruction which represent landmarks of emphysema, as well in modulation of augmentation therapy.

The early recognition and diagnostics of A1ATD is the most important in terms of prevention and delay of the onset of symptoms of emphysema. The optimal approach in therapy of A1ATD would be to inhibit the polymerization of the Z protein (intracellular and extracellular) and prevent oxidative stress, accompanied by standard augmentation therapy.

## **6. Acknowledgment**

This work was supported by grants 173008 from the Ministry of Education and Science, Republic of Serbia.

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Nukiwa, T., Satoh, K., Brantly, M.L., Ogushi, F., Fells, G.A., Courtney, M. & Crystal, R.G.

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## **Recent Advances in the Research and Development of Alpha-1 Proteinase Inhibitor for Therapeutic Use**

## Elena Karnaukhova

*Division of Hematology, Center for Biologics Evaluation and Research Food and Drug Administration USA* 

## **1. Introduction**

82 Lung Diseases – Selected State of the Art Reviews

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444.

239.

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Schluchter, M.D. & Stoller, J.K. (1996).Clinical features of individuals with PI\*SZ phenotype of -1 antitrypsin deficiency. *Am J Respir Crit Care Med* 154, 1718- 1725. Ueda, U., Mashiba, S. & Uchida, K. (2002). Evaluation of oxidized alpha-1-antitrypsin in

blood as an oxidative stress marker using anti-oxidative 1-AT monoclonal

inactivation of 1-proteinase inhibitor by alveolar macrophages from healthy smokers requires the presence of myeloperoxidase. *Am J Respir Cell Mol Biol* 5, 437-

pollution is associated with disease severity in 1-antitrypsin deficiency. *Eur Respir* 

obstructive pulmonary disease: effect of 1-antitrypsin deficiency and the role of

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abnormality of PI S variant of human alpha1-antitrypsin. *Am J Hum Genet* 29, 233-

S., Scabini, R., von Eckardstein, A., Berger, W., Brändli, O., Rochat, T., Luisetti, M. & Probst-Hensch, N. (2008). SERPINA1 gene variants in individuals from the general populationwith reduced 1-antitrypsin concentrations. *Clin Chem* 54, 1331Human alpha-1-proteinase inhibitor (1-PI) is a well-characterized multifunctional protease inhibitor, the major physiological role of which is inhibition of neutrophil elastase (NE) in the lungs. The importance of1-PI is underlined by its deficiency which is characterized by low levels of 1-PI in the circulation. Under such conditions, lower levels of 1-PI are transported to tissues, including the fragile alveoli of the lungs. 1-PI deficiency (with levels of 1-PI in blood below 11 M, insufficient for inhibition of proteolytic enzymes in the lungs) is a common genetic condition predisposing 1-PI-deficient individuals to the development of chronic obstructive pulmonary disease (COPD). Hereditary1-PI deficiency is classically associated with the development of premature, ultimately fatal, panacinar emphysema. To slow down the progression of emphysema, several licensed 1-PI concentrate preparations derived from pooled human plasma are currently available for intravenous augmentation therapy for patients with congenital 1-PI deficiency and clinically evident emphysema. In addition, and as an alternative to the plasma-derived 1-PI products, multiple efforts have been made to develop recombinant versions of human 1-PI over the last three decades. This review describes the recent advances in the research and development of human 1-PI for therapetic use and covers the following: characterization of human 1-PI; epidemiology of 1-PI deficiency and currently licensed treatment; summary of the manufacturing and recent quality improvements of the 1-PI plasma-derived products; safety and efficacy of 1-PI intravenous augmentation and alternative routes; development of recombinant versions of human 1-PI; conditions other than emphysema that are associated with 1-PI; and some other aspects related to the research and development of 1-PI for therapeutic use.

 The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy

## **2. Human 1-PI and 1-PI deficiency**

### **2.1 Structure and function of 1-PI**

Human alpha-1-proteinase inhibitor (α1−PI), also known as alpha-1-antitrypsin, is the most abundant inhibitor of serine proteases in plasma. It is predominantly synthesized in hepatocytes, but is also produced, to a lower extent, by alveolar macrophages, neutrophils, and some other cells (White et al., 1981; Carlson et al., 1988; Paakko et al., 1996). In healthy individuals, the concentration of α1−PI in blood normally varies from 20 µM to 53 µM (1.04- 2.76 g/L) (Brantly et al., 1988; Brantly et al., 1991) with a half-life in the circulation of about 3-5 days (Crystal, 1989; Kalsheker et al., 2002). Though α1−PI has a wide range of inhibitory activities, its main physiological role is known to be the inhibition of polymorphonuclear leukocyte (neutrophil) elastase (NE) in the lungs (Travis, 1988). In the lower respiratory tract of healthy lungs, 1–PI provides more than 90% of the anti–neutrophil elastase protection (Crystal, 1991; Crystal et al., 1989). Hereditary α1−PI deficiency (with levels of α1−PI in blood below 11 µM, insufficient for inhibition of NE) is classically associated with development of early-onset pulmonary emphysema, a hallmark of α1−PI deficiency (Crystal et al., 1989; Snider, 1992). Smoking is known to be the biggest risk factor for developing emphysema; in smokers with α1−PI deficiency a severe lung impairment is usually observed in their fourth decade of life.

α1-PI is encoded by a single 12.2 kb gene (Pi) located on the long arm of chromosome 14 (Long et al., 1984; Rabin et al., 1986). Over 120 alleles of α1−PI have been identified with approximately 35 of them being associated with α1−PI deficiency, including Z-allele, which is the most common cause of the deficiency when inherited in a homozygous fashion. Due to a single mutation in the mobile domain (Glu342Lys), the α1−PI Z-mutant undergoes aberrant conformational transitions that prompts the protein to aggregate. This results in retention of polymerized α1−PI Z mutant within hepatocytes, thus inducing disease conditions in the liver and causing α1−PI deficiency in the circulation (Ekeowa et al., 2011; Lomas, 2005; Volpert et al., 2000). The prevalence of three major α1−PI variants (PiM, PiS, and PiZ) defines the number of carriers (PiMZ and PiMS) and individuals with deficiency phenotypes ( PiZZ, PiSZ, and PiSS). The epidemiology of α1−PI deficiency and its clinical manifestations, including lung diseases and liver diseases, has been described in detail (Ekeowa et al., 2011; Luisetti & Seersholm, 2004; Needham & Stockley, 2004; Gooptu & Lomas, 2009). Based on the α1−PI serum concentration, a common classification to define α1−PI deficiency includes the four major categories: (1) normal (with α1−PI serum levels not lower than 20 M); (2) deficient (with α1−PI concentrations in serum lower than 20 M); (3) dysfunctional (with normal α1−PI level, but lost or lower inhibitory activity); and (4) null (with α1−PI serum concentrations below the detectable level).

α1−PI is a 52 kDa glycoprotein belonging to the serine protease inhibitor (serpin) superfamily, which in addition to α1-PI also includes 1-antichymotrypsin, antithrombin, plasminogen activator inhibitor, C1 esterase inhibitor, and many others (Stein & Carrell, 1995; Silverman et al., 2001). A single polypeptide chain of α1-PI is comprised of 394 amino acid residues, including one cysteine, 2 tryptophanes, and 9 methionine residues (Carp et al., 1982; Johnson & Travis, 1979). Three N-linked glycans attached to asparagine residues 46, 83, and 247 represent ~12% of α1−PI by molecular weight (Mega et al., 1980a,b;

Carrell et al., 1981, 1982). The carbohydrate moiety is comprised of biantennary N-glycans, but also triantennary and traces of tetraantennary structures grounded on the mannose fork core and containing N-acetyl glucosamine, galactose, and terminal negatively-charged sialic

Human alpha-1-proteinase inhibitor (α1−PI), also known as alpha-1-antitrypsin, is the most abundant inhibitor of serine proteases in plasma. It is predominantly synthesized in hepatocytes, but is also produced, to a lower extent, by alveolar macrophages, neutrophils, and some other cells (White et al., 1981; Carlson et al., 1988; Paakko et al., 1996). In healthy individuals, the concentration of α1−PI in blood normally varies from 20 µM to 53 µM (1.04- 2.76 g/L) (Brantly et al., 1988; Brantly et al., 1991) with a half-life in the circulation of about 3-5 days (Crystal, 1989; Kalsheker et al., 2002). Though α1−PI has a wide range of inhibitory activities, its main physiological role is known to be the inhibition of polymorphonuclear leukocyte (neutrophil) elastase (NE) in the lungs (Travis, 1988). In the lower respiratory tract of healthy lungs, 1–PI provides more than 90% of the anti–neutrophil elastase protection (Crystal, 1991; Crystal et al., 1989). Hereditary α1−PI deficiency (with levels of α1−PI in blood below 11 µM, insufficient for inhibition of NE) is classically associated with development of early-onset pulmonary emphysema, a hallmark of α1−PI deficiency (Crystal et al., 1989; Snider, 1992). Smoking is known to be the biggest risk factor for developing emphysema; in smokers with α1−PI deficiency a severe lung impairment is usually observed

α1-PI is encoded by a single 12.2 kb gene (Pi) located on the long arm of chromosome 14 (Long et al., 1984; Rabin et al., 1986). Over 120 alleles of α1−PI have been identified with approximately 35 of them being associated with α1−PI deficiency, including Z-allele, which is the most common cause of the deficiency when inherited in a homozygous fashion. Due to a single mutation in the mobile domain (Glu342Lys), the α1−PI Z-mutant undergoes aberrant conformational transitions that prompts the protein to aggregate. This results in retention of polymerized α1−PI Z mutant within hepatocytes, thus inducing disease conditions in the liver and causing α1−PI deficiency in the circulation (Ekeowa et al., 2011; Lomas, 2005; Volpert et al., 2000). The prevalence of three major α1−PI variants (PiM, PiS, and PiZ) defines the number of carriers (PiMZ and PiMS) and individuals with deficiency phenotypes ( PiZZ, PiSZ, and PiSS). The epidemiology of α1−PI deficiency and its clinical manifestations, including lung diseases and liver diseases, has been described in detail (Ekeowa et al., 2011; Luisetti & Seersholm, 2004; Needham & Stockley, 2004; Gooptu & Lomas, 2009). Based on the α1−PI serum concentration, a common classification to define α1−PI deficiency includes the four major categories: (1) normal (with α1−PI serum levels not lower than 20 M); (2) deficient (with α1−PI concentrations in serum lower than 20 M); (3) dysfunctional (with normal α1−PI level, but lost or lower inhibitory activity); and (4) null

α1−PI is a 52 kDa glycoprotein belonging to the serine protease inhibitor (serpin) superfamily, which in addition to α1-PI also includes 1-antichymotrypsin, antithrombin, plasminogen activator inhibitor, C1 esterase inhibitor, and many others (Stein & Carrell, 1995; Silverman et al., 2001). A single polypeptide chain of α1-PI is comprised of 394 amino acid residues, including one cysteine, 2 tryptophanes, and 9 methionine residues (Carp et al., 1982; Johnson & Travis, 1979). Three N-linked glycans attached to asparagine residues

Carrell et al., 1981, 1982). The carbohydrate moiety is comprised of biantennary N-glycans, but also triantennary and traces of tetraantennary structures grounded on the mannose fork core and containing N-acetyl glucosamine, galactose, and terminal negatively-charged sialic

46, 83, and 247 represent ~12% of α1−PI by molecular weight (Mega et al., 1980a,b;

(with α1−PI serum concentrations below the detectable level).

**2. Human 1-PI and 1-PI deficiency** 

**2.1 Structure and function of 1-PI** 

in their fourth decade of life.

(N-acetylneuraminic) acid (Mega et al., 1980b; Travis & Salvesen, 1983; Kolarich et al., 2006a). The glycosylation pattern is a major cause of the iso-electric focusing (IEF) pattern typical for α1−PI with major isoforms M2, M4, M6, and also M7 and M8 due to the Nterminal truncation (Jeppsson et al., 1985; Kolarich et al., 2006a,b). Some characteristics of human α1−PI are listed in Table 1. Like the majority of other native glycoproteins, α1-PI is intrinsically a highly heterogeneous moiety, mainly due to variably trimmed glycosylation and an N-terminal pentapeptide that can be absent (Hercz, 1985; Krasnewich et al., 1995; Vaughan et al., 1982).


a NLT, not lower than; b n.d., non-detectable; c See Table 3

Table 1. Characteristics of human α1-PI

Figure 1 shows a crystal structure of α1-PI, typical for serpins, which features 9 α-helices, 3 β-sheets (A, B, and C), and a mobile 15-residue reactive center loop (RCL) exposed for interaction with the target serine protease (Johnson & Travis, 1979; Lomas, 2005). Protease attack of the RCL results in cleavage at Met358-Ser359, formation of a covalent α1-PIprotease complex with the amino-terminal polypeptide inserted into the A β-sheet, and an overall dramatic conformational change (Huntington et al., 2000; Ludeman et al., 2001; Stratikos & Gettins, 1999; Wilczynska et al., 1997).

Unlike the majority of proteins, α1−PI is naturally folded in a metastable structure which is essential for its function. This is not the most thermodynamically stable form, and thus, α1- PI is prone to a variety of conformational transitions and modifications (Lomas, 2005; Lomas et al., 1995). Much like other serpins, α1-PI can intramolecularly convert into a more stable latent form, which is inactive, but the biological activity can be restored via denaturation and refolding (Lomas et al., 1995; Silverman et al., 2001).

Fig. 1. Crystal structure of α1-PI (PDB 1HP7) in two projections. (**A**) Front view at the α1-PI structure in respect to -sheet A, and (**B**) Side view obtained by 90o clockwise rotation of the molecule. The images were obtained using PyMOL (the PyMOL Molecular Graphics System, Version 1.1r1, Schrödinger, LLC).

In addition to its inhibitory antiprotease function, α1−PI exhibits a broad spectrum of noninhibitory activities (Brantly, 2002; Janciauskiene et al., 2011; Nita et al., 2005). Because of the nine methionine residues in α1−PI molecule, its plausible role as a putative antioxidant has been suggested (*e.g*., Levine et al., 1999, 2000).

Due to the abundance of α1−PI in human plasma and its conservative tertiary structure with hydrophobic cavities (Elliott et al., 2000; Lee et al., 2001; Parfrey et al., 2003), α1−PI has the capacity to bind small hydrophobic molecules. This property has been explored mainly with respect to the peptides and small molecules that may prevent the aggregation of the α1−PI Z mutant (Mahadeva et al. 2002; Mallya et al., 2007; Chang et al. 2009).

#### **2.2 The 1-PI deficiency and 1-PI replacement therapy**

There are approximately 60,000-100,000 severely deficient individuals in the United States which define α1-PI deficiency as a rare disease. However, according to several publications, α1-PI deficiency is widely under- and mis-diagnosed (*e.g*., de Serres, 2003; Bals et al., 2007). As reported by the World Health Organization (WHO, 1997), only 4% of the individuals with α1-PI deficiency cases are identified, and only a portion of them are receiving treatment. Currently licensed treatment of the patients with α1-PI deficiency and manifestation of pulmonary emphysema involves intravenous infusion of plasma-derived α1-PI preparations with the recommended dose of 60 mg of active α1-PI per kg of body weight administered once weekly. To maintain a threshold level of α1-PI (11µM), α1−PIdeficient patients should receive augmentation therapy for the duration of their lives, to slow the progression of emphysema. This nadir level has been determined based on α1-PI levels observed in the plasma of individuals who are heterozygous for Z-mutant α1−PI and who do not develop emphysema. Evaluation of the efficacy of α1-PI products used in clinical studies is based on surrogate markers: the infusion of α1-PI must elevate the circulating serum level of α1−PI above an epidemiologically established 'protective threshold' and the protein must be detectable in bronchoalveolar lavage fluid (Juvelekian & Stoller, 2004; Sandhaus, 2009). However, the ability of α1-PI augmentation therapy to reduce the progression of emphysema still remains to be proven. Safety and efficacy of intravenous α1- PI augmentation are considered in section 3.3.1. For other disease conditions that may possibly benefit from α1-PI therapy see section 3.3.3.

## **3. Research and development of 1-PI for therapeutic use**

## **3.1 Plasma-derived 1-PI products**

86 Lung Diseases – Selected State of the Art Reviews

et al., 1995). Much like other serpins, α1-PI can intramolecularly convert into a more stable latent form, which is inactive, but the biological activity can be restored via denaturation


RCL

Fig. 1. Crystal structure of α1-PI (PDB 1HP7) in two projections. (**A**) Front view at the α1-PI structure in respect to -sheet A, and (**B**) Side view obtained by 90o clockwise rotation of the molecule. The images were obtained using PyMOL (the PyMOL Molecular Graphics

In addition to its inhibitory antiprotease function, α1−PI exhibits a broad spectrum of noninhibitory activities (Brantly, 2002; Janciauskiene et al., 2011; Nita et al., 2005). Because of the nine methionine residues in α1−PI molecule, its plausible role as a putative antioxidant

Due to the abundance of α1−PI in human plasma and its conservative tertiary structure with hydrophobic cavities (Elliott et al., 2000; Lee et al., 2001; Parfrey et al., 2003), α1−PI has the capacity to bind small hydrophobic molecules. This property has been explored mainly with respect to the peptides and small molecules that may prevent the aggregation of the α1−PI Z

There are approximately 60,000-100,000 severely deficient individuals in the United States which define α1-PI deficiency as a rare disease. However, according to several publications, α1-PI deficiency is widely under- and mis-diagnosed (*e.g*., de Serres, 2003; Bals et al., 2007). As reported by the World Health Organization (WHO, 1997), only 4% of the individuals with α1-PI deficiency cases are identified, and only a portion of them are receiving treatment. Currently licensed treatment of the patients with α1-PI deficiency and manifestation of pulmonary emphysema involves intravenous infusion of plasma-derived α1-PI preparations with the recommended dose of 60 mg of active α1-PI per kg of body weight administered once weekly. To maintain a threshold level of α1-PI (11µM), α1−PIdeficient patients should receive augmentation therapy for the duration of their lives, to slow the progression of emphysema. This nadir level has been determined based on α1-PI

and refolding (Lomas et al., 1995; Silverman et al., 2001).

**A** RCL **<sup>B</sup>**

System, Version 1.1r1, Schrödinger, LLC).

has been suggested (*e.g*., Levine et al., 1999, 2000).

mutant (Mahadeva et al. 2002; Mallya et al., 2007; Chang et al. 2009).

**2.2 The 1-PI deficiency and 1-PI replacement therapy** 

## **3.1.1 Currently approved 1-PI products**

Currently there are six commercial plasma-derived α1-PI products (Table 2) licensed by the US FDA for intravenous treatment of patients with hereditary α1-PI deficiency who show evidence of emphysema. Prolastin® (registered trade name of Bayer Corporation since 1987) was the first α1-PI product to be approved. Since 2005, when Bayer Corporation was acquired by Talecris Biotherapeutics (Research Triangle Park, NC, USA; www.talecris.com), the product has been manufactured by Talecris. Aralast® (initially registered trademark of Alpha Therapeutic Corporation) was approved in 2003, and has been manufactured under the direction of Baxter Healthcare Corporation since then (Baxter, Westlake Village, CA, USA www.baxter.com). Zemaira® (registered trade name of Aventis Behring since 2003), another available product, is now manufactured by CSL Behring LLC (Kankakee, IL, USA; www.cslbehring-us.com). In 2007, the US FDA approved another of Baxter's preparations of α1-PI concentrate - Aralast NP® - that has the same formulation as its predecessor, but differs from the earlier approved product by having a significantly lower content of C-terminal lysine-truncated α1-PI (approximately 2% vs. 67%). In 2009, the US FDA approved Prolastin C®, the updated version of the earlier Talecris product that had been on the market for more than two decades. Due to more sophisticated purification and pathogen reduction steps, including two dedicated viral inactivation steps instead of heat treatment, the specific activity of Prolastin C® (above 0.7 mg of functional α1-PI per mg of total protein) is twice higher than that of Prolastin®, which means that lower volumes and shorter transfusion time are needed. Most recently, in July 2010, the FDA approved GlassiaTM (formerly Respira), a product manufactured by Kamada (Weizmann Science Park, Ness Ziona, Israel; www.kamada.com) and commercially launched by Baxter in the United States and some other countries. GlassiaTM is another highly purified α1-PI (with specific activity above 0.7 mg of active α1-PI per mg of total protein) and the only α1-PI product that is available in a ready-to-use liquid form with a shelf-life stability of two years under refrigerated conditions.

α1−PI products are manufactured as part of a complex plasma fractionation scheme which was originally developed for large-scale production of albumin, but now also yields many other plasma therapeutics . Since products are made from pooled human plasma, they may

The US FDA product approval information is available at

http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedPro ductsBLAs/FractionatedPlasmaProducts/default.htm


a Based on recent publications including (Stockley, 2010; Tonelli & Brantly, 2010)

b Reconstitution using Sterile Water for Injection is required

c Aralast®, previously known as Respitin, contains approximately 67% of α1-PI with the truncated C-terminal lysine (Lys394)

d Aralast NP® contains approximately 2% of α1-PI with truncation of C-terminal lysine residue

Table 2. The plasma-derived α1-PI therapeutic products approved by the US FDA for chronic augmentation and maintenance therapy in adults with congenital α1-PI deficiency and clinically evident emphysemaa

carry the risk of transmitting human infectious agents, *e.g*., some viruses, and theoretically, the Creutzfeldt-Jakob disease (CJD) agent or disease variant agents, as well as emerging or unknown infectious agents. To reduce the potential risk of transmitting infectious agents, the α1-PI preparations are manufactured using a number of viral inactivation and removal steps. Currently approved α1−PI products differ in the procedures used for pathogen reduction. For instance, the heat treatment procedure used in manufacturing of Prolastin was one of the reasons for higher content of inactive and aggregated α1−PI in the product. The manufacturing procedures of the later products (Table 2) include two dedicated steps that are specifically designed for inactivation and removal of viruses (Hotta et al., 2010). Thus, solvent/detergent treatment and nanofiltration are used as the dedicated pathogen reduction steps in the manufacturing of recently approved Prolastin C®, GlassiaTM and both Aralast products. Overall, the manufacturing history of plasma-derived α1-PI therapeutic products reflects a trend of continous improvement of product quality.

## **3.1.2 Heterogeneity of α1-PI products**

Heterogeneity of α1-PI therapeutic preparations is a complex phenomenon. First of all, heterogeneous nature of plasma α1-PI is an intrinsic property of the native glycoproten (see 2.1). Second, the presence of variously processed α1-PI forms including latent, cleaved, complexed or aggregated α1-PI species, is barely avoidable. However, it must be kept minimal as the inactive protein species have a direct influence on the product's specific activity. Third, α1-PI products purified from pooled human plasma contain certain impurities of other plasma proteins, including albumin, haptoglobin, α1-antichymotrypsin, α1-lipoprotein, antithrombin III, C1-esterase inhibitor, etc. The human origin of these

Biotherapeutics 12/2/1987 Lyophilized

Healthcare Co. 3/21/2003 Lyophilized

Healthcare Co. 5/4/2007 Lyophilized

Biotherapeutics 10/16/20099 Lyophilized

c Aralast®, previously known as Respitin, contains approximately 67% of α1-PI with the truncated

carry the risk of transmitting human infectious agents, *e.g*., some viruses, and theoretically, the Creutzfeldt-Jakob disease (CJD) agent or disease variant agents, as well as emerging or unknown infectious agents. To reduce the potential risk of transmitting infectious agents, the α1-PI preparations are manufactured using a number of viral inactivation and removal steps. Currently approved α1−PI products differ in the procedures used for pathogen reduction. For instance, the heat treatment procedure used in manufacturing of Prolastin was one of the reasons for higher content of inactive and aggregated α1−PI in the product. The manufacturing procedures of the later products (Table 2) include two dedicated steps that are specifically designed for inactivation and removal of viruses (Hotta et al., 2010). Thus, solvent/detergent treatment and nanofiltration are used as the dedicated pathogen reduction steps in the manufacturing of recently approved Prolastin C®, GlassiaTM and both Aralast products. Overall, the manufacturing history of plasma-derived α1-PI therapeutic

Heterogeneity of α1-PI therapeutic preparations is a complex phenomenon. First of all, heterogeneous nature of plasma α1-PI is an intrinsic property of the native glycoproten (see 2.1). Second, the presence of variously processed α1-PI forms including latent, cleaved, complexed or aggregated α1-PI species, is barely avoidable. However, it must be kept minimal as the inactive protein species have a direct influence on the product's specific activity. Third, α1-PI products purified from pooled human plasma contain certain impurities of other plasma proteins, including albumin, haptoglobin, α1-antichymotrypsin, α1-lipoprotein, antithrombin III, C1-esterase inhibitor, etc. The human origin of these

d Aralast NP® contains approximately 2% of α1-PI with truncation of C-terminal lysine residue Table 2. The plasma-derived α1-PI therapeutic products approved by the US FDA for chronic augmentation and maintenance therapy in adults with congenital α1-PI deficiency

Zemaira® CSL Behring 7/8/2003 Lyophilized

GlassiaTM Kamada 7/1/2010 Ready-to-use

b Reconstitution using Sterile Water for Injection is required

a Based on recent publications including (Stockley, 2010; Tonelli & Brantly, 2010)

products reflects a trend of continous improvement of product quality.

licensure Product form Major steps of viral

powderb

powder

powder

powder

powder

liquid

inactivation/removal

Depth Filtration Heat

Solvent/Detergent & Nanofiltration

Solvent/Detergent & Nanofiltration

Solvent/Detergent & Nanofiltration

Solvent/Detergent & Nanofiltration

Heat Treatment & Ultrafiltration

Treatment

Drug

product Manufacturer Date of

Prolastin® Talecris

Aralast®c Baxter

Aralast NP®d Baxter

Prolastin C® Talecris

C-terminal lysine (Lys394)

and clinically evident emphysemaa

**3.1.2 Heterogeneity of α1-PI products** 

impurities ensures their tolerability, however, the level of these plasma proteins in α1-PI concentrate may significantly increase the non-therapeutic protein load in the α1-PI preparation intended for transfusion. In addition to all that, multistep manufacturing procedures are known to induce various protein alterations, such as aggregation and chemical modifications (*e.g.*, deamidation, cysteinylation, and C-terminal truncation). Some modifications can be observed by IEF and other techniques (Cowden et al., 2005; Kolarich et al., 2006a, 2006b) and reflected in the product specifications. Currently there are no data that would demonstrate whether these alterations affect the *in vivo* activity, safety, efficacy or immunogenicity of α1-PI therapeutic preparations. In general, commercial plasma-derived α1-PI products differ in terms of their purity, specific activity, modifications, and excipients (Lomas et al., 1997; Cowden et al., 2005; Stockley, 2010; Tonelli & Brantly, 2010).

#### **3.2 Research and development of the recombinant versions of human 1-PI 3.2.1 Advances in the development of recombinant α1-PI**

The plasma supply *per se* is a limited source and appears to be insufficient to meet anticipated clinical demand. Moreover, despite effective viral inactivation/removal steps in the manufacturing of plasma proteins (Cai et al., 2005; Hotta et al., 2010), the risk of contamination with new and unknown pathogens may still exist. Therefore, recombinant technology has been widely explored as an alternative approach for the production of human α1-PI since the pioneering works of the early 1980s (Bollen et al., 1983; Cabezon et al., 1984; Rosenberg et al., 1984). As evident from numerous reports, both from academic research and industry, the human gene for α1-PI has been expressed in virtually all available hosts (*E. coli*, various yeasts, fungi, insect cells, CHO cells, human neuronal cells, and produced in transgenic plants and animals). For more details on research and development of recombinant α1-PI (r-α1-PI) in different systems and advances and limitations of the recombinant approach for production of stable and biologically active α1-PI, see our comprehensive 2006 review (Karnaukhova et al., 2006). More recently, the human gene for α1-PI has been expressed in filamentous fungi (Chill et al., 2009; Karnaukhova et al., 2007), transgenic tomato plants (Agarwal et al., 2009), tobacco cell cultures (Huang et al., 2009; Nadai et al., 2009), and human neuronal cell lines (Blanchard et al., 2011). Nevertheless, no rα1-PI is available as a licensed therapeutic treatment. In general, the essential criteria for the development of therapeutics for human use are safety, optimal clinical efficacy, and maximum cost-effectiveness. Among many efforts to develop r-α1-PI of therapeutic quality (see Karnaukhova et al., 2006), there appear to be only two examples of the r-α1-PIs for which development went far enough to get to clinical trials. The first was r-α1-PI produced in the yeasts *Saccromyces cerevisiae* and manufactured by Arriva Pharmaceuticals Inc. (Arriva) for several indications. A nebulized formulation of this non-glycosylated r-α1-PI preparation has been intended for the treatment of respiratory disorders including emphysema and COPD (phase II clinical trials), and asthma (pre-clinical studies) (Brown, 2006a). Although animal studies have been considered to be successful (Pemberton et al., 2006), human trials have not been recommended (see review by Stokley, 2010). A topical gel formulation of r-α1-PI has been intended for the treatment of dermatitis and other severe dermatological disorders in phase II clinical trials (see Brown, 2006b).

The second example of the advanced development of recombinant human α1-PI is large scale production performed in transgenic dairy animals (t-α1-PI): sheep [by PPL Therapeutics (UK) in partnership with Bayer Biologicals (USA), (Dalrymple & Garner, 1998; Wright et al., 1991)], and goats [by Genzyme Transgenics Corporation (USA), (Ziomek, 1998)]. The transgenic α1-PI recovered from sheep milk was purified to 99.9% purity. Even so, sheep native α1-PI and sheep α1-antichymotrypsin were major impurities, at 6.7-18.7 mg/L and 60.3-75.8 parts per million, respectively. Two sequential clinical studies were performed to evaluate the safety and immunogenicity of aerosolized transgenic human α1- PI. None of the subjects had an antibody response to human t-α1-PI (Tebbutt, 2000; Spencer et al., 2005); however, antibody responses were observed to sheep α1-PI and to sheep α1 antichymotrypsin (Spencer et al., 2005). Four patients withdrew from the study due to the development of dyspnea and a decline in lung function, and the later product development was terminated.

#### **3.2.2 Pitfalls in the development of r-α1-PI for therapeutic use**

The general regulatory requirements for biologicals intended for therapeutic use, including r-α1-PI, are purity, safety, and efficacy. In order to be effective, therapeutic proteins have to be stable *in vivo* and *in vitro* (Karnaukhova et al., 2006). Reviewing the work performed over the last two decades to produce stable and biologically active r-α1-PI of therapeutic quality, one can see basically two major factors that were impeding the progress: (1) impurities that could induce antibody responses and cause adverse reactions in patients, and (2) lower stability than that of plasma counterpart, mainly caused by the lack of glycosylation or nonhuman type of glycosylation (the latter may also induce immune responses). Although presently the first reason can be technically better solved, removal of trace amounts of nonhuman native proteins derived from the host, *e.g.,* sheep α1-PI, from the human r-α1-PI to exclude further adverse reactions, requires a much higher level of purification than was possible at the time of that development. As for the second reason, indeed, glycosylation is considered to be a cause of rapid clearance of r-α1-PI from the circulation (Casolaro et al., 1987; Cantin et al., 2002a). Aberrant glycosylation (or lack of glycans) does not neccessarily affect biological activity of the recombinant protein, but it is important for its stability. According to recently published data, glycosylation of α1-PI does not interfere with the serpin native state flexibility (or instability) essential for its efficient function, though it may confer resistance to degradation by proteases and thus extend its half-life in the circulation (Sarkar & Wintrode, 2011). Extensive work performed over decades for the development of viable r-α1-PI of therapeutic quality and lessons learned from these experiences truly paved the way for other protein therapeutics. It is worthwhile to mention two serpins produced in transgenic animals that were recently approved. In 2009, the US FDA approved recombinant antithrombin (ATryn®) produced in the milk of transgenic goats (Fyfe & Tait, 2009). In 2010, another serpin, recombinant human C1-esterase inhibitor (Ruconest®) produced in the milk of transgenic rabbits was granted European marketing authorization (Varga & Farkas, 2011). Both pharmaceutical proteins show a faster clearance, yet it may not be an issue depending on the intended use. For instance, Ruconest® was approved for the treatment of acute attacks of hereditary angioedema, and therefore there is no need to maintain its higher level in blood longer than its action is required. Given a shorter *in vivo* half-life of recombinant α1- PI, it has been considered for other administration routes and applications, such as inhalation for the treatment of emphysematous condition, and topical application for various skin diseases. However, a convincing proof of the recombinant product efficacy and safety in appropriate clinical trials is as problematic as it is for plasma-derived α1-PI; large clinical trials in the cases of rare diseases are difficult to perform because of small geographically dispersed patient populations. In addition, a limited population means a limited market, which is less attractive for large investments. No doubt, these reasons markedly slow down the development of r-α1-PI.

## **3.3 1-PI –based therapies**

90 Lung Diseases – Selected State of the Art Reviews

Wright et al., 1991)], and goats [by Genzyme Transgenics Corporation (USA), (Ziomek, 1998)]. The transgenic α1-PI recovered from sheep milk was purified to 99.9% purity. Even so, sheep native α1-PI and sheep α1-antichymotrypsin were major impurities, at 6.7-18.7 mg/L and 60.3-75.8 parts per million, respectively. Two sequential clinical studies were performed to evaluate the safety and immunogenicity of aerosolized transgenic human α1- PI. None of the subjects had an antibody response to human t-α1-PI (Tebbutt, 2000; Spencer et al., 2005); however, antibody responses were observed to sheep α1-PI and to sheep α1 antichymotrypsin (Spencer et al., 2005). Four patients withdrew from the study due to the development of dyspnea and a decline in lung function, and the later product development

The general regulatory requirements for biologicals intended for therapeutic use, including r-α1-PI, are purity, safety, and efficacy. In order to be effective, therapeutic proteins have to be stable *in vivo* and *in vitro* (Karnaukhova et al., 2006). Reviewing the work performed over the last two decades to produce stable and biologically active r-α1-PI of therapeutic quality, one can see basically two major factors that were impeding the progress: (1) impurities that could induce antibody responses and cause adverse reactions in patients, and (2) lower stability than that of plasma counterpart, mainly caused by the lack of glycosylation or nonhuman type of glycosylation (the latter may also induce immune responses). Although presently the first reason can be technically better solved, removal of trace amounts of nonhuman native proteins derived from the host, *e.g.,* sheep α1-PI, from the human r-α1-PI to exclude further adverse reactions, requires a much higher level of purification than was possible at the time of that development. As for the second reason, indeed, glycosylation is considered to be a cause of rapid clearance of r-α1-PI from the circulation (Casolaro et al., 1987; Cantin et al., 2002a). Aberrant glycosylation (or lack of glycans) does not neccessarily affect biological activity of the recombinant protein, but it is important for its stability. According to recently published data, glycosylation of α1-PI does not interfere with the serpin native state flexibility (or instability) essential for its efficient function, though it may confer resistance to degradation by proteases and thus extend its half-life in the circulation (Sarkar & Wintrode, 2011). Extensive work performed over decades for the development of viable r-α1-PI of therapeutic quality and lessons learned from these experiences truly paved the way for other protein therapeutics. It is worthwhile to mention two serpins produced in transgenic animals that were recently approved. In 2009, the US FDA approved recombinant antithrombin (ATryn®) produced in the milk of transgenic goats (Fyfe & Tait, 2009). In 2010, another serpin, recombinant human C1-esterase inhibitor (Ruconest®) produced in the milk of transgenic rabbits was granted European marketing authorization (Varga & Farkas, 2011). Both pharmaceutical proteins show a faster clearance, yet it may not be an issue depending on the intended use. For instance, Ruconest® was approved for the treatment of acute attacks of hereditary angioedema, and therefore there is no need to maintain its higher level in blood longer than its action is required. Given a shorter *in vivo* half-life of recombinant α1- PI, it has been considered for other administration routes and applications, such as inhalation for the treatment of emphysematous condition, and topical application for various skin diseases. However, a convincing proof of the recombinant product efficacy and safety in appropriate clinical trials is as problematic as it is for plasma-derived α1-PI; large clinical trials in the cases of rare diseases are difficult to perform because of small geographically dispersed patient populations. In addition, a limited population means a

**3.2.2 Pitfalls in the development of r-α1-PI for therapeutic use** 

was terminated.

#### **3.3.1 Safety and efficacy of intravenous 1-PI augmentation**

The intravenous augmentation of α1-PI was shown to be safe and well tolerated over a long history of the replacement therapy. However, its impact on disease progression and mortality still remains to be convincingly proven. α1-PI augmentation is assumed to slow down the rate of emphysema development and progression and, thus, to improve the life quality and duration of α1-PI deficient patients, yet the essential proof of efficacy is missing. According to Hubbard & Crystal (1990), only approximately 2-3% of infused α1−PI actually reaches the lungs; and the effictiveness of α1-PI replacement therapy has been evaluated mainly on the bases of biochemical (not clinical) criteria (Tonelli & Brantly, 2010). For recently approved α1-PI products, their pharmacokinetic equivalence and comparable safety profile to Prolastin were demonstrated (*e.g*., Stocks et al., 2010). α1-PI therapy is a life-long and very expensive treatment that may cost up to \$150,000 (Silverman, 2009) in the United States. Whether this therapy decreases mortality also remains unknown, as there are no reliable data on mortality, as well as morbidity and survival (Gøtzsche & Johansen, 2010a). Some observational studies support the idea that augmentation therapy may help to slow the decline in lung function (Seersholm et al., 1997; Wencker et al., 2001; Kueppers, 2011). But there are also more critical evaluations including the opinion that α1-PI augmentation therapy cannot be recommended due to lack of evidence of clinical benefit and the cost of treatment (Gøtzsche & Johansen, 2010a, 2010b). It is currently widely admitted that the efficacy of α1-PI augmentation therapy has never been persuasively demonstrated and must be proven in a proper clinical trial. Due to the widespread and small clusters of patients all over the country, conducting a prospective, randomized, placebo-controlled clinical trial is challenging. In addition, the development of emphysema proceeds slowly, creating the additional difficulties of monitoring lung function decline and mortality data (Hutchinson & Hughes, 1997; Schluchter et al., 2000).

#### **3.3.2 Alternative routes of administration of 1-PI products**

Due to the inconvenience of life-time intravenous augmentation therapy and low levels of α1−PI reaching lungs, the inhalation of aerosolized α1-PI has been suggested as a less invasive and more efficient way to deliver large amounts of α1-PI directly to the lungs where it is most needed (Hubbard et al., 1989; McElvaney et al., 1991; Cockett, 1999). Although strategies for aerosol therapy of α1-PI deficiency has been proposed two decades ago (Hubbard et al., 1989; Hubbard & Crystal, 1990), there is still no α1-PI aerosolized treatment approved. Several studies examined efficiency of the α1-PI inhalation therapy in animals and in humans (Kropp et al., 2001; Siekmeier, 2010). It was demonstrated (Kropp et al., 2001) that significantly more α1-PI was deposited in the lungs through the inhalational route than via intravenous infusion (14.6% vs. 2%). Although the inhalation route seems attractive, nevertheless, enabling the inhaled material to reach the lung interstitium, the most important to the emphysematous process region, is still problematic. With regards to recombinant versions of α1-PI, it is generally assumed that products directly delivered to the lungs may not require the same degree of stability as α1-PI given intravenously. However, as mentioned above, human studies using r-α1-PI from transgenic sheep were associated with adverse reactions due to impurities derived from the host (Spenser et al., 2005). Thus, higher levels of purification and more clinical studies are required.

#### **3.3.3 Other 1-PI applications**

Currently, α1-PI therapeutic preparations are licensed exclusively for one indication, *i.e*., chronic augmentation and maintenance therapy in individuals with emphysema due to congenital α1-PI deficiency. Previously unrecognized inherited disorder, α1-PI deficiency was first described in 1963 (Laurell & Eriksson, 1963) based on the serum electrophoretic analysis that revealed five individuals deficient of α1–fraction; three of those patients had developed emphysematous conditions. Six years later, in 1969, cirrosis associated with α1-PI deficiency was described (Sharp et al., 1969). These findings initiated a concept of linkage between α1-PI deficiency and pulmonary and liver diseases. As evident from the available literature, due to the multiple biological activities of α1-PI, it has been associated with other lung diseases (first of all, cystic fibrosis) and many non-pulmonary diseases (Table 3). Some of these conditions may possibly benefit from α1-PI augmentation therapy (see recent reviewes by Blanco et al., 2011 and Janciauskiene et al., 2011).

According to Blanco et al. (2011), α1-PI therapy has proven remarkable efficacy in small cohorts of α1-PI-deficient patients who also suffer from fibromyalgia, systemic vasculitis, relapsing panniculitis and bronchial asthma. Although the putative benefits of α1-PI therapy for treatment of additional rare diseases (some are listed in Table 3) requires much more clinical data than are currently available to support clinical efficacy and safety of α1-PI treatment, in general it indicates a clear potential for additional α1-PI supply to satisfy the anticipated clinical demand in near future. Because of controversy related to the additional clinical implications of 1-PI deficiency, more clinical data are needed to verify whether the reported links between 1-PI deficiency and other rare diseases are real or accidental.

As a potent anti-inflammatory agent, 1-PI has been investigated in clinical studies for treatment of cystic fibrosis (Jones & Helm, 2009). Whereas patients with emphysematous conditions suffer from the hereditary 1-PI deficiency and, thus, insufficient levels of the protease inhibitor in the lungs due to impaired 1-PI synthesis in hepatocytes, patients with cystic fibrosis may have normal synthesis of 1-PI and suffer from severe pulmonary inflammation due to high excess of NE in the lungs, leading to a progressive loss of lung function (Allen, 1996; Siekmeier, 2010). Therefore, it has been proposed that both groups of patients may benefit from 1-PI augmentation therapy to prevent the deleterious effect of free protease (Allen, 1996; Birrer, 1995; Birrer et al, 1996) However, intravenous administration of 1-PI did not result in a suppression of the respiratory neutrophil elastase burden (McElvaney et al, 1991). Several studies have been conducted using inhalation of an aerosolized 1-PI in cystic fibrosis and 1-PI deficiency (Hubbard et al., 1989; Griese et al, 2001, 2007; Martin et al, 2006; Brand et al, 2009).

Whereas several studies that investigated the efficacy of treatment with an aerosolized 1-PI both in patients with cystic fibrosis and in those with 1-PI deficiency came to positive conclusions regarding deposition of inhaled 1-PI in the lungs and its anti-elastase activity (see review by Siekmeier, 2010), the conclusion from other studies was that treatment with 1-PI did not demonstrate any clinical improvements (Martin, 2006). If further clinical studies support the safety and efficacy of an aerosolized1-PI, and it is approved for treatment of cystic fibrosis, the demand for therapeutic 1-PI preparations could be significantly increased.

with adverse reactions due to impurities derived from the host (Spenser et al., 2005). Thus,

Currently, α1-PI therapeutic preparations are licensed exclusively for one indication, *i.e*., chronic augmentation and maintenance therapy in individuals with emphysema due to congenital α1-PI deficiency. Previously unrecognized inherited disorder, α1-PI deficiency was first described in 1963 (Laurell & Eriksson, 1963) based on the serum electrophoretic analysis that revealed five individuals deficient of α1–fraction; three of those patients had developed emphysematous conditions. Six years later, in 1969, cirrosis associated with α1-PI deficiency was described (Sharp et al., 1969). These findings initiated a concept of linkage between α1-PI deficiency and pulmonary and liver diseases. As evident from the available literature, due to the multiple biological activities of α1-PI, it has been associated with other lung diseases (first of all, cystic fibrosis) and many non-pulmonary diseases (Table 3). Some of these conditions may possibly benefit from α1-PI augmentation therapy (see recent

According to Blanco et al. (2011), α1-PI therapy has proven remarkable efficacy in small cohorts of α1-PI-deficient patients who also suffer from fibromyalgia, systemic vasculitis, relapsing panniculitis and bronchial asthma. Although the putative benefits of α1-PI therapy for treatment of additional rare diseases (some are listed in Table 3) requires much more clinical data than are currently available to support clinical efficacy and safety of α1-PI treatment, in general it indicates a clear potential for additional α1-PI supply to satisfy the anticipated clinical demand in near future. Because of controversy related to the additional clinical implications of 1-PI deficiency, more clinical data are needed to verify whether the

reported links between 1-PI deficiency and other rare diseases are real or accidental.

As a potent anti-inflammatory agent, 1-PI has been investigated in clinical studies for treatment of cystic fibrosis (Jones & Helm, 2009). Whereas patients with emphysematous conditions suffer from the hereditary 1-PI deficiency and, thus, insufficient levels of the protease inhibitor in the lungs due to impaired 1-PI synthesis in hepatocytes, patients with cystic fibrosis may have normal synthesis of 1-PI and suffer from severe pulmonary inflammation due to high excess of NE in the lungs, leading to a progressive loss of lung function (Allen, 1996; Siekmeier, 2010). Therefore, it has been proposed that both groups of patients may benefit from 1-PI augmentation therapy to prevent the deleterious effect of free protease (Allen, 1996; Birrer, 1995; Birrer et al, 1996) However, intravenous administration of 1-PI did not result in a suppression of the respiratory neutrophil elastase burden (McElvaney et al, 1991). Several studies have been conducted using inhalation of an aerosolized 1-PI in cystic fibrosis and 1-PI deficiency (Hubbard et al., 1989; Griese et al,

Whereas several studies that investigated the efficacy of treatment with an aerosolized 1-PI both in patients with cystic fibrosis and in those with 1-PI deficiency came to positive conclusions regarding deposition of inhaled 1-PI in the lungs and its anti-elastase activity (see review by Siekmeier, 2010), the conclusion from other studies was that treatment with 1-PI did not demonstrate any clinical improvements (Martin, 2006). If further clinical studies support the safety and efficacy of an aerosolized1-PI, and it is approved for treatment of cystic fibrosis, the demand for therapeutic 1-PI preparations could be

higher levels of purification and more clinical studies are required.

reviewes by Blanco et al., 2011 and Janciauskiene et al., 2011).

2001, 2007; Martin et al, 2006; Brand et al, 2009).

significantly increased.

**3.3.3 Other 1-PI applications** 


Table 3. Conditions other than emphysema and liver disease possibly associated with α1-PI

### **3.3.4 Research toward the enhancement of 1-PI-therapies**

During last decade various approaches have been considered for the enhancement of 1-PIbased therapies. For instance, to prolong a short half-life of r-1-PI in the circulation, Cantin and co-workers hypothesized that conjugation of r-1-PI with polyethylene glycol (PEG) at Cys232 could extend the *in vivo* half-life of recombinant protein in blood and lung (Cantin et al., 2002b). According to their data, the site-specific conjugation with either 20 or 40 kD PEG at Cys232 of nonglycosylated r-1-PI (human) results in an active inhibitor with extended *in vivo* stability.Moreover, 72 h later after airway instillation, the PEG-r-1-PI seemedto be significantly better than glycosylated 1-PI at protectingthe lung against elastase–induced lung hemorrhage. As an example of the *in vitro* biochemical evaluation of the concept, α1−PI has been considered for its affinity to various small ligands and drugs for different reasons. Mainly this approach has been explored with respect to the peptides and small molecules in order to prevent the aggregation of Z mutant (*e.g*., Mallya et al., 2007; Chang et al. 2009). In the meantime, the protein's potential for binding small ligands of pharmaceutical interest has been proposed as a promising approach that is directed at, and may ultimately enhance, currently existing α1−PI therapies (Karnaukhova et al., 2010). For instance, α1−PI's affinity to retinoic acid, which is known for a wide range of physiological activities including alveolar repair and regrowth (Roche clinical studies, see Stockley, 2010; Massaro & Massaro, 1996, 1997) and tissue rejuvenation in various dermatologic diseases, has been convincingly demonstrated in biochemical experiments *in vitro* (Karnaukhova et al., 2010). As α1−PI augmentation therapy cannot cure, but may only slow down, the progression of emphysema, its complexation with retinoic acid could be more efficient for treatment than α1−PI alone. It is noteworthy that the interactions of α1−PI with several other physiologically active ligands (including porphyrins) may reveal additional properties of this multifunctional serpin.

## **4. Conclusions**

Since 1-PI deficiency was first described by Carl-Bertil Laurell and Sten Eriksson (Laurell & Eriksson, 1963) as a condition that could lead to the development of severe obstructive pulmonary disease, our knowledge about 1-PI structure-function relationships and clinical manifestations of 1-PI deficiency has increased tremendously. Moreover, multi-disciplinary research efforts prompted the development of 1-PI-based augmentation therapy to maintain the inhibitor level above the protective threshold. Since 1987, several 1-PI products derived from pooled human plasma have been approved and are currently available to slow down the progression of emphysematous conditions in 1-PI-deficient patients. In addition, due to its multiple physiological activities, 1-PI has been identified for its putative involvement in several other rare diseases, the treatment of which may possibly benefit from 1-PI-based therapies. As an alternative to intravenous administration that may improve the efficacy of1-PI treatment, the inhalation of aerosolized 1-PI preparations has been in clinical trials. Recombinant versions of human 1-PI have been produced in all available hosts and in several transgenic animals. These efforts made a remarkable impact on the research realm of recombinant protein therapeutics, but did not yet bring any viable version of recombinant 1-PI to the treatment. In regards to therapeutic preparations and their use, there are several questions to be addressed when looking to the future. Keeping in mind the long history of replacement therapy using currently approved plasma-derived 1- PI products, it is essential that the efficacy of 1-PI replacement therapy be clearly demonstrated in prospective, randomized, placebo-controlled trials. Will the efficacy of inhalation therapy using aerosolized 1-PI preparations be proven to be superior to that of the intravenous route? Will the recombinant/transgenic versions of human 1-PI be optimized to meet the requirements for protein therapeutics? Will other rare diseases currently implicated in association with 1-PI and 1-PI deficiency be clearly proven to benefit from 1-PI treatment? From the standpoint of product quality, safety and efficacy, the current state of research and development of 1-PI for therapeutic use demonstrates a symbiosis of the recent achievements and controversies, hopefully typical of our progress.

#### **5. References**


pulmonary disease, our knowledge about 1-PI structure-function relationships and clinical manifestations of 1-PI deficiency has increased tremendously. Moreover, multi-disciplinary research efforts prompted the development of 1-PI-based augmentation therapy to maintain the inhibitor level above the protective threshold. Since 1987, several 1-PI products derived from pooled human plasma have been approved and are currently available to slow down the progression of emphysematous conditions in 1-PI-deficient patients. In addition, due to its multiple physiological activities, 1-PI has been identified for its putative involvement in several other rare diseases, the treatment of which may possibly benefit from 1-PI-based therapies. As an alternative to intravenous administration that may improve the efficacy of1-PI treatment, the inhalation of aerosolized 1-PI preparations has been in clinical trials. Recombinant versions of human 1-PI have been produced in all available hosts and in several transgenic animals. These efforts made a remarkable impact on the research realm of recombinant protein therapeutics, but did not yet bring any viable version of recombinant 1-PI to the treatment. In regards to therapeutic preparations and their use, there are several questions to be addressed when looking to the future. Keeping in mind the long history of replacement therapy using currently approved plasma-derived 1- PI products, it is essential that the efficacy of 1-PI replacement therapy be clearly demonstrated in prospective, randomized, placebo-controlled trials. Will the efficacy of inhalation therapy using aerosolized 1-PI preparations be proven to be superior to that of the intravenous route? Will the recombinant/transgenic versions of human 1-PI be optimized to meet the requirements for protein therapeutics? Will other rare diseases currently implicated in association with 1-PI and 1-PI deficiency be clearly proven to benefit from 1-PI treatment? From the standpoint of product quality, safety and efficacy, the current state of research and development of 1-PI for therapeutic use demonstrates a symbiosis of the recent achievements and controversies, hopefully typical of our progress.

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## **Mechanisms Promoting Chronic Lung Diseases: Will Targeting Stromal Cells Cure COPD and IPF?**

Lynne A. Murray1 and Cory M. Hogaboam2

*1Respiratory, Inflammation and Autoimmunity, MedImmune Ltd, Cambridge, 2Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 1UK 2USA* 

#### **1. Introduction**

104 Lung Diseases – Selected State of the Art Reviews

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Colman, A. (1991). High level of expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. *Bio/Technology,* Vo.9, No.9, (September 1991), pp. Tissue remodeling is a common pathology in many diseases. The reparative processes of wound healing result in an increase in extracellular matrix (ECM) generation, serving to restore barrier protection and normal tissue architecture. However in the lung, increased formation of ECM results in decreased lung compliance and impaired gas exchange. The chronic lung diseases COPD (chronic obstructive pulmonary disease) and IPF (idiopathic pulmonary fibrosis) both exhibit increased extracellular matrix (ECM) deposition within the lung due to increased stromal cell number and activation. The chronic nature of the disease is hypothesized to correlate with the extent of scarring and remodeling, with greater evidence being available for COPD rather than IPF. The potential underlying causes of both diseases are numerous, including direct insults to the lung such as cigarette smoke or exhaust particles; as well as underlying autoimmune conditions such as scleroderma or collagen vascular disease. Moreover, a correlation between COPD and smoking is well established; however, any causes for IPF, apart from a familial link, are currently not understood. To date there are no approved anti-fibrotic therapies that target the underlying remodeling in either disease. In order to treat this pathology, understanding the mechanisms promoting continual ECM deposition may elucidate novel therapeutic approaches that could provide clinical benefit to patients suffering from these debilitating diseases. This Chapter will focus on the cellular mechanisms and interactions within the fibrotic lung including resident fibroblast-epithelial cells as well as the cross-talk between fibroblasts and recruited bone marrow derived cells such as fibrocytes and monocytes. Also, the soluble mediators that have been associated with disease and how these can directly and indirectly modulate stromal cell activation will be discussed.

#### **2. COPD and IPF disease pathogenesis**

One of the common features between COPD and IPF is the heterogeneous pathology observed in the lung at the macroscopic level in both disease settings. Both diseases exhibit patchy areas of pathology, consisting of ECM proteins or inflammatory infiltrates, with fibrotic regions being juxtaposed to regions of normal alveolar tissue or areas with interstitial leukocyte accumulation. Grossly, COPD contains regions dense in bronchiolar inflammation and consolidated lung tissue, as well as emphysematous areas, due to alveolar destruction 1,2. In IPF, salient hallmarks of disease included profound collagen deposition, as well as regions of honeycombed lung due to a collapse of alveolar walls 3. COPD has been clinically separated into distinct GOLD (Global initiative for chronic Obstructive Lung Disease) Stages based primarily on a key parameter of lung function, Forced Expiratory Volume in one second (FEV1). Here, patients with more significant impairment in FEV1 have a higher GOLD Stage status. Supporting these changes in lung function analysis of the underlying pathologies of patients within each GOLD Stage has also highlighted that, as the disease progresses, there is an increase in airway wall thickness 4. Even though the GOLD staging system is universally recognized, there are efforts underway to also separate COPD patients in to fast decliners and slow decliners based on lung function parameters. Extensive work is currently underway to try and stage IPF in an equivalent manner. In this disease, retrospective analysis of patient survival has indicated that there is both a rapidly progressing phenotype and a more slowly progressing phenotype of IPF patients. In the 'rapid' IPF progressors, approximately 50% of patients will die within 6-8 months, whereas this is greatly extended in slow progressors 5,6.

Chronic remodeling is a common feature between IPF and COPD. In order to understand the mechanisms underlying the maintenance, progression and staging of disease, this Book Chapter will focus on the similarities and differences between stromal cells in these patients. We will focus on the phenotypic differences in fibroblasts and myofibroblasts as well as how these cell types interact within the lung (Fig. 1). We will also describe some of the key families of mediators that are found to be elevated in disease and how their mechanism may be directly promoting chronic lung disease. Lastly, we will discuss the potential therapeutic approaches to targeting stromal cells clinically.

#### **3. Profibrotic role of fibroblasts**

Fibroblasts play a myriad of important roles in normal tissue function. In the lung they coordinate organogenesis and budding of the lung from the foregut through intimate bidirectional communication with adjacent epithelial cells. Myofibroblasts are "smooth muscle-like cells" that are morphologically similar to fibroblasts, but also express alphasmooth muscle actin (-SMA) 7. Fibroblasts and myofibroblasts are also key cells in the production and homeostatic maintenance of the ECM of the tissue or organ in which they reside. They are metabolically active cells, capable of synthesizing and secreting ECM components such as collagens and proteoglycans. Fibroblasts continually synthesize ECM proteins although the amount they secrete is tightly regulated, with up to 90% of all procollagen molecules being degraded intracellularly prior to secretion, depending on tissue and age. Further, fibroblasts generate both matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases, thus controlling homeostatic tissue architecture.

Myofibroblasts were first described by Gabbiani and colleagues as cells central to wound healing 8. The actin filaments result in myofibroblasts having contractile properties, which, at sites of wound healing serve to close the wound. However, the presence of contractile myofibroblasts in the interstitium of the lung may cause a retraction of parenchymal tissue resulting in alveolar collapse giving the characteristic honeycombing as observed in the lungs of IPF patients, or add to the increase in alveolar size, which is characteristic of COPD 9.

fibrotic regions being juxtaposed to regions of normal alveolar tissue or areas with interstitial leukocyte accumulation. Grossly, COPD contains regions dense in bronchiolar inflammation and consolidated lung tissue, as well as emphysematous areas, due to alveolar destruction 1,2. In IPF, salient hallmarks of disease included profound collagen deposition, as well as regions of honeycombed lung due to a collapse of alveolar walls 3. COPD has been clinically separated into distinct GOLD (Global initiative for chronic Obstructive Lung Disease) Stages based primarily on a key parameter of lung function, Forced Expiratory Volume in one second (FEV1). Here, patients with more significant impairment in FEV1 have a higher GOLD Stage status. Supporting these changes in lung function analysis of the underlying pathologies of patients within each GOLD Stage has also highlighted that, as the disease progresses, there is an increase in airway wall thickness 4. Even though the GOLD staging system is universally recognized, there are efforts underway to also separate COPD patients in to fast decliners and slow decliners based on lung function parameters. Extensive work is currently underway to try and stage IPF in an equivalent manner. In this disease, retrospective analysis of patient survival has indicated that there is both a rapidly progressing phenotype and a more slowly progressing phenotype of IPF patients. In the 'rapid' IPF progressors, approximately 50% of patients will die within 6-8 months, whereas

Chronic remodeling is a common feature between IPF and COPD. In order to understand the mechanisms underlying the maintenance, progression and staging of disease, this Book Chapter will focus on the similarities and differences between stromal cells in these patients. We will focus on the phenotypic differences in fibroblasts and myofibroblasts as well as how these cell types interact within the lung (Fig. 1). We will also describe some of the key families of mediators that are found to be elevated in disease and how their mechanism may be directly promoting chronic lung disease. Lastly, we will discuss the potential therapeutic

Fibroblasts play a myriad of important roles in normal tissue function. In the lung they coordinate organogenesis and budding of the lung from the foregut through intimate bidirectional communication with adjacent epithelial cells. Myofibroblasts are "smooth muscle-like cells" that are morphologically similar to fibroblasts, but also express alphasmooth muscle actin (-SMA) 7. Fibroblasts and myofibroblasts are also key cells in the production and homeostatic maintenance of the ECM of the tissue or organ in which they reside. They are metabolically active cells, capable of synthesizing and secreting ECM components such as collagens and proteoglycans. Fibroblasts continually synthesize ECM proteins although the amount they secrete is tightly regulated, with up to 90% of all procollagen molecules being degraded intracellularly prior to secretion, depending on tissue and age. Further, fibroblasts generate both matrix metalloproteinases (MMPs) and tissue

inhibitors of metalloproteinases, thus controlling homeostatic tissue architecture.

Myofibroblasts were first described by Gabbiani and colleagues as cells central to wound healing 8. The actin filaments result in myofibroblasts having contractile properties, which, at sites of wound healing serve to close the wound. However, the presence of contractile myofibroblasts in the interstitium of the lung may cause a retraction of parenchymal tissue resulting in alveolar collapse giving the characteristic honeycombing as observed in the lungs of IPF patients, or add to the increase in alveolar size, which is characteristic of COPD 9.

this is greatly extended in slow progressors 5,6.

approaches to targeting stromal cells clinically.

**3. Profibrotic role of fibroblasts** 

#### Fig. 1. *Cellular Pathways in Lung Fibrosis: Multi-Faceted Aberrant Response*

Chronic lung remodelling is hypothesized to occur following repeated trauma or injury to the lung. Multiple cellular pathways and interactions which can all promote ECM deposition have been described in lung fibrosis which promote fibroblast and myofibroblast activation, proliferation and survival. Following injury to the epithelium, apoptotic and necrotic signals stimulate collagen producing cells. These resident epithelial cells can also differentiate into a collagen-producing phenotype during EMT. The immune response in the lung is also altered in chronic lung diseases, with pro-fibrotic M2 macrophages and type 2 T cells predominating. These cells generate soluble mediators such as TGF, CCL2 and IL13 which directly activates collagen producing cells.

Key: EMT: epithelial to mesenchymal transition; Mac: Macrophage; Treg: regulatory T cell

Under normal conditions, myofibroblasts sequentially perpetuate and then dampen inflammation via the secretion of chemokines, cytokines, arachidonic acid metabolites and protease inhibitors. When activated, they express cell surface adhesion molecules allowing specific interactions with immune and inflammatory cells, including lymphocytes, mast cells and neutrophils. If these processes become dysregulated, fibrosis may ensue with catastrophic consequences for lung function.

Most insight into the potential role of fibroblasts at driving pulmonary remodeling, as well as phenotypic differences in fibroblasts found in fibrotic regions versus those located in normal tissue has been garnered from *in vitro* studies using fibroblasts isolated from IPF lungs and animal models. Fibroblasts isolated from fibrotic environments are phenotypically different than non-fibrotic fibroblasts 10-12. Fibroblasts from a profibrotic environment exhibit altered responsiveness to growth factors, express enhanced receptor levels for chemokines, which has also been observed in murine models of pulmonary remodeling. These studies suggest a distinct heterogeneity in fibroblast function and phenotype in the fibrotic lung.

We and other investigators have reported that fibroblasts derived from IPF lungs proliferate faster than cells derived from normal lung tissue. In contrast, others have shown that the growth rate of IPF fibroblasts was significantly slower than normal fibroblasts. This discrepancy may be due to the site in the lung from which fibroblasts are harvested, since the magnitude of inflammation and fibrosis are heterogeneous in distribution. Thus, areas of active fibrosis may yield hyperproliferative fibroblasts compared to areas of established fibrosis in which cells may be hypoproliferative.

To begin to address this diversity, recent studies have used microarray technologies to profile global gene expression in pulmonary fibrosis in man and mouse models. These studies have showed expression of almost 500 genes are increased more than two- fold in fibrotic lungs, including many genes related to cytoskeletal reorganization, ECM, cellular metabolism and protein biosynthesis, signaling, proliferation and survival 13,14. There was excellent concordance between gene expression in human and experimental models, giving us some confidence in the value of our efforts to model human disease. Studies examining human lung fibroblast global gene expression in response to TGF1 have shown almost 150 genes upregulated, representing several functional categories described above. This included 80 genes that were not previously known to be TGF -responsive.

The progression and severity of many lung diseases, most notably IPF, are tightly associated with regions of fibroblast accumulation and proliferation, to the extent that these regions have become a reliable indicator of survival. The increased number of (myo)fibroblasts seen in these diseases, implies that they are either hyper-proliferative and/or resistant to apoptosis. However, most studies suggest that these cells proliferate faster than normal 10,15,16

### **4. Sources and fates of fibroblasts and myofibroblasts**

As remodeling of the lung is associated with accumulation and activation of fibroblasts and myofibroblasts, the derivation of both cell types is currently under examination. Reports indicate that there may be multiple pathways through which fibroblasts and myofibroblasts are derived 17. Fibroblasts isolated from human or murine fibrotic tissue exhibit enhanced basal proliferation. Therefore an increase in the fibroblast pool could be due to local proliferation. Myofibroblasts express a panel of markers and these markers have been correlated with site of derivation. For example, myofibroblasts found in the peripheral and subpleural regions of fibrosis express -SMA, vimentin and desmin, whereas cells found in other regions of the lung do not express desmin 18.

#### **4.1 Epithelial to mesenchymal transition**

Another potential source of fibroblasts is by a process called epithelial to mesenchymal transition (EMT). EMT is a dynamic process by which epithelial cells undergo phenotypic transition to fully differentiated motile mesenchymal cells, such as fibroblasts and myofibroblasts. This process occurs normally during early fetal development where there is seamless plasticity between epithelial and mesenchymal cells 19. Furthermore, this phenomenon is well accepted in cancer as a key mechanism that supports tumor metastases 20. However, the process of EMT in development and cancer differ greatly in that, unlike developmental EMT, the tumorigenic EMT process is poorly regulated 21. During the process of EMT, the downregulation of epithelial and tight junction proteins is associated with a concomitant increase in mesenchymal cell markers 22,23. In chronic lung diseases, the dual expression of epithelial and mesenchymal markers in the same cells has led investigators to

We and other investigators have reported that fibroblasts derived from IPF lungs proliferate faster than cells derived from normal lung tissue. In contrast, others have shown that the growth rate of IPF fibroblasts was significantly slower than normal fibroblasts. This discrepancy may be due to the site in the lung from which fibroblasts are harvested, since the magnitude of inflammation and fibrosis are heterogeneous in distribution. Thus, areas of active fibrosis may yield hyperproliferative fibroblasts compared to areas of established

To begin to address this diversity, recent studies have used microarray technologies to profile global gene expression in pulmonary fibrosis in man and mouse models. These studies have showed expression of almost 500 genes are increased more than two- fold in fibrotic lungs, including many genes related to cytoskeletal reorganization, ECM, cellular metabolism and protein biosynthesis, signaling, proliferation and survival 13,14. There was excellent concordance between gene expression in human and experimental models, giving us some confidence in the value of our efforts to model human disease. Studies examining human lung fibroblast global gene expression in response to TGF1 have shown almost 150 genes upregulated, representing several functional categories described above. This

The progression and severity of many lung diseases, most notably IPF, are tightly associated with regions of fibroblast accumulation and proliferation, to the extent that these regions have become a reliable indicator of survival. The increased number of (myo)fibroblasts seen in these diseases, implies that they are either hyper-proliferative and/or resistant to apoptosis.

As remodeling of the lung is associated with accumulation and activation of fibroblasts and myofibroblasts, the derivation of both cell types is currently under examination. Reports indicate that there may be multiple pathways through which fibroblasts and myofibroblasts are derived 17. Fibroblasts isolated from human or murine fibrotic tissue exhibit enhanced basal proliferation. Therefore an increase in the fibroblast pool could be due to local proliferation. Myofibroblasts express a panel of markers and these markers have been correlated with site of derivation. For example, myofibroblasts found in the peripheral and subpleural regions of fibrosis express -SMA, vimentin and desmin, whereas cells found in

Another potential source of fibroblasts is by a process called epithelial to mesenchymal transition (EMT). EMT is a dynamic process by which epithelial cells undergo phenotypic transition to fully differentiated motile mesenchymal cells, such as fibroblasts and myofibroblasts. This process occurs normally during early fetal development where there is seamless plasticity between epithelial and mesenchymal cells 19. Furthermore, this phenomenon is well accepted in cancer as a key mechanism that supports tumor metastases 20. However, the process of EMT in development and cancer differ greatly in that, unlike developmental EMT, the tumorigenic EMT process is poorly regulated 21. During the process of EMT, the downregulation of epithelial and tight junction proteins is associated with a concomitant increase in mesenchymal cell markers 22,23. In chronic lung diseases, the dual expression of epithelial and mesenchymal markers in the same cells has led investigators to

included 80 genes that were not previously known to be TGF -responsive.

However, most studies suggest that these cells proliferate faster than normal 10,15,16

**4. Sources and fates of fibroblasts and myofibroblasts** 

other regions of the lung do not express desmin 18.

**4.1 Epithelial to mesenchymal transition** 

fibrosis in which cells may be hypoproliferative.

postulate that EMT is a mechanism resulting in more ECM-generating mesenchymal cells in the lung 24,25. The differentiation of airway epithelial cells has been previously described, for example, type I pneumocytes transitioning into goblet cells 26-29. However, the switching of an epithelial cell into a phenotype that moves beyond the original cell's embryonic lineage, has only recently been suggested to be a causative factor in fibrosis 23,30,31.

The initial events of EMT include the loss of cell polarity and the induction of matrix metalloproteinases (MMPs) that promote basement membrane degradation and cell detachment. The cells also undergo cytoskeletal changes, as well as altered expression of surface molecules which allow for the migration and transition of these cells to a mesenchymal phenotype 32,33. The majority of the work evaluating EMT has been performed *in vitro;* however the full extent of this pathogenic pathway *in vivo* is currently being evaluated. In animal models of kidney fibrosis, it has been estimated that up to 20% of the fibroblasts found in the fibrotic lesions were derived from the epithelium through EMT 30,34-37.

The idea of EMT promoting the fibrosis observed in COPD and IPF is rapidly beginning to evolve 29. Several recent studies have also shown that EMT occurs in lung epithelial cells both *in vitro* and *in vivo*, supporting the concept of EMT contributing to the fibrosis observed in IPF 29,38,39. Moreover, we have recently published an alteration in the microRNA regulation of genes associated with EMT in IPF40. The potential of EMT promoting COPD requires further investigation. Nevertheless, there is an emerging link between COPD and lung cancer, thus increasing the likelihood of EMT being present in the COPD lung. The link between smoking, lung cancer and COPD is also apparent, in that smoking is a risk factor for COPD and also for lung cancer 41,42. An underlying response to cigarette smoke is generating an altered inflammatory environment in the lung 43, which can then be susceptible to either COPD or lung cancer development 44. Therefore, future work correlating the timecourse of EMT induction with COPD and IPF disease staging will be insightful to determine the extent of contribution mediated by this process.

### **4.2 Circulating progenitor mesenchymal cells**

Along with the epithelium, studies have also highlighted a role for bone marrow-derived circulating progenitor mesenchymal cells, or fibrocytes, in promoting lung fibrosis by differentiating into fibroblasts or myofibroblasts 45,46,47 (Fig. 1). Fibrocytes have been observed at sites of active fibrosis and increased numbers of these cells in the circulation correlate with mortality in IPF 5. They are induced by pro-fibrotic mediators such as TGF1 and Th2 cytokines 48; and the cell markers include leukocyte markers (CD45, CD34), mesenchymal markers (collagen I, fibronectin) and chemokine receptors (CCR3, CCR5, CCR7 and CXCR4) 49. Human and mouse studies have demonstrated that fibrocytes from peripheral blood migrate to skin wound chambers 49-51 and bronchial mucosa after antigen challenge 52. Furthermore, these cells have been reported in disease states with fibrotic pathologies including hypertrophic scars, asthma and IPF 52-55.

Fibrocytes are pleiotropic and may contribute to fibrogenesis by directly producing collagen, as well as inflammatory cytokines, hematopoietic growth factors, and chemokines 54-58 (Fig. 2). In a study performed in collaboration between AstraZeneca and Malmö University Hospital, increased fibrocyte numbers were observed in the circulation of COPD patients, where patients with mild COPD had the most elevated number of fibrocytes in comparison to moderate COPD, severe COPD or healthy patients 59. Although there was no correlation of fibrocyte number with any lung function parameter, inhaled glucocorticosteroid use


Fig. 2. Overlapping Differentiation Pathways and Functions of Fibroblasts, Fibrocytes and Macrophages

Fibroblasts, fibrocytes and macrophages share many *in vitro* and *in vivo* functions such as mediator production, host defence and extracellular matrix deposition. Monocytes can differentiate into fibrocytes or macrophages depending on the environment and the nature of the stimulus. *In vitro* studies have highlighted a role for various mediators such as cytokines, TLR signals, cellular debris and cell function at promoting differentiation. However, it is likely a combination of these pathways *in situ* which dictates the fate of the monocyte. Fibrocytes have been shown to differentiate into fibroblasts, in part through losing CD45 expression but retaining collagen I expression. Moreover in vitro, fibrocytes stimulated with TGF or ET-1 can promote fibrocyte to fibroblast differentiation. Recent evidence has indicated that fibroblasts can differentiate into haematopoietic cells when stimulated with appropriate growth factors.

Key: TGF: transforming growth factor ; GM-CSF: granulocyte macrophage colonystimulating factor; M-CSF: macrophage colony-stimulating factor; TLR: toll-like receptors; ET-1: endothelin-1; OCT4: octamer-binding transcription factor 4); ECM: extracellular matrix; SMA: -smooth muscle actin.

Fig. 2. Overlapping Differentiation Pathways and Functions of Fibroblasts, Fibrocytes and

Fibroblasts, fibrocytes and macrophages share many *in vitro* and *in vivo* functions such as mediator production, host defence and extracellular matrix deposition. Monocytes can differentiate into fibrocytes or macrophages depending on the environment and the nature of the stimulus. *In vitro* studies have highlighted a role for various mediators such as cytokines, TLR signals, cellular debris and cell function at promoting differentiation. However, it is likely a combination of these pathways *in situ* which dictates the fate of the monocyte. Fibrocytes have been shown to differentiate into fibroblasts, in part through losing CD45 expression but retaining collagen I expression. Moreover in vitro, fibrocytes stimulated with TGF or ET-1 can promote fibrocyte to fibroblast differentiation. Recent evidence has indicated that fibroblasts can differentiate into haematopoietic cells when

Key: TGF: transforming growth factor ; GM-CSF: granulocyte macrophage colonystimulating factor; M-CSF: macrophage colony-stimulating factor; TLR: toll-like receptors; ET-1: endothelin-1; OCT4: octamer-binding transcription factor 4); ECM: extracellular

Macrophages

stimulated with appropriate growth factors.

matrix; SMA: -smooth muscle actin.

significantly inhibited circulating fibrocyte numbers, corroborating the pro-inflammatory association of fibrocytes 59. While it was originally thought that fibrocytes promote fibrosis through production of ECM components, it is becoming increasingly hypothesized that their primary role in tissue remodelling may be through secretion of soluble factors.

Fibrocytes have pleuripotent potential to differentiate into other cell lineages, as has been demonstrated with fibrocyte-derived adipocytes 60. Furthermore, the extreme plasticity of these cells *in vitro*, makes both the derivation and characterization of these cells difficult. Exposure of fibrocytes to TGF1 *in vitro* results in the cells transitioning into a myofibroblast phenotype that expresses both fibronectin and type III collagen 52. Using an adoptive transfer model of bone marrow cells from green fluorescent protein (GFP) transgenic mice into mice challenged with intratracheal bleomycin to initiate lung injury, recruited GFP+ fibrocytes were shown to differentiate into fibroblasts while resident lung fibroblasts differentiated into myofibroblasts 56.

At sites of normal wound healing, once sufficient ECM has been deposited, fibroblasts and myofibroblasts undergo apoptosis 61,62. This serves to limit the excessive deposition of ECM and also dampen the pro-inflammatory and pro-fibrotic milieu. However, myofibroblasts persist in fibrotic conditions. Moreover, IPF fibroblasts are relatively resistant to apoptosis *in vitro*, with IPF cells inducing a different pattern of pro-apoptotic enzymes in response to Fas-L stimulation compared to normal fibroblasts 63.

A recently described study showed an alternate potential fate of fibroblasts in that they can de-differentiation or transition into haematopoietic cells64. Here the investigators showed that, in the presence of haematopoietic transcription factors, fibroblasts can convert into haematopoietic progenitor cells, indicating a stem-like potential of cells that have traditionally thought to be resident to the lung 64. Therefore, expanding our understanding on the pathways that control fibroblast to myofibroblast fate, as well as fibroblast to nonfibroblast cell fate, particularly in disease, will greatly expand our understanding of disease.

## **5. Fibroblast cell: Cell interactions**

In the lung, fibroblasts are found in the greatest number in the subepithelial layer of the conducting airways and the interstitium of the lung parenchyma (Fig. 1). Here they are in a prime location to interact with the epithelial and endothelial cells, as well as leukocytes in the airspaces, interstitium and vasculature. One of the key inflammatory cells that is found in the lung, and one that is becoming more associated with the maintenance and progression of fibrosis, is the alternatively activated (M2) macrophage. The M2 macrophage is the predominant macrophage found in the lungs of IPF patients 65. Moreover, M2 macrophages have been associated with COPD 66,67. In healthy tissue, alveolar macrophages are known to remove apoptotic and necrotic debris and pathogens via phagocytosis in a non-phlogistic mechanism, in that downstream inflammation is limited and the inflammatory process is attenuated 68.

In chronic lung disease, the predominant M2 macrophage phenotype is inefficient at clearing debris. M2 macrophages are defective in phagocytosis and do not dampen the inflammatory response 69. These macrophages are capable of synthesizing pro-fibrotic mediators, which supports their role in wound healing, yet this cell type is inefficient at supporting host defense 69. This may explain why COPD and IPF patients are susceptible to repeated bouts of pulmonary infections or exacerbation of disease. These cells express elevated levels of scavenger receptors such as macrophage scavenger receptor (MSR) and mannose receptor C (MRC/ CD206) 69,70. Assessing circulating primary cells from IPF patients, we have determined an elevation of CD163+CD14+ cells and M2-associated soluble mediators in the circulation, which was more pronounced in progressive IPF patients, suggestive of an overall elevated M2 background in these patients 71. We have also shown that peripheral blood monocytes from patients with scleroderma-related lung disease display a pro-fibrotic phenotype, characterized by increased CD163 expression and CCL18 production 72. Interestingly, macrophages in the lungs of COPD patients, as well as smokers with or without COPD, also demonstrate a skewing towards an M2 phenotype, with a deactivated M1 phenotype 73,74. Moreover *in vitro*, cigarette smoke induces a M2-type phenotype and this result was also seen in an *in vivo* cigarette smoke model in mice 73. *In vitro*, macrophage polarization to an M2 phenotype has only been shown robustly with mouse cells. Here the differentiation of a monocyte to an M2 phenotype requires a cocktail of cytokines including IL13 and CCL17/TARC75. Therefore the Th2 cytokine profile observed in remodeled lungs contributes to the appearance of M2 macrophages.

Studies of bleomycin-induced fibrosis have assessed either M2 macrophage or fibrocyte number 71,76. It is increasingly recognized that there is some overlap between these cell subsets in terms of both function and markers 76. However, although both fibrocytes and M2 macrophages can be derived from monocytes, they are not completely redundant in function. Recently, using a lung-specific TGF1 over-expression model of lung fibrosis, we determined that depletion of lung monocyte/macrophages using liposomal clodronate reduced collagen accumulation, but this had no effect on the TGF-induced fibrocyte recruitment 77. Therefore, for novel therapeutic approaches such as cell depletion or specific targeting, the M2 macrophage may be a more compelling target for chronic lung remodeling.

In COPD, alveolar macrophages have been hypothesized to have reduced anti-inflammatory properties, as well as a reduced capacity to turn over matrix 78. However a recent study looking at macrophage number, MMP expression and emphysema determined a correlation between a greater infiltration of macrophage to the lung and emphysema in COPD patients 79. *Ex vivo*, cigarette smoke induces a wide array of changes in the inflammatory profile and the host defence potential of COPD alveolar macrophages 80. Moreover, microarray analysis indicated a correlation between alveolar macrophage gene expression, circulating monocyte gene expression and lung function 81. The investigators hypothesized that there is a "COPDrelated gene expression pattern" as many genes differentially expressed in COPD alveolar macrophages were also expressed in COPD circulating monocytes 81.

#### **6. Activators of fibroblasts/ mesenchymal cells**

Fibroblasts are activated by numerous signals including: mechanical forces such as those imposed during bronchoconstriction; matrix interactions; hypoxia and resultant changes in pH levels; and soluble mediators (Fig. 1). The large variety of soluble mediators capable of activating mesenchymal cells are produced by many different cell types found in fibrotic regions. Furthermore, proteases of the coagulation cascade and other serum factors also promote fibroblast proliferation, collagen synthesis, migration and differentiation.

#### **6.1 Growth factors**

Growth factors are the most recognized mediators that activate mesenchymal cells. Transforming growth factor β (TGFβ) is one of the most potent profibrotic mediators *in vitro* and is a central driver in the remodeling process. TGF1 regulates numerous biologic

mannose receptor C (MRC/ CD206) 69,70. Assessing circulating primary cells from IPF patients, we have determined an elevation of CD163+CD14+ cells and M2-associated soluble mediators in the circulation, which was more pronounced in progressive IPF patients, suggestive of an overall elevated M2 background in these patients 71. We have also shown that peripheral blood monocytes from patients with scleroderma-related lung disease display a pro-fibrotic phenotype, characterized by increased CD163 expression and CCL18 production 72. Interestingly, macrophages in the lungs of COPD patients, as well as smokers with or without COPD, also demonstrate a skewing towards an M2 phenotype, with a deactivated M1 phenotype 73,74. Moreover *in vitro*, cigarette smoke induces a M2-type phenotype and this result was also seen in an *in vivo* cigarette smoke model in mice 73. *In vitro*, macrophage polarization to an M2 phenotype has only been shown robustly with mouse cells. Here the differentiation of a monocyte to an M2 phenotype requires a cocktail of cytokines including IL13 and CCL17/TARC75. Therefore the Th2 cytokine profile

observed in remodeled lungs contributes to the appearance of M2 macrophages.

macrophage may be a more compelling target for chronic lung remodeling.

macrophages were also expressed in COPD circulating monocytes 81.

**6. Activators of fibroblasts/ mesenchymal cells** 

**6.1 Growth factors** 

Studies of bleomycin-induced fibrosis have assessed either M2 macrophage or fibrocyte number 71,76. It is increasingly recognized that there is some overlap between these cell subsets in terms of both function and markers 76. However, although both fibrocytes and M2 macrophages can be derived from monocytes, they are not completely redundant in function. Recently, using a lung-specific TGF1 over-expression model of lung fibrosis, we determined that depletion of lung monocyte/macrophages using liposomal clodronate reduced collagen accumulation, but this had no effect on the TGF-induced fibrocyte recruitment 77. Therefore, for novel therapeutic approaches such as cell depletion or specific targeting, the M2

In COPD, alveolar macrophages have been hypothesized to have reduced anti-inflammatory properties, as well as a reduced capacity to turn over matrix 78. However a recent study looking at macrophage number, MMP expression and emphysema determined a correlation between a greater infiltration of macrophage to the lung and emphysema in COPD patients 79. *Ex vivo*, cigarette smoke induces a wide array of changes in the inflammatory profile and the host defence potential of COPD alveolar macrophages 80. Moreover, microarray analysis indicated a correlation between alveolar macrophage gene expression, circulating monocyte gene expression and lung function 81. The investigators hypothesized that there is a "COPDrelated gene expression pattern" as many genes differentially expressed in COPD alveolar

Fibroblasts are activated by numerous signals including: mechanical forces such as those imposed during bronchoconstriction; matrix interactions; hypoxia and resultant changes in pH levels; and soluble mediators (Fig. 1). The large variety of soluble mediators capable of activating mesenchymal cells are produced by many different cell types found in fibrotic regions. Furthermore, proteases of the coagulation cascade and other serum factors also

Growth factors are the most recognized mediators that activate mesenchymal cells. Transforming growth factor β (TGFβ) is one of the most potent profibrotic mediators *in vitro* and is a central driver in the remodeling process. TGF1 regulates numerous biologic

promote fibroblast proliferation, collagen synthesis, migration and differentiation.

activities such as proliferation, apoptosis, and differentiation 82,83. TGF1 is upregulated in the lungs of IPF patients and patients with other chronic lung diseases 84-89. Interestingly, expression of TGF1 is nearly absent in the bronchial epithelial cells but is highly expressed in inflammatory cells beneath the basement membrane where subepithelial fibrosis predominates 90. In COPD, TGF has also been reported to be produced by circulating and interstitial T cells and monocytes 91. Polymorphisms in the promoter region of TGF1 have been reported in COPD patients 92. However, TGF SNPs have been associated with protection in COPD 93 or have been linked to COPD but not related to worsening of disease 94.

Transient overexpression of TGF1, or pulmonary delivery of this cytokine to mouse lungs, induces a pronounced interstitial fibrosis mediated by excess ECM generation and deposition, as well as the presence of myofibroblasts95. Using a transgenic mouse model of SMAD-3 deficiency, TGF/SMAD-3 signaling has been shown to be required for alveolar integrity and ECM homeostasis and that this pathway is involved in pathogenic mechanisms mediating both tissue destruction and fibrogenesis.

Due to TGF1 being such a potent growth factor, the release and activation of this growth factor is tightly regulated. TGF is released in a latent complex, associated with LAP (latency activated peptide) and LTBP. There are several mechanisms by which TGF is then activated and these include integrin-mediated cell cytoskeletal rearrangement in the case of integrin v696; cell membrane MMP recruitment for enzymatic cleavage in the case of integrin v897 and other protease-related mechanisms exhibited by components of the coagulation cascade98 (discussed later). The integrin v6 is upregulated in IPF99 and neutralization of this integrin *in vivo* reduces fibrosis in various experimental models 99-101.

There are also endogenous inhibitors of TGF, the largest being the BMP (bone morphogenic protein) family. The BMP family is structurally and functionally related to the TGF superfamily 102. BMPs inhibit TGF signaling through either inhibiting Smad 2/3 phosphorylation or competing for Smad 4 or both 103,104. Recombinant BMP7 has been shown to inhibit TGF-induced EMT *in vitro*, as well as reducing renal fibrosis 105. However, we have shown that BMP7 had no anti-fibrotic effect in models of lung fibrosis, nor were there effects on lung epithelial cells *in vitro* 106.

## **6.2 Th2-associated mediators**

Interleukin 13 (IL13) and IL4 are two pleiotropic, Th2-associated cytokines, with numerous distinct and overlapping functions. They share overlapping roles due to the shared IL13R1 receptor subunit. However they also have unique subunits that confer the distinct functions. IL13 activates epithelial cells and goblet cells causing mucous production, goblet cell hyperplasia and EMT 107,108. Various animal models of pulmonary fibrosis have indicated a more pro-fibrotic role for IL13 than IL4. Indeed it has been hypothesized that IL4 is involved in the initiation of fibrosis whereas IL13 is central to the maintenance of the fibrotic response.

We have published that IL-13 is elevated in the lungs of IPF patients 71 and this protein is associated with fibrotic pathologies and aberrant remodeling at various tissue sites 65,109. There is also elevated expression of the two IL13 receptor subunits IL13R1 and IL13R2 which are prominant on fibroblasts 110,111. More recently we have shown that IPF fibroblasts are hyper-responsive to IL13 in comparison to non-fibrotic fibroblasts58. It was originally hypothesized that IL13R2 is a decoy receptor as it has a short cytoplasmic tail and although it is the higher affinity receptor subunit, it is found at high levels in a soluble form, but only in murine models of fibrosis112 and not in humans113. However, recent data has suggested that signaling of IL13 through IL13R2 is pro-fibrotic, resulting in TGF1 production 114.

Lung-specific over-expression of IL13 in mice results in remodeling and emphysema 115. Moreover, various MMPs and cathepsins that are associated with COPD1 are also induced in the IL13-transgenic lung 116. However robust detection of IL13 has not been consistently reported. IL13 producing macrophages and NKT cells have been detected in the COPD lung 117. Also, the presence of the cytokine and IL13 positive cells have been shown in the bronchial epithelium of smokers with chronic bronchitis 118. In contrast, decreased IL13 has been measured in emphysema compared to non-emphysematous lungs 119. An inverse correlation between plasma IL13 and the lung diffusion capacity for carbon monoxide (DLCO) parameter, used to determine the efficiency of gas exchange in COPD has been reported 120.

Another Th2-associated mediator that is being actively researched in fibrosis is the chemokine CCL18/ PARC. In IPF, CCL18 has been associated with poor outcome, namely, patients with CCL18 levels greater than 150ng/ml in the circulation will typically have more progressive disease, compared to those with CCL18 levels below 150ng/ml 6. CCL18 expression is also a marker for M2 macrophages 121, therefore linking the profibrotic cell phenotype with IPF progression. In the recent ECLISPE study, elevated CCL18 levels in COPD patients has been associated with increased chance of COPD exacerbation 122. This association of CCL18 with COPD exacerbations was in stark contrast to TNF, IL6 or CXCL8/ IL8 levels, or the number of pack years of smoking 122. These observations may change the understanding of the pleiotropic nature of the underlying mechanisms for COPD, as CCL18 is not associated with smoking or with neutrophil accumulation and activation.

#### **6.3 Coagulation cascade**

Thrombin is a serine protease generated during activation of the coagulation pathway 123. Thrombin has been implicated in a number of pulmonary fibrotic diseases such as acute lung injury (ALI) 124,125 acute respiratory distress syndrome (ARDS) 126, interstitial lung disease (ILD) 127,128 and IPF 127,129-132 and IPF BAL fluid thrombin has been shown to promote fibroblast proliferation 131. In COPD, elevated procoagulant activity has been observed in the serum of patients with moderate to severe disease 133. Animal models of fibrosis have strengthened the connection between thrombin and fibrosis. Increased thrombin is found in the lungs of mice challenged with bleomycin and pharmacological inhibition of thrombin significantly reduced the collagen deposition 130.

At the cellular level, thrombin has numerous biologic effects that are in addition to its role as a coagulation pathway proteinase. It has been shown to promote inflammation and fibrosis through inducing chemokine and growth factor production from fibroblasts. Many of the cellbased activities of thrombin are mediated through a family of receptors termed proteinaseactivated receptors (PARs). PARs are G-protein coupled receptors (GPCR), with 4 known subtypes identified to date. A defining feature of these receptors is that they are activated by proteases that cleave a portion of the extracellular amino terminus to unmask a new Nterminal sequence, which then functions as a tethered ligand that autoactivates the receptor. PAR-1-deficient mice are protected from bleomycin induced lung fibrosis 134. Further, PAR-2 deficient mice had decreased eotaxin/CCL11 and reduced eosinophilia in the lungs following antigen challenge in an allergen sensitization and challenge model of asthma 135,136. Furthermore, thrombin and other PAR1 agonists promote the integrin-mediated activation latent TGF in a model of acute lung injury 98 . Thus, the local activation of TGF at sites of fibrosis may be enhanced by the coagulation cascade in both IPF and COPD.

#### **6.4 Pentraxins**

114 Lung Diseases – Selected State of the Art Reviews

in murine models of fibrosis112 and not in humans113. However, recent data has suggested that signaling of IL13 through IL13R2 is pro-fibrotic, resulting in TGF1 production 114. Lung-specific over-expression of IL13 in mice results in remodeling and emphysema 115. Moreover, various MMPs and cathepsins that are associated with COPD1 are also induced in the IL13-transgenic lung 116. However robust detection of IL13 has not been consistently reported. IL13 producing macrophages and NKT cells have been detected in the COPD lung 117. Also, the presence of the cytokine and IL13 positive cells have been shown in the bronchial epithelium of smokers with chronic bronchitis 118. In contrast, decreased IL13 has been measured in emphysema compared to non-emphysematous lungs 119. An inverse correlation between plasma IL13 and the lung diffusion capacity for carbon monoxide (DLCO) parameter,

Another Th2-associated mediator that is being actively researched in fibrosis is the chemokine CCL18/ PARC. In IPF, CCL18 has been associated with poor outcome, namely, patients with CCL18 levels greater than 150ng/ml in the circulation will typically have more progressive disease, compared to those with CCL18 levels below 150ng/ml 6. CCL18 expression is also a marker for M2 macrophages 121, therefore linking the profibrotic cell phenotype with IPF progression. In the recent ECLISPE study, elevated CCL18 levels in COPD patients has been associated with increased chance of COPD exacerbation 122. This association of CCL18 with COPD exacerbations was in stark contrast to TNF, IL6 or CXCL8/ IL8 levels, or the number of pack years of smoking 122. These observations may change the understanding of the pleiotropic nature of the underlying mechanisms for COPD, as CCL18 is not associated with

Thrombin is a serine protease generated during activation of the coagulation pathway 123. Thrombin has been implicated in a number of pulmonary fibrotic diseases such as acute lung injury (ALI) 124,125 acute respiratory distress syndrome (ARDS) 126, interstitial lung disease (ILD) 127,128 and IPF 127,129-132 and IPF BAL fluid thrombin has been shown to promote fibroblast proliferation 131. In COPD, elevated procoagulant activity has been observed in the serum of patients with moderate to severe disease 133. Animal models of fibrosis have strengthened the connection between thrombin and fibrosis. Increased thrombin is found in the lungs of mice challenged with bleomycin and pharmacological

At the cellular level, thrombin has numerous biologic effects that are in addition to its role as a coagulation pathway proteinase. It has been shown to promote inflammation and fibrosis through inducing chemokine and growth factor production from fibroblasts. Many of the cellbased activities of thrombin are mediated through a family of receptors termed proteinaseactivated receptors (PARs). PARs are G-protein coupled receptors (GPCR), with 4 known subtypes identified to date. A defining feature of these receptors is that they are activated by proteases that cleave a portion of the extracellular amino terminus to unmask a new Nterminal sequence, which then functions as a tethered ligand that autoactivates the receptor. PAR-1-deficient mice are protected from bleomycin induced lung fibrosis 134. Further, PAR-2 deficient mice had decreased eotaxin/CCL11 and reduced eosinophilia in the lungs following antigen challenge in an allergen sensitization and challenge model of asthma 135,136. Furthermore, thrombin and other PAR1 agonists promote the integrin-mediated activation latent TGF in a model of acute lung injury 98 . Thus, the local activation of TGF at sites of

used to determine the efficiency of gas exchange in COPD has been reported 120.

smoking or with neutrophil accumulation and activation.

inhibition of thrombin significantly reduced the collagen deposition 130.

fibrosis may be enhanced by the coagulation cascade in both IPF and COPD.

**6.3 Coagulation cascade** 

Pentraxins comprise a highly conserved superfamily of cyclic pentameric proteins. These proteins interact with numerous ligands, including selected pathogens and apoptotic cells137, and are recognized by macrophages via mannose 6P and Fc gamma () receptors, ultimately leading to complement activation, pathogen recognition, and apoptotic cell clearance 138-140. Pentraxins are subdivided into the short pentraxins that include C-reactive protein (CRP) and serum amyloid P component (SAP, PTX2), and the long pentraxin 3 (PTX3) 141,142. SAP appears to be uniquely involved in the resolution or repair phase of tissue injury via its modulator effects on resident and bone marrow derived collagen producing cells 76,143. SAP binds to Fc receptors144 and the anti-fibrotic activities of SAP have been shown to be mediated through Fc receptors143 which affect peripheral blood monocyte differentiation and activation states.

SAP promotes the differentiation of M1 classically activated macrophages in a tuberculosis model of lung infection 145. We have recently demonstrated that SAP has prominent immunomodulatory effects on mouse macrophages, thereby providing a mechanism for its ability to prevent the development and reverse established experimental fungal airway disease 75. Moreover, human SAP has been shown to potently inhibit the differentiation of monocytes into fibrocytes146 and it has consequently been used therapeutically in animal models to inhibit lung fibrosis and fibrosis in a number of organ sites. In addition, SAP causes an inhibition of the differentiation of peripheral blood mononuclear cells into CD45+/collagen I+ cells called fibrocytes76,146,147, as well as reducing M2 macrophage number 75,77.

#### **6.5 Matrix Metalloproteinases and Chitinases**

Active remodeling of the ECM is dependent on the coordinated activities of proteases and protease inhibitors 148,149. Fibroblasts generate metalloproteinases (MMPs), which are elevated in asthma and COPD (reviewed in 150). MMPs are a family of proteins that exert proteolytic activities on various proteins including ECM components and are thus central to ECM formation and organization 150. Tissue inhibitors of metalloproteinases (TIMP)s are endogenous inhibitors of MMPs that bind to the catalytic site on these proteinases 151. In IPF, MMP1, 2 and 9 were co-localized to the epithelium surrounding fibrotic lesions, whereas increased TIMP2 was also observed suggesting that the MMP activity may be inhibited and that the fibrotic region not degraded 152.

Another family of enzymes associated with fibrosis and remodeling is the chitinase family. Chitinases are proteolytic enzymes that bind, but do not cleave chitin 153. Chitin is the main component of the insect exoskeleton, thought to be absent in human tissue. However, these proteins are postulated to play important, yet currently undefined roles in biology. The prototypic chitinase-like protein is derived from the chitinase 3 like 1 (*Ch3l1*) gene, called YKL40 in humans and BRP39 in mice. YKL40/BRP39 is a circulating regulator of apoptosis, and shown to have a role in M2 macrophage activation, TGF1 induction and tissue fibrosis 154. An early report in asthmatics indicated that YKL40 is elevated in the circulation and is associated with asthma severity 155. Chitinolytic activity has also been demonstrated in COPD patients 156,157, as well as in models of cigarette smoke induced emphysema 157. Interestingly, BRP39 gene deficient mice have increased epithelial cell apoptosis and alveolar destruction in response to cigarette smoke, but worsened pathology in hyperoxia-induced lung injury which is a more acute ARDS-like model 157,158. Increased YKL40 has also been detected in the lungs and circulation of IPF patients, with levels correlating with survival 159,160. Thus, there is clear disease association with chitinase activity, however because of discrepancies in the different animal models, whether these chitinases are promoting disease or uniquely serve as a marker of disease activity still requires further elucidation.

## **7. Therapeutic options for targeting pro-fibrotic cells**

IPF is a disease that is driven by continual parenchymal ECM deposition, which reduces lung compliance and effective gas exchange. The fibrosis observed in the lungs of COPD patients predominates around small airways4,161 and may not be the main driving factor causing the loss in lung function and death. Emphysema and lung tissue destruction is often observed in COPD patients and honeycombing is a salient feature in IPF patient lungs 162. Overall this suggests an imbalance in repair processes within the lung and targeting the underlying pro-fibrotic mechanisms may impact COPD and IPF in a positive way. In IPF, the capacity of the lungs (FVC, forced vital capacity); as well as the efficiency of gas exchange (DLCO) may improve with a reduction in fibrosis. Both of these parameters are used clinically in IPF patients to measure lung function and monitor disease progression162. In COPD, anti-fibrotic strategies directed at the same cells and/or mediators may attenuate the obstructive nature in the airways and this may be the most discernible improvement in lung function. This would translate to an improvement in forced expiratory volume in a short time frame (FEV1). Again, FEV1 is commonly used in COPD patient management and is used to segregate patients into the various GOLD stages of disease4.

As has been highlighted in this Chapter, monocytes and mesenchymal cells express a variety of receptor that can promote recruitment, proliferation or activation, as well as differentiation into other phenotypes (Fig. 2). In experimental mouse models of lung fibrosis, blocking fibrocyte recruitment through chemokine ligand/ receptor blockade significantly attenuated ECM deposition 54-56,163. However, these chemokine receptors are not uniquely expressed on fibrocytes164, so the anti-fibrotic effects observed following blockade of these G-protein coupled receptors might extend beyond impaired fibrocyte recruitment. Also, we have demonstrated that depletion of lung monocyte/ macrophages inhibits TGF-induced lung fibrosis, but has no effect on lung fibrocyte number, suggesting a redundant role for fibrocyte recruitment in promoting TGF-induced lung fibrosis 77.

One other mechanism to consider in any therapeutic approach in pulmonary fibrosis is fibrocyte activation. A prototypic activator of fibrocytes, fibroblasts and myofibroblasts is TGF. Adenoviral-mediated over-expression of TGF in the lungs of mice or rats, or lungspecific transgenic over-expression in mice induces significant lung pathology95,165,166. In the lung specific TGF over expression, active TGF is expressed by airway epithelial cells. At early timepoints following over-expression, apoptosis is observed, following by interstitial leukocyte accumulation and then increased collagen deposition, predominating around the airways and then gradually increasing in the parenchyma77,165,167. At later stages of lungspecific TGF over-expression, alveolar collapse is observed77 whereas adenoviral TGF expression results in a prolonged, severe fibrosis95. These apparent chronic differences may be due to the initial extent of acute lung injury immediately following TGF overexpression, in that transgenic mediated TGF induces extensive apoptosis, whereby blocking apoptosis inhibits subsequent fibrosis165. Blockade of TGF in experimental models of lung fibrosis through either antibody neutralization, receptor inhibition of TGF activation has been shown to attenuate lung remodelling 99,100,168-170. However, TGF1 gene deficient mice die within 3-4 weeks of age and demonstrate significant autoimmune pathologies, potentially due to the lack of immunoregulation in the absence of TGF 171,172. Therefore direct neutralization of this target might have significant safety concerns in IPF or COPD patients.

Other factors that work in concert with TGF- include IL4 and IL13. In order to examine the role of IL4- and IL13-responsive cells in pulmonary fibrosis, we have conducted preclinical studies using IL13 conjugated to a Pseudomonas exotoxin, IL13-PE173, which targets cells expressing the type 2 IL4 receptor and IL13R2. This protein-toxin conjugate selectively targets IPF fibroblasts because they express these receptor subunits, resulting in specific cell death. Moreover, *in vivo* use in a murine model of bleomycin-induced lung fibrosis showed that selective targeting of fibroblasts at the end stages or maintenance phase of fibrosis, attenuated remodelling. In contrast, IL13-PE delivered to the lung during the onset or initiation of disease had no therapeutic benefit173. This suggests that targeting these cells in established disease may allow for the resolution of lung pathology that is observed in chronic lung disease patients.

Taken together, targeting stromal cells to dampen the extent of activation and the amount of ECM deposition, regardless of disease, or region within the lung may have an impact in lung function that can either halt disease progression or promote resolution and restoration of lung function.

## **8. Acknowledgements**

We also thank Dr. Judith Connett for her critical reading of the manuscript.

## **9. References**

116 Lung Diseases – Selected State of the Art Reviews

animal models, whether these chitinases are promoting disease or uniquely serve as a marker

IPF is a disease that is driven by continual parenchymal ECM deposition, which reduces lung compliance and effective gas exchange. The fibrosis observed in the lungs of COPD patients predominates around small airways4,161 and may not be the main driving factor causing the loss in lung function and death. Emphysema and lung tissue destruction is often observed in COPD patients and honeycombing is a salient feature in IPF patient lungs 162. Overall this suggests an imbalance in repair processes within the lung and targeting the underlying pro-fibrotic mechanisms may impact COPD and IPF in a positive way. In IPF, the capacity of the lungs (FVC, forced vital capacity); as well as the efficiency of gas exchange (DLCO) may improve with a reduction in fibrosis. Both of these parameters are used clinically in IPF patients to measure lung function and monitor disease progression162. In COPD, anti-fibrotic strategies directed at the same cells and/or mediators may attenuate the obstructive nature in the airways and this may be the most discernible improvement in lung function. This would translate to an improvement in forced expiratory volume in a short time frame (FEV1). Again, FEV1 is commonly used in COPD patient management and

As has been highlighted in this Chapter, monocytes and mesenchymal cells express a variety of receptor that can promote recruitment, proliferation or activation, as well as differentiation into other phenotypes (Fig. 2). In experimental mouse models of lung fibrosis, blocking fibrocyte recruitment through chemokine ligand/ receptor blockade significantly attenuated ECM deposition 54-56,163. However, these chemokine receptors are not uniquely expressed on fibrocytes164, so the anti-fibrotic effects observed following blockade of these G-protein coupled receptors might extend beyond impaired fibrocyte recruitment. Also, we have demonstrated that depletion of lung monocyte/ macrophages inhibits TGF-induced lung fibrosis, but has no effect on lung fibrocyte number, suggesting a redundant role for fibrocyte recruitment in promoting TGF-induced lung fibrosis 77. One other mechanism to consider in any therapeutic approach in pulmonary fibrosis is fibrocyte activation. A prototypic activator of fibrocytes, fibroblasts and myofibroblasts is TGF. Adenoviral-mediated over-expression of TGF in the lungs of mice or rats, or lungspecific transgenic over-expression in mice induces significant lung pathology95,165,166. In the lung specific TGF over expression, active TGF is expressed by airway epithelial cells. At early timepoints following over-expression, apoptosis is observed, following by interstitial leukocyte accumulation and then increased collagen deposition, predominating around the airways and then gradually increasing in the parenchyma77,165,167. At later stages of lungspecific TGF over-expression, alveolar collapse is observed77 whereas adenoviral TGF expression results in a prolonged, severe fibrosis95. These apparent chronic differences may be due to the initial extent of acute lung injury immediately following TGF overexpression, in that transgenic mediated TGF induces extensive apoptosis, whereby blocking apoptosis inhibits subsequent fibrosis165. Blockade of TGF in experimental models of lung fibrosis through either antibody neutralization, receptor inhibition of TGF activation has been shown to attenuate lung remodelling 99,100,168-170. However, TGF1 gene deficient mice die within 3-4 weeks of age and demonstrate significant autoimmune pathologies, potentially due to the lack of immunoregulation in the absence of TGF 171,172.

of disease activity still requires further elucidation.

**7. Therapeutic options for targeting pro-fibrotic cells** 

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## **Cancer Stem Cells (CSCs) in Lung Cancer**

Hiroyuki Sakashita, Yuki Sumi and Naohiko Inase

*Department of Integrated Pulmonology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University Japan* 

#### **1. Introduction**

The cancer stem cell model for tumor progression is the first model of its type to suggest that only one subpopulation of cancer cells is capable of proliferating indefinitely. These cells resemble normal stem cells in their capacity for self-renewal and multi-potential differentiation, and can both initiate and maintain tumors. The prevailing names for these cells are "cancer stem cells (CSCs)" and "cancer initiating cells (CICs)." The CSC model implies a hierarchical organization within the tumor in which a limited number of CSCs represents the apex of the hierarchy. CSCs are chemo-resistant, radio-resistant, and quiescent, and have been shown to cause both metastasis and relapse.

CSCs were first described in patients with acute myeloid leukemia (AML) by Dick et al. (Lapidot et al., 1994). As to be expected from leukemia stem cells, these CSCs exhibited the properties of self-renewal, proliferation and multipotency. The frequency of these leukemiainitiating cells in the peripheral blood of those AML patients was one engraftment unit in 250,000 cells. Dick et al. identified the leukemia-initiating cells as CD34+CD38- .

 In the years since, CSCs have been identified in cancers of the breast(Al-Hajj et al., 2003), brain (Singh et al., 2003) and prostate, pancreas and lung (Eramo et al., 2008). In this chapter we review CSCs in lung cancer.

## **2. Overview of CSCs in lung cancer**

#### **2.1 Concept of CSCs**

Two major models have been described for tumor propagation: the clonal evolution model, which involves a stochastic component, and the CSC model, which is defined as hierarchical. According to the clonal evolution model, a neoplasm arises from a single cell of origin, whereupon an acquired genetic variability within the original clone allows a sequential selection to more aggressive clones, thereby allowing the tumor to progress. According to the CSC model, tumor cells are heterogeneous, and only the CSC subset has the ability to proliferate extensively and form new tumors (Wicha et al., 2006). Yet neither model alone can adequately explain the complex biology of tumor progression, resistance, and metastasis. Fig. 1 describes a new CSC hypothesis model that encompasses both the CSC hierarchical and clonal evolution components, a model in which pre-existing CSCs can transform into secondary CSCs (Takebe & Ivy, 2003).

Fig. 1. CSC development.

A combination of DNA hits and niche microenvironmental factors can transform normal stem cells into primary CSCs or more differentiated cancer progenitor cells. Primary tumors are formed mostly from bulk tumor cells together with a small percentage of CSCs. The accumulation of additional DNA hits plus an altered niche microenvironment may drive primary CSCs to evolve into a genetically distinct population of secondary CSCs. Metastatic CSCs have the potential to proliferate and form metastatic tumors at distant sites composed mostly of bulk tumor cells together with a minority of metastatic CSCs (Takebe & Ivy, 2003).

#### **2.2 Origins of CSCs**

It remains uncertain whether CSCs originate from normal (somatic) stem cells that acquire oncogenic mutations or from non-stem cells of more differentiated forms that dedifferentiate and acquire stem-cell-like properties through mutation and reprogramming. CSCs can convert into differentiated cells, (Fig. 2) and evidence has suggested that these differentiated cells can acquire stem-cell-like properties via exogenous circumstances (including the niche), with plasticity. (Fig. 2) Cancer stem cells, for example, might be supplied from cancer cells of a non-metastatic epithelial form through a process referred to as "epithelial– mesenchymal transition" (EMT). Besides tissue stem cells, bone marrow-derived cells (BMDCs) may also represent a potential source of malignancy (Fig. 2). Houghton et al.

A combination of DNA hits and niche microenvironmental factors can transform normal stem cells into primary CSCs or more differentiated cancer progenitor cells. Primary tumors are formed mostly from bulk tumor cells together with a small percentage of CSCs. The accumulation of additional DNA hits plus an altered niche microenvironment may drive primary CSCs to evolve into a genetically distinct population of secondary CSCs. Metastatic CSCs have the potential to proliferate and form metastatic tumors at distant sites composed mostly of bulk tumor cells together with a minority of metastatic CSCs (Takebe & Ivy, 2003).

It remains uncertain whether CSCs originate from normal (somatic) stem cells that acquire oncogenic mutations or from non-stem cells of more differentiated forms that dedifferentiate and acquire stem-cell-like properties through mutation and reprogramming. CSCs can convert into differentiated cells, (Fig. 2) and evidence has suggested that these differentiated cells can acquire stem-cell-like properties via exogenous circumstances (including the niche), with plasticity. (Fig. 2) Cancer stem cells, for example, might be supplied from cancer cells of a non-metastatic epithelial form through a process referred to as "epithelial– mesenchymal transition" (EMT). Besides tissue stem cells, bone marrow-derived cells (BMDCs) may also represent a potential source of malignancy (Fig. 2). Houghton et al.

Fig. 1. CSC development.

**2.2 Origins of CSCs** 

showed that chronic infection of C57BL/6 mice with Helicobacter, a known carcinogen, repopulated the stomach with BMDCs (Houghton et al., 2004). Not long after, these cells progressed through metaplasia and dysplasia to intraepithelial cancer. These findings have broad implications for the multistep model of cancer progression, as they suggest that epithelial cancers can originate from bone-marrow-derived sources. The BMDCs may also be the precursors to CSCs in lung cancer.

Fig. 2. Origins of CSCs

## **2.3 How CSCs can be identified?**

Cancer stem cells are difficult to isolate in solid cancers, though several possible methodologies for attempting isolation are available. The process can be attempted with a surface marker (CD44 or CD133), or with non-adherent cells cultured in a specific condition (sphere-forming), or with side population (SP) cells identified by efflux of dye and an intracellular enzyme activity (aldehyde dehydrogenase, ALDH). As yet, there is no apparent consensus about the 'best marker' by which to identify CSCs. The gold standard assay *in vivo*, the assessment that isolates what most closely fits the definition of CSCs, may be serial transplantation in animal models.

## **2.3.1 Surface marker**

CD44 or CD133 have served as CSC markers in many solid cancers. In a lung cancer study by Eramo et al., some tumors contained a rare population of CD133+ cancer stem-like cells that could both self-renew and generate an unlimited progeny of non-tumorigenic cells (Eramo et al., 2008). Eramo's group found that the tumorigenic cells in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) consisted of rare populations of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. In their cultures, lung cancer CD133+ cells were able to grow indefinitely as 'tumor spheres.' Meanwhile, Bertolini et al. independently reported similar findings using CD133+ cells isolated from 60 samples of human lung cancer (Bertolini et al., 2009). In their experiments, a CD133+ population was increased in primary NSCLC compared with normal lung tissue. Importantly, the expression of CD133 in tumors was linked to shorter progression-free survival of NSCLC patients treated with platinum-based regimens. The proliferative potential, invasiveness, and chemoresistance of CD133+ cells isolated from human lung tumors are reported to depend on the expression of Oct-4 (Chen et al., 2008), a protein important in embryonic stem cell development. Lung cancer CD133+ cells have higher Oct-4 expression, can self-renew, and robustly resist both chemotherapy agents and radiotherapy. Leung et al. found that stem cell-like properties are enriched in CD44+ subpopulations of some lung cancer cell lines, while most cancer cell lines showed no significant CD133 expression (Leung et al., 2010).

Urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation. Both take part in cell migration and invasion under physiological and pathological conditions. uPAR/CD87 was identified as one of the candidate CSC markers in SCLC (Gutova et al., 2007). uPAR+ cells exhibited multi-drug resistance, high clonogenic activity, and co-expression of the putative cancer stem cell markers CD44 and MDR1 in all of the SCLC cell lines examined.

#### **2.3.2 Sphere formation in culture**

Primary cancer cells can be propagated in spheroid cultures (Reynolds & Weiss, 1992 ), and doing so may allow extensive CSC characterization *in vitro*. The cells are grown *in vitro* as tumor spheres under nonadherent conditions using a serum-free medium supplemented with growth factors. Once cultured by this technique, they exhibit high clonogenic potential and readily renew themselves, generate differentiated progeny, and generate tumors *in vivo* (Singh et al., 2003; Ricci-Vitiani et al., 2007). From the major subtypes of lung cancer, 'tumor spheres' were found to possess CSC properties, both *in vitro* (expression of the CSC marker CD133, unlimited proliferative potential, extended abilities to self-renew and differentiate ) and *in vivo* (high tumorigenic potential, capacity to recapitulate tumor heterogeneity and mimic the histology of the specific tumor subtype from which CSCs were derived). Lung cancer 'spheres' are also extremely resistant to most conventional drugs currently used to treat lung cancer patients. This spheroid culture method serves adequately in isolating CSCs from clinical samples.

#### **2.3.3 Side population (SP) on flow cytometry**

SP cells are a subpopulation of cells rich in stem-cell-like characteristics. This subpopulation was first identified by flow-cytometer-based cell sorting defined by Hoechst 33342 dye exclusion (Goodell et al., 1996). Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescentactivated cell sorting dot plots. This test is based on ABCG2 transporter, the second member of the G subfamily of ATP binding cassette (ABC) transporters. ABCG2 is one of the most important multidrug-resistance transporters, and its substrates include Hoeschst 33342 (Ding et al., 1996). SP cells were isolated from several solid cancers and certified to have stemness. Regarding the lung, SP cells isolated from six lung cancer cell lines exhibited higher invasiveness, higher resistance to chemotherapeutic drugs, and higher tumorigenicity *in vivo* compared with non-SP cells (Ho et al., 2007). Most of the SP fraction appeared to be in the G(0) quiescent state. Several SCLC cell lines examined by Salcido et al. contained a consistent SP fraction comprising <1% of the bulk population. SP cells had higher proliferative capacity *in vitro*, were able to efficiently self-renew, and exhibited reduced cell surface expression of differentiation markers. These cells also over-expressed many genes associated with cancer stem cells, drug resistance, and angiogenesis (Salcido et al., 2010).

#### **2.3.4 ALDH activity**

132 Lung Diseases – Selected State of the Art Reviews

Importantly, the expression of CD133 in tumors was linked to shorter progression-free survival of NSCLC patients treated with platinum-based regimens. The proliferative potential, invasiveness, and chemoresistance of CD133+ cells isolated from human lung tumors are reported to depend on the expression of Oct-4 (Chen et al., 2008), a protein important in embryonic stem cell development. Lung cancer CD133+ cells have higher Oct-4 expression, can self-renew, and robustly resist both chemotherapy agents and radiotherapy. Leung et al. found that stem cell-like properties are enriched in CD44+ subpopulations of some lung cancer cell lines, while most cancer cell lines showed no significant CD133

Urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation. Both take part in cell migration and invasion under physiological and pathological conditions. uPAR/CD87 was identified as one of the candidate CSC markers in SCLC (Gutova et al., 2007). uPAR+ cells exhibited multi-drug resistance, high clonogenic activity, and co-expression of the putative cancer stem cell

Primary cancer cells can be propagated in spheroid cultures (Reynolds & Weiss, 1992 ), and doing so may allow extensive CSC characterization *in vitro*. The cells are grown *in vitro* as tumor spheres under nonadherent conditions using a serum-free medium supplemented with growth factors. Once cultured by this technique, they exhibit high clonogenic potential and readily renew themselves, generate differentiated progeny, and generate tumors *in vivo* (Singh et al., 2003; Ricci-Vitiani et al., 2007). From the major subtypes of lung cancer, 'tumor spheres' were found to possess CSC properties, both *in vitro* (expression of the CSC marker CD133, unlimited proliferative potential, extended abilities to self-renew and differentiate ) and *in vivo* (high tumorigenic potential, capacity to recapitulate tumor heterogeneity and mimic the histology of the specific tumor subtype from which CSCs were derived). Lung cancer 'spheres' are also extremely resistant to most conventional drugs currently used to treat lung cancer patients. This spheroid culture method serves adequately in isolating CSCs

SP cells are a subpopulation of cells rich in stem-cell-like characteristics. This subpopulation was first identified by flow-cytometer-based cell sorting defined by Hoechst 33342 dye exclusion (Goodell et al., 1996). Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescentactivated cell sorting dot plots. This test is based on ABCG2 transporter, the second member of the G subfamily of ATP binding cassette (ABC) transporters. ABCG2 is one of the most important multidrug-resistance transporters, and its substrates include Hoeschst 33342 (Ding et al., 1996). SP cells were isolated from several solid cancers and certified to have stemness. Regarding the lung, SP cells isolated from six lung cancer cell lines exhibited higher invasiveness, higher resistance to chemotherapeutic drugs, and higher tumorigenicity *in vivo* compared with non-SP cells (Ho et al., 2007). Most of the SP fraction appeared to be in the G(0) quiescent state. Several SCLC cell lines examined by Salcido et al. contained a consistent SP fraction comprising <1% of the bulk population. SP cells had higher proliferative capacity *in vitro*, were able to efficiently self-renew, and exhibited

markers CD44 and MDR1 in all of the SCLC cell lines examined.

expression (Leung et al., 2010).

**2.3.2 Sphere formation in culture** 

from clinical samples.

**2.3.3 Side population (SP) on flow cytometry** 

Aldehyde dehydrogenase (ALDH) is a detoxifying enzyme known for its role in the oxidation of intracellular aldehydes and for its contribution to the oxidation of retinol to retinoic acid in early stem cell differentiation (Jiang et al., 2009). Class 1 of the ALDH family (ALDH1) is the predominant ALDH isoform in mammals, and ALDH1 activity might serve as a common marker for both normal and malignant stem cell populations. Jiang et al. used the Aldefluor assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1-positive cells from human lung cancer cell lines. The ALDH1-positive cancer cells they isolated exhibited several of the important CSC properties: self-renewal, differentiation, multidrug resistance and expression of stem cell marker *in vitro*; tumor initiation and occurrence of a heterogeneous population of cancer cells *in vivo*. Jiang et al. also found that relatively high ALDH1 protein levels were positively associated with the stage and grade of the tumors, and inversely related to patient survival.

#### **2.3.5 Surface marker may vary even when the cells originate from the same tumor subtype**

The marked heterogeneity within CSC sub-populations underlines the need to find more specific single markers or to define new marker combinations for the prospective isolation of CSCs in solid tumors. CD133 is generally considered a stem cell marker, but CD133 tumors also contain cells with CSC activity. Independent studies have shown that CD133 glioblastoma cells can establish tumors in recipient mice with efficiencies comparable to those of CD133+ cells (Beier et al., 2007). In a study by Meng et al., CD133+ and CD133 subpopulations in lung cancer cells exhibited similar levels of colony formation, selfrenewal, proliferation, differentiation, and invasion, as well as similar resistance to chemotherapy drugs (Meng et al., 2009). As such, these CD133+ and CD133- subpopulations can be assumed to have contained similar numbers of cancer stem cells. In some cases, CD133 is undetectable among lung cancer samples. In a study by Tirino et al., for example, CD133+ was found in only 72% of 89 fresh specimens (Tirino et al., 2009). As to be expected from heterogeneous populations, the CSC phenotype is less than uniform, even when the cells originate from the same tumor subtype. Primary tumors with different genotypes at just one locus can have tumor-propagating cell populations with distinct markers. Fig. 3 (Curtis et al., 2010)

Transgenic mice carrying mutant Kras (left), mutant Kras with p53 deficiency (center), and mutant EGFR (right) all develop lung adenocarcinomas that harbor similar proportions of cells expressing the mouse stem cell marker Sca-1 (blue cells). The tumor-propagating capacity of Sca-1+ and Sca-1- cells from each primary tumor genotype was tested by implanting small numbers of sorted cells into the lungs of recipient mice. When isolated from primary Kras tumors, both Sca-1+ and Sca-1- cells generated secondary tumors that recapitulated the Sca-1 cell heterogeneity found in the primary tumor (left). Yet in tumors with the mutant Kras and p53 deficiency, Sca-1+ cells were better able to form secondary tumors (represented as larger tumors) than Sca-1- cells (center). Further, the secondary tumors derived from Sca-1+ cells harbored Sca-1+ cells in a proportion comparable to the

Fig. 3. Tumor Propagating Capacity of Sca-1+ Lung Cancer Cells

primary tumor, whereas the few small tumors derived from Sca-1 cells had no detectable Sca-1+ population. These data suggest that the Sca-1+ cells in this tumor genotype are enriched in lung cancer stem cells. The opposite appears to be true in mutant EGFR adenocarcinomas, in which Sca-1- cells exhibited a greater capacity for generating secondary tumors (right). The distribution of Sca-1+ cells in secondary mutant EGFR tumors remains undetermined. Adapted with permission from Sullivan JP, Minna JD. Tumor oncogenotypes and lung cancer stem cell identity. Cell Stem Cell. 2010 Jul 2;7(1):2-4.

### **2.4 Serial transplantation is the gold standard to validate CSCs**

Serial transplantation in animal models has been the gold standard to certify stemness, but animal models fail to mimic the human tumor microenvironment as closely as desired. This can be overcome, however, by orthotopic transplantation of candidate cancer-initiating cells back into their normal microenvironment. When working with putative lung cancerinitiating cells, this can be achieved by intratracheal delivery into the lung cavity using a methodology employed for the delivery of an adeno-Cre virus in sporadic murine models of human lung cancer (Meuwissen et al., 2001, 2003). In serial transplantation in animal models, the presence of residual immune effector cells in recipient mice may influence the efficiency of human cell engraftment in NOD/SCID mice. Shultz et al. showed that NOD*scid IL2R*γ*null* (NSG) mice engrafted with human hemopoietic stem cells generate a 6-fold higher percentage of human CD45+ cells in host bone marrow compared to similarly treated NOD-*scid* mice (Shultz et al., 2005).

### **2.5 How do we overcome obstacles of researching CSCs?**

It would be impossible to extensively investigate CSCs without expanding the cell populations *in vitro*. Given the low frequency of lung CSCs within primary tumor tissues, we have difficulty in finding agents which can kill CSCs with strong selective toxicity. There are two methods to surmount this obstacle.

#### **2.5.1 Sphere formation is the best way to obtain CSCs from patients**

This experimental strategy is the best approach so far developed to obtain the unlimited expansion of a tumorigenic lung cancer cell population from primary patients. As such, it

Sca-1+ population. These data suggest that the Sca-1+ cells in this tumor genotype are enriched in lung cancer stem cells. The opposite appears to be true in mutant EGFR adenocarcinomas, in which Sca-1- cells exhibited a greater capacity for generating secondary tumors (right). The distribution of Sca-1+ cells in secondary mutant EGFR tumors remains undetermined. Adapted with permission from Sullivan JP, Minna JD. Tumor oncogenotypes

Serial transplantation in animal models has been the gold standard to certify stemness, but animal models fail to mimic the human tumor microenvironment as closely as desired. This can be overcome, however, by orthotopic transplantation of candidate cancer-initiating cells back into their normal microenvironment. When working with putative lung cancerinitiating cells, this can be achieved by intratracheal delivery into the lung cavity using a methodology employed for the delivery of an adeno-Cre virus in sporadic murine models of human lung cancer (Meuwissen et al., 2001, 2003). In serial transplantation in animal models, the presence of residual immune effector cells in recipient mice may influence the efficiency of human cell engraftment in NOD/SCID mice. Shultz et al. showed that NOD*scid IL2R*γ*null* (NSG) mice engrafted with human hemopoietic stem cells generate a 6-fold higher percentage of human CD45+ cells in host bone marrow compared to similarly treated

It would be impossible to extensively investigate CSCs without expanding the cell populations *in vitro*. Given the low frequency of lung CSCs within primary tumor tissues, we have difficulty in finding agents which can kill CSCs with strong selective toxicity. There

This experimental strategy is the best approach so far developed to obtain the unlimited expansion of a tumorigenic lung cancer cell population from primary patients. As such, it

cells had no detectable

Fig. 3. Tumor Propagating Capacity of Sca-1+ Lung Cancer Cells primary tumor, whereas the few small tumors derived from Sca-1-

and lung cancer stem cell identity. Cell Stem Cell. 2010 Jul 2;7(1):2-4.

**2.4 Serial transplantation is the gold standard to validate CSCs** 

**2.5 How do we overcome obstacles of researching CSCs?** 

**2.5.1 Sphere formation is the best way to obtain CSCs from patients** 

NOD-*scid* mice (Shultz et al., 2005).

are two methods to surmount this obstacle.

serves as a powerful enabler for extensive studies on these cells. Yet CSC spheres are difficult to establish from epithelial tumors, particularly in the case of lung cancer. Indeed, only a few specialized laboratories in the world are able to use CSC spheroids from primary tumors. And as another potential limiting factor, CSCs constitute 5 - 30% of the cells in an average tumor sphere (Eramo et al., 2008).

#### **2.5.2 Inducing CSCs by EMT**

Another potential solution is the generation of 'induced' CSCs (iCSC). The induction of an epithelial-mesenchymal transition (EMT) in normal or neoplastic mammary epithelial cell populations has been shown to enrich the cells with stem-like properties. Gupta et al. demonstrated that normal and cancer cell populations experimentally induced into an EMT also exhibited an increased resistance to chemotherapy drug treatment. When cancer cell populations are induced to pass through an EMT, the proportion of CSCs could increase (Gupta et al., 2009).

#### **2.6 Lung stem cells and lung cancer stem cells**

The lung is a complex organ made up of regionally and functionally distinct cell phenotypes. A diverse class of lung stem cells drives the development and turnover of these populations. The epithelium of the adult airways consists of three distinct compartments arranged along a proximal-distal axis. One factor impeding efforts to demonstrate the existence of adult lung stem cells has been the slow turnover rates in the adult epithelium. Yet in spite of this factor, findings from new studies on pollutant- and pathogen-induced injuries leading to massive lung cell proliferation suggest that adult stem cells are present in each of the epithelial compartments. These different tumor subclasses may arise from distinct cells of origin localized within a defined regional compartment/microenvironment. (Fig. 4)

Fig. 4. Airway stem cell microenvironments and associated human carcinomas.

A schematic diagram of the mouse lung highlighting the spatially distinct cellular environments shown to harbour airway stem/progenitor cells. Candidate epithelial niches (depicted on the right hand side) have been identified and found to exist in spatially defined regions: the tracheal submucosal gland ducts, neuroendocrine bodies (NEB) of the bronchi/bronchioles, and the broncholalveolar duct junction (BADJ). Adapted with permission from Sutherland KD, Berns A. Cell of origin of lung cancer. Mol Oncol. 2010 Oct;4(5):398.

## **2.6.1 Adenocarcinoma**

The distal airways are composed of respiratory bronchioles and alveoli lined with cuboidal epithelium. The bronchioalveolar duct junction (BADJ) has been identified as a microenvironment harboring airway stem cells (Giangreco et al., 2002). With regard to tumorigenicity, Jackson et al. reported that lung tumors were suggested to initiate by oncogenic K-Ras activation appeared to be derived from targeted cells located in the BAD (Fig. 5A) (Jackson et al., 2001). Kim et al. have isolated bronchioalveolar stem cells (BASCs), a regional pulmonary stem cell population, in BADJ, and have identified the candidate origins of CSCs in lung adenocarcinomas (Kim et al., 2005). The BASCs in their experiments resisted bronchiolar and alveolar damage and proliferated during epithelial cell renewal *in vivo*. The BASCs also exhibited self-renewal capability and multipotent properties in clonal assays, and expanded in response to oncogenic K-ras in culture and developed to the lung tumor precursors *in vivo*.

## **2.6.2 Squamaous carcinoma**

The histopathology and gene expression patterns of mouse lung SCC-like lesions frequently resemble those of tracheal basal cell progenitors. This presents the appealing prospect that these are the target cells of origin in this subclass of lung cancer (Fig. 5C). Keratin (K) 5/14 expressing basal cells are located at the submucosal gland duct junctions or intracartilaginous boundaries and are capable of self-renewal, proliferation, and multipotency (Borthwick et al., 2001; Hong et al., 2004a, 2004b; Rawlins et al., 2008; Rock et al., 2009). As such, K 5/14-expressing cells are the putative major airway stem cells. But the clear relationship between basal progenitors and lung SCC has not been established yet.

#### **2.6.3 Small cell carcinoma**

The specific cell population that gives rise, upon genetic alteration, to SCLC remains to be identified. Human and mouse SCLC predominately localize to the midlevel bronchioles and typically express a range of neuroendocrine markers, including calcitonin-gene related peptide (CGRP) and other neuropeptides normally expressed within pulmonary neuroendocrine cells (PNECs) (Meuwissen et al., 2003). Some investigators have hypothesized, based on these observations, that a rare population of PNECs are the progenitors of SCLC. (Fig. 5B) In the mouse lung, microenvironments found in close proximity to neuroepithelial bodies (NEB) have been shown to maintain putative stem cell populations containing both PNECs and variant CCSP-expressing (vCE) cells (Reynolds et al., 2000a). These may be the CSCs of small cell carcinoma.

#### **2.6.4 Human lung stem cells**

Kajstura et al. recently identified a set of potential stem cells in the human lung (Kajstura et al., 2011). These cells were self renewing, clonogenic and multipotent *in vitro*. And when

A schematic diagram of the mouse lung highlighting the spatially distinct cellular environments shown to harbour airway stem/progenitor cells. Candidate epithelial niches (depicted on the right hand side) have been identified and found to exist in spatially defined regions: the tracheal submucosal gland ducts, neuroendocrine bodies (NEB) of the bronchi/bronchioles, and the broncholalveolar duct junction (BADJ). Adapted with permission from Sutherland KD, Berns A. Cell of origin of lung cancer. Mol Oncol. 2010

The distal airways are composed of respiratory bronchioles and alveoli lined with cuboidal epithelium. The bronchioalveolar duct junction (BADJ) has been identified as a microenvironment harboring airway stem cells (Giangreco et al., 2002). With regard to tumorigenicity, Jackson et al. reported that lung tumors were suggested to initiate by oncogenic K-Ras activation appeared to be derived from targeted cells located in the BAD (Fig. 5A) (Jackson et al., 2001). Kim et al. have isolated bronchioalveolar stem cells (BASCs), a regional pulmonary stem cell population, in BADJ, and have identified the candidate origins of CSCs in lung adenocarcinomas (Kim et al., 2005). The BASCs in their experiments resisted bronchiolar and alveolar damage and proliferated during epithelial cell renewal *in vivo*. The BASCs also exhibited self-renewal capability and multipotent properties in clonal assays, and expanded in response to oncogenic K-ras in culture and developed to the lung tumor precursors *in vivo*.

The histopathology and gene expression patterns of mouse lung SCC-like lesions frequently resemble those of tracheal basal cell progenitors. This presents the appealing prospect that these are the target cells of origin in this subclass of lung cancer (Fig. 5C). Keratin (K) 5/14 expressing basal cells are located at the submucosal gland duct junctions or intracartilaginous boundaries and are capable of self-renewal, proliferation, and multipotency (Borthwick et al., 2001; Hong et al., 2004a, 2004b; Rawlins et al., 2008; Rock et al., 2009). As such, K 5/14-expressing cells are the putative major airway stem cells. But the clear relationship between basal progenitors and lung SCC has not been established yet.

The specific cell population that gives rise, upon genetic alteration, to SCLC remains to be identified. Human and mouse SCLC predominately localize to the midlevel bronchioles and typically express a range of neuroendocrine markers, including calcitonin-gene related peptide (CGRP) and other neuropeptides normally expressed within pulmonary neuroendocrine cells (PNECs) (Meuwissen et al., 2003). Some investigators have hypothesized, based on these observations, that a rare population of PNECs are the progenitors of SCLC. (Fig. 5B) In the mouse lung, microenvironments found in close proximity to neuroepithelial bodies (NEB) have been shown to maintain putative stem cell populations containing both PNECs and variant CCSP-expressing (vCE) cells (Reynolds et

Kajstura et al. recently identified a set of potential stem cells in the human lung (Kajstura et al., 2011). These cells were self renewing, clonogenic and multipotent *in vitro*. And when

Oct;4(5):398.

**2.6.1 Adenocarcinoma** 

**2.6.2 Squamaous carcinoma** 

**2.6.3 Small cell carcinoma** 

**2.6.4 Human lung stem cells** 

al., 2000a). These may be the CSCs of small cell carcinoma.

Fig. 5. Schematic overview of the putative role of normal tissue stem/progenitor cells in lung cancer.

(A) BADJ contains a rare cell population that expresses both Clara-specific and alveolarspecific markers. These cells are BASCs. K-Ras activation enhances the proliferation of BASCs. (B) Two hypotheses on the origin of lung NE tumors. NEBs in the epithelial lining of the bronchi harbor PNECs associated with vCEs. The first hypothesis proposes that (i) NE tumors arise from these PNECs. The second hypothesis proposes (ii) that NE hyperplasia and SCLC arise from a less-differentiated progenitor-like cell (for example vCE). (C) Given the basal-like phenotype of SCC, one could hypothesize that squamous cell tumors arise from these basal stem cells. Adapted with permission from Sutherland KD, Berns A. Cell of origin of lung cancer. Mol Oncol. 2010 Oct;4(5):397-403.

injected into a mouse model of lung injury, they regenerated bronchioles, alveoli, smooth muscle, pulmonary vessels and many other lung components. The experiments were performed using c-kit as a stem cell marker. These results are exciting, though rigorous validation will be required. CSCs of the human lung with the potential to differentiate into NSCLCs and SCLCs may originate from not only regional lung stem cells, but also multipotent stem cells.

#### **2.6.5 Analysis of important molecules and pathways of CSCs in the mouse model**

Several papers have reported analyses of important molecules related with CSCs in mouse models.

## **2.6.5.1 Bmi1**

Bmi1 is requisite for K-ras–induced tumorigenesis in the mouse model (Dovey et al., 2008). Loss of Bmi1 in K-ras transgenic mice decreased the prevalence and progression of lung tumors and impaired BADJ stem cell proliferation and self-renewal *in vivo* and *in vitro*.

## **2.6.5.2 PI3K/PTEN/Akt**

The phosphoinositide 3-kinases (PI3K)/phosphatase and tensin homolog (PTEN)/protein kinase B (Akt) pathway is requisite for normal stem cell function. The tumor suppressor PTEN encodes a lipid phosphatase that negatively regulates the PI3K /Akt cell survival pathway. In NSCLC, loss of PTEN protein expression occurs frequently (Marsit et al., 2005).

Fig. 6. Loss of PTEN function results in accumulation of PIP3, which activates a cascade of signaling molecules.

AKT activation inhibits pro-apoptotic factors and stimulates cell cycle progression. The loss of PTEN function leads to increased cell survival and proliferation. RTK, receptor tyrosine kinase. Adapted with permission from Hill R, Wu H. PTEN, stem cells, and cancer stem cells. J Biol Chem. 2009 May 1;284(18):11755-9.

Recent work has linked the PI3K/PTEN/Akt pathway to lung cancer stem cells and to the suppression of K-ras mutations. (Fig. 6) Spontaneous lung adenocarcinomas develop in transgenic mice with an inducible loss of PTEN expression in bronchioalveolar cells (Yanagi et al., 2007). Interestingly, a loss of PTEN expression resulted in K-ras mutations in 33% of mice and developed spontaneous adenocarcinomas. A loss of PTEN expression and PI3K activation may elicit increases in BADJ stem cells, side-population (SP) cells, and the frequency of K-ras mutations, and thereby initiate the development of lung adenocarcinoma over time.

## **2.6.5.3 Hedgehog**

The Hedgehog (Hh) signaling pathway acts as an important regulator of stem cell fates during embryonic development and has been linked to SCLC (Watkins et al., 2003a, 2003b). The observation that intraepithelial Hh signaling is increased after naphthalene-induced airway injury suggests that progenitor cells activate Hh signaling in response to injury. Increased Hh signaling is also observed in the lungs of PTEN-deficient mice that develop spontaneous lung adenocarcinomas in conjunction with BADJ stem cell expansion, and thus may play a causal role in this process (Yanagi et al., 2007).

## **2.6.5.4 Wnt**

138 Lung Diseases – Selected State of the Art Reviews

Bmi1 is requisite for K-ras–induced tumorigenesis in the mouse model (Dovey et al., 2008). Loss of Bmi1 in K-ras transgenic mice decreased the prevalence and progression of lung tumors and impaired BADJ stem cell proliferation and self-renewal *in vivo* and *in vitro*.

The phosphoinositide 3-kinases (PI3K)/phosphatase and tensin homolog (PTEN)/protein kinase B (Akt) pathway is requisite for normal stem cell function. The tumor suppressor PTEN encodes a lipid phosphatase that negatively regulates the PI3K /Akt cell survival pathway. In NSCLC, loss of PTEN protein expression occurs frequently (Marsit et al., 2005).

Fig. 6. Loss of PTEN function results in accumulation of PIP3, which activates a cascade of

AKT activation inhibits pro-apoptotic factors and stimulates cell cycle progression. The loss of PTEN function leads to increased cell survival and proliferation. RTK, receptor tyrosine kinase. Adapted with permission from Hill R, Wu H. PTEN, stem cells, and cancer stem

Recent work has linked the PI3K/PTEN/Akt pathway to lung cancer stem cells and to the suppression of K-ras mutations. (Fig. 6) Spontaneous lung adenocarcinomas develop in transgenic mice with an inducible loss of PTEN expression in bronchioalveolar cells (Yanagi et al., 2007). Interestingly, a loss of PTEN expression resulted in K-ras mutations in 33% of mice and developed spontaneous adenocarcinomas. A loss of PTEN expression and PI3K activation may elicit increases in BADJ stem cells, side-population (SP) cells, and the frequency of K-ras mutations, and thereby initiate the development of lung adenocarcinoma

The Hedgehog (Hh) signaling pathway acts as an important regulator of stem cell fates during embryonic development and has been linked to SCLC (Watkins et al., 2003a, 2003b). The observation that intraepithelial Hh signaling is increased after naphthalene-induced airway injury suggests that progenitor cells activate Hh signaling in response to injury. Increased Hh signaling is also observed in the lungs of PTEN-deficient mice that develop

**2.6.5.1 Bmi1** 

**2.6.5.2 PI3K/PTEN/Akt** 

signaling molecules.

over time.

**2.6.5.3 Hedgehog** 

cells. J Biol Chem. 2009 May 1;284(18):11755-9.

The Wnt developmental pathway is an another critical regulator of embryonic lung stem cells (Reynolds et al., 2008; Zhang et al., 2008). In adult mice, conditional deletion of p38+, a known downstream target of noncanonical Wnt signaling (Ma & Wang, 2007), leads to an expansion of CCSP+SP-C+ stem cells, hyperproliferation, and increased sensitization to Kras–induced tumorigenesis (Ventura et al., 2007).

## **2.7 Therapeutic strategy against CSCs**

CSCs are thought to be responsible for disease relapse or metastasis, and also for resistance to radiation or conventional chemotherapy. CSCs showed increased quiescence *in vivo* and *in vitro*, which suggests that they may respond poorly to conventional treatments designed to mainly kill the proliferating cells or terminally differentiated cells forming the bulk of tumors (Guan et al., 2003). Several therapies against CSCs have been considered, but none have been fully developed for lung cancer CSCs. The remainder of this chapter will discuss strategies for targeting CSCs in various organs.

#### **2.7.1 Targeting the key molecule (stem cell marker), key gene & key signaling pathways**

Given that CSCs share many of the same features as normal stem cells, there is a potential risk of killing normal stem cells while targeting CSCs. It thus becomes important to further characterize the similarities and differences between these two types of stem cells. New therapeutic approaches to selectively target CSC-specific markers, genes, and pathways are needed.

#### **2.7.1.1 Antibody-based treatment against surface markers**

One recently introduced therapeutic approach for human AML employs an activating mAb directed to the adhesion molecule CD44, a known CSC marker of AML (Jin et al., 2006). The *in vivo* administration of this Ab to NOD/SCID mice transplanted with human AML markedly reduced leukemic repopulation. Mechanistically, CD44-specific Ab treatment induced differentiation to a more mature cancer cell progeny that were unable to establish robust leukemia upon xenotransplantation. For solid tumors, CD13, a CSC marker of hepatocellular carcinoma, is expected to become a target of CSC therapy (Haraguchi et al., 2010).

#### **2.7.1.2 Inhibition of essential pathways in the CSCs function**

The eventual goal is to generate targeted therapeutics that inhibit essential pathways in the CSC fraction. It will probably be complicated to target these pathways, as the same pathways are also pivotal in normal stem cell function. The Wnt pathway, hedgehog pathway, and notch pathway are all reportedly essential for maintaining stemness. The blocking of these pathways thus holds promise as a therapeutic approach. Many reports and target drugs have been published and developed in pursuit of such a therapy, but only few of these reports have touched upon lung cancer CSCs. The hedgehog pathway may play an important role in lung CSCs, as the pharmacological inhibition of this pathway reduced the growth of lung tumor cells in xenograft models (Watkins et al., 2003a). Suppression of the Notch pathway by treatment with either a -secretase inhibitor or stable expression of shRNA against NOTCH3 significantly decreased ALDH+ lung cancer cells, and commensurate reductions in tumor cell proliferation and clonogenicity were observed (Sullivan et al., 2010). Notch signaling thus appears to take part in lung cancer stem cell maintenance.

#### **2.7.1.3 Blocking of the stemness gene**

CD133+ cells in lung cancer exhibit higher Oct-4 expression. Oct-4 plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of CD133+ cells. Knock-down of Oct-4 expression can significantly inhibit the abilities of tumor invasion and colony formation, increase apoptotic activities, and enhance the treatment effect of chemoradiotherapy (Chen et al., 2008). The downregulation of transcription factor SOX in lung CSCs reportedly suppresses growth and metastasis (Xiang et al., 2011), and the blocking of the SCF/c-kit signaling pathway inhibits CSC proliferation and survival after chemotherapy exposure in human lung cancer cell lines (Levina et al., 2010). Cisplatin treatment eliminated most of the tumor cells, but unlike the blocking of c-kit, it was unsuccessful in eliminating CSCs. A combination treatment with cisplatin and c-kit blocking prevented the growth of both tumor cell subpopulations.

#### **2.7.2 Regulation of micro RNA**

Micro RNAs can affect the signaling pathways that influence stem cell self-renewal. A lack of let-7 is required for self renewal *in vitro* and for tumorigenicity *in vivo*. In other words, an overexpression of let-7a reduces self renewal and proliferative capacity and converts highly malignant and metastasizing CSCs into less malignant cells (Yu et al., 2007).

## **2.7.3 Induction of diffentiation of CSCs**

Differentiation therapy aims at converting tumorigenic CSCs in their non-tumorigenic progeny. Treatment with bone morphogenic protein 4 (BMP4) reduced the tumor-initiating cell pool in a glioma model and markedly slowed down tumor growth *in vivo* without toxic side effects (Piccirillo et al., 2006).

#### **2.7.4 Altering the CSCs' environment (niche)**

The stem cell niche plays an important role in maintaining CSCs and apparently enhances the therapy resistance of CSCs by sheltering the cells from diverse insults (Folkins et al., 2007). Perivascular, hypoxic, premetastatic and stromal myofibroblast niches have all been reported. The perivascular niche in brain tumors has been shown to contribute directly to the generation of CSCs and tumor growth. Anti-angiogenic therapy using vascular endothelial growth factor inhibitors not only depleted tumor vascularization, but also ablated CSCs in the xenograft (Calabrese et al., 2007). While some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients (Parmar et al., 2007). However, the underlying mechanisms are still unclear. O2 availability may have a direct role in stem cell regulation through the HIF-1 modulation of Wnt/-catenin signaling (Mazumdar et al., 2010). These niches are expected to become targets of CSC therapy. In experiments with colon adenocarcinomas, Vermeulen et al. found that hepatocyte growth factor and other myofibroblast-secreted factors activate CSC clonogenicity (Vermeulen et al., 2010). More significantly, myofibroblast-secreted factors

Notch pathway by treatment with either a -secretase inhibitor or stable expression of shRNA against NOTCH3 significantly decreased ALDH+ lung cancer cells, and commensurate reductions in tumor cell proliferation and clonogenicity were observed (Sullivan et al., 2010). Notch signaling thus appears to take part in lung cancer stem cell

CD133+ cells in lung cancer exhibit higher Oct-4 expression. Oct-4 plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of CD133+ cells. Knock-down of Oct-4 expression can significantly inhibit the abilities of tumor invasion and colony formation, increase apoptotic activities, and enhance the treatment effect of chemoradiotherapy (Chen et al., 2008). The downregulation of transcription factor SOX in lung CSCs reportedly suppresses growth and metastasis (Xiang et al., 2011), and the blocking of the SCF/c-kit signaling pathway inhibits CSC proliferation and survival after chemotherapy exposure in human lung cancer cell lines (Levina et al., 2010). Cisplatin treatment eliminated most of the tumor cells, but unlike the blocking of c-kit, it was unsuccessful in eliminating CSCs. A combination treatment with cisplatin and c-kit blocking

Micro RNAs can affect the signaling pathways that influence stem cell self-renewal. A lack of let-7 is required for self renewal *in vitro* and for tumorigenicity *in vivo*. In other words, an overexpression of let-7a reduces self renewal and proliferative capacity and converts highly

Differentiation therapy aims at converting tumorigenic CSCs in their non-tumorigenic progeny. Treatment with bone morphogenic protein 4 (BMP4) reduced the tumor-initiating cell pool in a glioma model and markedly slowed down tumor growth *in vivo* without toxic

The stem cell niche plays an important role in maintaining CSCs and apparently enhances the therapy resistance of CSCs by sheltering the cells from diverse insults (Folkins et al., 2007). Perivascular, hypoxic, premetastatic and stromal myofibroblast niches have all been reported. The perivascular niche in brain tumors has been shown to contribute directly to the generation of CSCs and tumor growth. Anti-angiogenic therapy using vascular endothelial growth factor inhibitors not only depleted tumor vascularization, but also ablated CSCs in the xenograft (Calabrese et al., 2007). While some stem cells are perivascular, others may occupy hypoxic niches and be regulated by O2 gradients (Parmar et al., 2007). However, the underlying mechanisms are still unclear. O2 availability may have a direct role in stem cell regulation through the HIF-1 modulation of Wnt/-catenin signaling (Mazumdar et al., 2010). These niches are expected to become targets of CSC therapy. In experiments with colon adenocarcinomas, Vermeulen et al. found that hepatocyte growth factor and other myofibroblast-secreted factors activate CSC clonogenicity (Vermeulen et al., 2010). More significantly, myofibroblast-secreted factors

malignant and metastasizing CSCs into less malignant cells (Yu et al., 2007).

maintenance.

**2.7.1.3 Blocking of the stemness gene** 

**2.7.2 Regulation of micro RNA** 

side effects (Piccirillo et al., 2006).

**2.7.3 Induction of diffentiation of CSCs** 

**2.7.4 Altering the CSCs' environment (niche)** 

prevented the growth of both tumor cell subpopulations.

restored the CSC phenotype in more differentiated tumor cells both *in vitro* and *in vivo*. Vermeulen's group therefore propose that the stemness of colon cancer cells is in part orchestrated by the microenvironment.

#### **2.7.5 Activation of reactive oxygen species (ROS)**

CSCs have the ability to keep ROS levels low. Subsets of CSCs in some tumors contain lower ROS levels and enhanced ROS defenses compared to their non-tumorigenic progeny. This may contribute to tumor radioresistance. Overcoming low ROS levels within CSCs may be a useful method for improving local and systemic oncologic therapies (Diehn et al., 2009). Haraguchi et al. found that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, a combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. Thus, the combination of a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer (Haraguchi et al., 2010). CD44 is an adhesion molecule expressed in cancer stem-like cells in gastric cancer. Ishimoto et al. showed that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH) (Ishimoto et al., 2011). Human gastrointestinal cancer cells with strong CD44 expression showed an enhanced capacity for GSH synthesis and defense against ROS. Ablation of CD44 induced a loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. The activation of ROS may be viable as another target therapy for CSCs.

#### **2.7.6 Overcoming of chemoresistance and radioresistance in CSCs**

Reversing chemoresistance in CSC populations can be achieved through a specific blockade of multidrug resistance ABC transporters (Frank et al., 2005). Enhanced drug efflux mediated by ABCB1 P-glycoprotein and related ATP-binding cassette transporters is one of several mechanisms of multidrug resistance thought to impair chemotherapeutic success in human cancers. In CD133+ CSC in malignant melanoma, ABCB5, a novel human ABC transporter, mediates melanoma resistance to the chemotherapeutic agent doxorubicin, and this effect is reversible by both mAb-mediated inhibition of ABCB5-dependent drug efflux. In addition, ABCB5 gene silencing substantially increases the sensitivity of human melanoma cells to the anticancer chemotherapeutics 5-fluorouracil (5-FU) and camptothecin (Huang et al., 2004). CSC-targeted therapeutic approaches might also include strategies directed at reversal of radioresistance. Bao et al. reported that CD133+ human glioma CSCs contributed to tumor radioresistance by preferentially activating the DNA damage checkpoint response and enhancing the DNA repair capacity (Bao et al., 2006). An inhibition of the Chk1 and Chk2 checkpoint kinases reversed the radioresistance of CD133+ glioma CSCs in their experiments.

#### **2.7.7 Supressing EMT and inducing MET**

EMT in carcinoma seems to be associated with the acquisition of a CSC phenotype endowed with a more invasive and metastatic phenotype. As such, a new drug to suppress EMT is expected as a target therapy for CSCs. Metastatic progression might involve the dissemination of CSCs at tumor margins that have undergone EMT. Thus, the mesenchymal-epithelial transition (MET) seems to hold promise as a therapy. Gupta et al. identified small molecules (salinomycin) that specifically inhibit cancer stem cell proliferation through the induction of MET (Gupta et al., 2009).

#### **3. Conclusion**

Though targeted therapies have been developed, we have witnessed only limited improvement in the prognosis of lung cancer patients. Ultimately, patient cure will require the eradication of all cells within a cancer. From this standpoint, combination therapies targeting both CSCs and bulk cancer populations hold promise. In the coming years we must clarify the origin of CSCs, find more specific CSC markers, elucidate the CSC niche, and develop more effective innovative agents against resistant tumorigenic lung CSCs. CSCs may vary in different lung cancers, so personalized CSC therapy may be needed.

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## **Oncogenes and Tumor Suppressor Genes in Small Cell Lung Carcinoma**

Pankaj Taneja1,2, Robert D. Kendig1,2, Sinan Zhu1,3, Dejan Maglic1,2,3,

Elizabeth A. Fry1,2 and Kazushi Inoue1,2,3,\* *1The Departments of Pathology, 2Cancer Biology, 3Graduate Program in Molecular Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem USA* 

## **1. Introduction**

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epithelial progenitors and in small-cell lung cancer. *Nature,* 422, 6929, (Mar 20), pp.

cancer stem cells suppresses growth and metastasis of lung cancer. *Br J Cancer,* 104,

bronchioalveolar stem cells, and onset of lung adenocarcinomas in mice. *J Clin*

stem cell development and airway regeneration. *Nat Genet,* 40, 7, (Jul), pp. 862-70,

Small cell lung cancer (SCLC) makes up almost 15% of all cases of lung cancer and occurs almost exclusively in individuals with a history of smoking (Blackhall & Faivre-Finn, 2011; Meyerson et al., 2004; Tamasi and Muller, 2011; Walenkamp et al., 2009). However, SCLCs differ significantly from NSCLCs in specific genetic alterations that occur. Moreover, smoking-damaged bronchial epithelia accompanying SCLCs appears to have undergone significantly more acquired genetic damage than is frequently found in NSCLCs. Two subtypes of SCLC exist: homogeneous small cell carcinoma and combined SCLC (mixture of any non-small cell type) (Meyerson et al., 2004; Tamasi and Muller, 2011). SCLC in its advanced stage has an aggressive clinical course and is commonly accompanied by paraneoplastic syndromes. Autocrine growth factors, such as neuroendocrine regulatory peptides (e.g. bombesin/gastrin-releasing peptide), are prominent in SCLC.

SCLC is categorized as limited stage disease (LS) when confined to the ipsilateral hemithorax and within a single radiation port, while extensive stage disease (ES) includes metastatic disease outside the ipsilateral hemithorax (Blackhall & Faivre-Finn, 2011; Meyerson et al., 2004; Tamasi and Muller, 2011; Walenkamp et al., 2009). SCLC is sensitive to chemotherapy; response rates to front-line agents are often in the range of 60%, with approximately 10% of patients achieving a complete response, even in the setting of metastatic disease (Brambilla et al., 2009 Jemal et al., 2006). Despite this, the relapse rates are quite high and survival with currently available salvage therapy is quite modest. With current therapy, patients with LS-SCLC have a median survival of 17 months and a 5-year overall survival rate of 12% , while patients with ES-SCLC have a median survival of 8.9 months, and a 5-year survival rate of approximately 2%. (Brambilla et al., 2009 Jemal et al., 2006; Tamasi and Muller, 2011). This article will review the molecular targeted agents, the genetic abnormalities, and therapeutic efficacy in SCLC.

<sup>\*</sup> Corresponding Author

## **2.** *Trp53* **gene**

The *p53* gene located on chromosome 17p13.1 encodes a nuclear protein that acts as a transcription factor and causes cell cycle arrest or apoptosis. Mutations of this gene lead to the loss of tumor suppressor function, thereby promoting cellular proliferation. The majority of the mutations seen in lung cancers are G to T transversions on the non-transcribed strand, suggesting mutagenesis secondary to tobacco smoke. Wild-type p53 protein is present in very low levels in normal cells, whereas mutant p53 is present in much greater quantities in tumor cells due to its prolonged half-life. In fact, 40–70% of SCLC express abnormal p53 protein (Wistuba et al., 2001). Inactivating mutations of *p53*, seen in approximately 90% of SCLC, are typically missense mutations in the DNA binding domain and, to a lesser degree, homozygous deletions (Demirhan et al., 2010). Preclinical studies have shown that a vaccine composed of dendritic cells transduced with a human wild-type *P53* containing recombinant adenovirus (DC-Ad-p53) causes an antitumor response (Ishihda et al., 1999). The differential expression of the mutant *P53* gene between normal and tumor cells could provide a basis for vaccine therapy.

Antonia et al. (2006) treated 29 patients with relapsed/refractory ES-SCLC with a vaccine consisting of dendritic cells transduced with the full-length wild-type *P53* gene delivered via an adenoviral vector. Only one patient showed a clinical response to the vaccine therapy. Interestingly, there was a high rate of objective clinical responses to chemotherapy (61.9%) that immediately followed vaccination. This clinical response to subsequent chemotherapy was closely associated with induction of immunologic response to vaccination. Hence, it is likely that vaccine therapy could serve as an adjunct to chemotherapy, rather than a primary treatment modality. Using this rationale, a current phase I/II trial is evaluating an autologous dendritic cell-adenovirus p53 vaccine following standard platinum-etoposide chemotherapy in patients with ES- SCLC (Horn et al., 2011). Another approach towards vaccination therapy is to target the ganglioside GD3, a cell surface glycosphingolipid antigen that is typically expressed on cells of neuroectodermal origin and a subset of Tlymphocytes (Grant et al., 1999). Studies using SCLC cell lines suggest that these cell lines express significant levels of GD3. The anti-idiotypic antibody BEC-2, which mimics the structure of the GD3 ganglioside, showed promising results in a pilot study (Grant et al., 1999). Based on these results, the European Organization for Research and Treatment of Cancer (EORTC) performed a randomized phase III study in 515 patients with LS-SCLC who were randomized to receive BEC-2 or not as a maintenance treatment following standard induction chemotherapy (Giaccone et al., 2005). Although there was no improvement in overall survival, progression-free survival, or quality of life in the vaccination arm, there was a trend toward prolonged survival in patients who had a humoral response. The authors concluded that vaccination strategies may be warranted with vaccines that were better able to induce a humoral immune response.

Yang et al. (2011) evaluated the role of genetic *P53* polymorphism in radiation-induced pneumonitis (RP), a common dose-limiting toxicity of radiotherapy. In a cohort comprised of 253 (188 NSCLC and 65 SCLC) lung cancer patients receiving thoracic irradiation, the *P53* 72Arg/Arg genotype was associated with increased radiation-induced pneumonitis risk compared with the 72Pro/Pro genotype. Furthermore, the *P53* Arg72Pro and ATM-111G>A polymorphisms displayed an additive effect in intensifying the risk of developing RP. The cross-validation test showed that 63.2% of RP cases could be identified by *P53* and *ATM* genotypes. Thus, genotyping *P53* and *ATM* polymorphisms might help proactively identify

The *p53* gene located on chromosome 17p13.1 encodes a nuclear protein that acts as a transcription factor and causes cell cycle arrest or apoptosis. Mutations of this gene lead to the loss of tumor suppressor function, thereby promoting cellular proliferation. The majority of the mutations seen in lung cancers are G to T transversions on the non-transcribed strand, suggesting mutagenesis secondary to tobacco smoke. Wild-type p53 protein is present in very low levels in normal cells, whereas mutant p53 is present in much greater quantities in tumor cells due to its prolonged half-life. In fact, 40–70% of SCLC express abnormal p53 protein (Wistuba et al., 2001). Inactivating mutations of *p53*, seen in approximately 90% of SCLC, are typically missense mutations in the DNA binding domain and, to a lesser degree, homozygous deletions (Demirhan et al., 2010). Preclinical studies have shown that a vaccine composed of dendritic cells transduced with a human wild-type *P53* containing recombinant adenovirus (DC-Ad-p53) causes an antitumor response (Ishihda et al., 1999). The differential expression of the mutant *P53* gene between normal and tumor cells could

Antonia et al. (2006) treated 29 patients with relapsed/refractory ES-SCLC with a vaccine consisting of dendritic cells transduced with the full-length wild-type *P53* gene delivered via an adenoviral vector. Only one patient showed a clinical response to the vaccine therapy. Interestingly, there was a high rate of objective clinical responses to chemotherapy (61.9%) that immediately followed vaccination. This clinical response to subsequent chemotherapy was closely associated with induction of immunologic response to vaccination. Hence, it is likely that vaccine therapy could serve as an adjunct to chemotherapy, rather than a primary treatment modality. Using this rationale, a current phase I/II trial is evaluating an autologous dendritic cell-adenovirus p53 vaccine following standard platinum-etoposide chemotherapy in patients with ES- SCLC (Horn et al., 2011). Another approach towards vaccination therapy is to target the ganglioside GD3, a cell surface glycosphingolipid antigen that is typically expressed on cells of neuroectodermal origin and a subset of Tlymphocytes (Grant et al., 1999). Studies using SCLC cell lines suggest that these cell lines express significant levels of GD3. The anti-idiotypic antibody BEC-2, which mimics the structure of the GD3 ganglioside, showed promising results in a pilot study (Grant et al., 1999). Based on these results, the European Organization for Research and Treatment of Cancer (EORTC) performed a randomized phase III study in 515 patients with LS-SCLC who were randomized to receive BEC-2 or not as a maintenance treatment following standard induction chemotherapy (Giaccone et al., 2005). Although there was no improvement in overall survival, progression-free survival, or quality of life in the vaccination arm, there was a trend toward prolonged survival in patients who had a humoral response. The authors concluded that vaccination strategies may be warranted

with vaccines that were better able to induce a humoral immune response.

Yang et al. (2011) evaluated the role of genetic *P53* polymorphism in radiation-induced pneumonitis (RP), a common dose-limiting toxicity of radiotherapy. In a cohort comprised of 253 (188 NSCLC and 65 SCLC) lung cancer patients receiving thoracic irradiation, the *P53* 72Arg/Arg genotype was associated with increased radiation-induced pneumonitis risk compared with the 72Pro/Pro genotype. Furthermore, the *P53* Arg72Pro and ATM-111G>A polymorphisms displayed an additive effect in intensifying the risk of developing RP. The cross-validation test showed that 63.2% of RP cases could be identified by *P53* and *ATM* genotypes. Thus, genotyping *P53* and *ATM* polymorphisms might help proactively identify

**2.** *Trp53* **gene** 

provide a basis for vaccine therapy.

patients susceptible to developing RP when receiving radiotherapy. A recent report by Garcia and co-workers (2010) describe the association of SCLC with ovarian metastases. A 54-year-old woman with SCLC presented with a left ovarian mass, 4.8 cm in diameter, the microscopic appearance of which was identical to the previous bronchoscopic biopsy. Molecular analysis of *P53* demonstrated an identical point mutation (S215) in both tumor sites. Moreover, a *P53* DNA polymorphism (P52R) was identified in normal tissue, but present in homozygosity in both tumor sites.

#### **3. Retinoblastoma (***RB***) gene**

The *RB* gene, located on chromosome 13q14.11, has been implicated in the regulation of cell cycle progression, particularly the G1 to S-phase transition, in part, through inactivation of members of the E2F transcription factor family (Modi et al., 2000; Schaffer et al., 2010; Wikman et al., 2006). Hypophosphorylated RB is the growth suppressing form that controls the transcription factors E2F1, E2F2 and E2F3, which are necessary for the G1/S transition. (Modi et al., 2000; Wikman et al., 2006). When bound to hypophosphorylated RB, E2F is in its inactive form, causing cell arrest in the G1 phase. The cyclin D1/CDK4 complex phosphorylates RB, which in turn releases E2F, allowing its activation and promoting entry into S phase. During S phase, cyclin E and CDK2 assert control over the phosphorylation of RB (Wikman et al., 2006; Xue et al., 2003). Inactivation of pRB by gross structural alterations or point mutations in the *RB-1* gene has been described in >90% of all SCLC (Wikman et al., 2006; Xue et al., 2003). The types of mutations that occur in the *RB* gene include deletions, nonsense mutations and splicing abnormalities. Phosphorylated RB suppresses apoptosis by repressing other pro-apoptotic target genes, including apoptotic protease activating factor-1 (Apaf-1) and caspases.

There have been numerous reports of RB protein expression in lung cancer (Wikman & Kettunen, 2006). Similar to human tumor entities in general, different lung cancer types show extensively varying expression patterns: SCLC and large cell neuroendocrine carcinomas (LCNEC) are mostly characterized by loss of RB expression (~90%). Conversely, altered expression of RB is rare (~25%) in squamous cell carcinoma (SCC) and adenocarcinoma (AC), which comprise most cases of NSCLC (Leversha et al., 2003; Gouyer et al., 1998). Rather, NSCLC is attributed to *p16INK4a* loss or CCND1 overexpression (Schauer et al., 1994). Interestingly, a few cases of SCLC display p16INK4a alterations, but retain normal RB. Preinvasive bronchial lesions and carcinoid tumors also rarely exhibit abnormal RB expression. It is thought that SCLC and other neuroendocrine lung tumors originate from the progenitor neural crest that is similar to the origin of RB.

*RB* is altered by mutations (20–30%) or small deletions and chromosomal loss (80–90%) in SCLC (Mori et al., 1990; Kashii et al., 1994). Even though 58% of neuroendocrine lung tumors show low or absent mRNA levels, no hypermethylation has been observed in these tumors. In NSCLC, some reports have detected no *RB* DNA alterations, whereas others, somewhat controversially, have found frequent loss of heterozygosity (LOH) (up to 75%) and mutations (33%) of *RB* in SCC (Gouyer et al., 1998; Leversha et al., 2003). DNA Alterations of *RB* have rarely bee described in AC (Sachse et al., 1994). In summary, complete loss of *RB* or a mutant form of *RB* are present in greater than 90% of SCLC cases (Modi et al., 2000). As all normal cells express functional RB, drugs that target cells with inactived or deleted *RB* would be appropriate candidates for testing in patients with SCLC. Such drugs include heat shock protein-90 (Hsp90) inhibitors (Rodina et al., 2007).

## **4.** *BCL2* **gene**

*BCL2* is an oncogene that plays a major role in suppressing apoptosis and thus in treatment resistance (Ilievska et al., 2008; Tudor et al., 2000). Therefore, suppression of BCL2 may increase therapeutic efficacy (Ilievska et al., 2008; Lawson et al., 2010; Ziegler et al., 1997). Since BCL2 is expressed in the vast majority of SCLC cases, it represents a potential therapeutic target in this disease. G3139 (oblimersen) is an 18-base antisense phosphorothioate oligonucleotide complementary to the *BCL2* mRNA in the region encoding the first six amino acids (Reed et al., 1990). Preclinical and clinical studies have demonstrated that intravenous administration of G3139 reduces BCL2 protein production (Waters et al., 2000). The combination of oblimersen and paclitaxel was evaluated in a phase II trial of 12 patients with chemo-refractory SCLC (Rudin et al., 2002). There were no objective responses, but four patients had stable disease. The low yield of this small study was attributed to the relatively low doses of both agents; the dose of oblimersen chosen may have been insufficient to suppress expression of the target gene *BCL2*, while paclitaxel had to be given at a dose clearly below that routinely used. Also, since inhibition of BCL2 expression may increase therapeutic efficacy of cytotoxic agents, this strategy may be more beneficial in patients who responded positively to chemotherapy. In order to test this hypothesis, Rudin et al. (2004) combined oblimersen with etoposide and carboplatin in 16 patients with newly diagnosed extensive stage SCLC. This combination yielded promising results with reasonable toxicity and is currently being studied in a randomized phase III trial to define an exact role for this molecule. Additionally, a small molecule BCL2 inhibitor, AT101, is currently being evaluated in combination with topotecan in patients with relapsed/refractory SCLC. ABT-263 has also been identified as a Bcl-2 inhibitor in numerous SCLC and leukemia/lymphoma cell lines *in vitro* and *in vivo* (Tahir et al., 2010). In another study, Knoefel et al. (2011) studied the single-nucleotide polymorphism C-938A to assess the potential impact as a genetic marker for response to chemotherapy and outcome prediction in 188 Caucasian SCLC patients. Patients carrying the *BCL2-938CC* genotype showed significantly worse time to progression and overall survival than those with the *BCL2- 938AA* genotype. This genetic marker might particularly impact on treatment strategies using *BCL2* antisense approaches.

## *5. MYC* **genes**

*c-MYC, N-MYC* and *L-MYC* are proto-oncogenes that code for proteins involved in the regulation of proliferation, differentiation, and apoptosis (Komiya et al., 2011; Paulson et al., 2009; Xion et al., 2011). Amplification of the *c-MYC* oncogene has been observed in various human malignancies (Komiya et al., 2011). Studies in small cell lung cancer have suggested that although amplification of the *MYC* family genes is seen in only about 10% of patients with newly diagnosed SCLC, this proportion increases following treatment (Komiya et al., 2011; Paulson et al., 2009; Xion et al., 2011 ; Barr et al., 1998), suggesting that MYC expression increases with resistance of SCLC to therapy. Other studies have suggested that higher levels of expression of the *MYC* gene family may play a significant role in the carcinogenesis of SCLC (Kumimoto et al., 2002). Protein overexpression is seen with *MYC*  activation by gene amplification or transcriptional dysregulation (Komiya et al., 2011). In addition, amplification of these genes may have a predictive value, since tumors with *N-MYC* amplification have been associated with poor response to chemotherapy, rapid tumor growth, and short survival (Komiya et al., 2011).

## **6. c-KIT receptor**

150 Lung Diseases – Selected State of the Art Reviews

*BCL2* is an oncogene that plays a major role in suppressing apoptosis and thus in treatment resistance (Ilievska et al., 2008; Tudor et al., 2000). Therefore, suppression of BCL2 may increase therapeutic efficacy (Ilievska et al., 2008; Lawson et al., 2010; Ziegler et al., 1997). Since BCL2 is expressed in the vast majority of SCLC cases, it represents a potential therapeutic target in this disease. G3139 (oblimersen) is an 18-base antisense phosphorothioate oligonucleotide complementary to the *BCL2* mRNA in the region encoding the first six amino acids (Reed et al., 1990). Preclinical and clinical studies have demonstrated that intravenous administration of G3139 reduces BCL2 protein production (Waters et al., 2000). The combination of oblimersen and paclitaxel was evaluated in a phase II trial of 12 patients with chemo-refractory SCLC (Rudin et al., 2002). There were no objective responses, but four patients had stable disease. The low yield of this small study was attributed to the relatively low doses of both agents; the dose of oblimersen chosen may have been insufficient to suppress expression of the target gene *BCL2*, while paclitaxel had to be given at a dose clearly below that routinely used. Also, since inhibition of BCL2 expression may increase therapeutic efficacy of cytotoxic agents, this strategy may be more beneficial in patients who responded positively to chemotherapy. In order to test this hypothesis, Rudin et al. (2004) combined oblimersen with etoposide and carboplatin in 16 patients with newly diagnosed extensive stage SCLC. This combination yielded promising results with reasonable toxicity and is currently being studied in a randomized phase III trial to define an exact role for this molecule. Additionally, a small molecule BCL2 inhibitor, AT101, is currently being evaluated in combination with topotecan in patients with relapsed/refractory SCLC. ABT-263 has also been identified as a Bcl-2 inhibitor in numerous SCLC and leukemia/lymphoma cell lines *in vitro* and *in vivo* (Tahir et al., 2010). In another study, Knoefel et al. (2011) studied the single-nucleotide polymorphism C-938A to assess the potential impact as a genetic marker for response to chemotherapy and outcome prediction in 188 Caucasian SCLC patients. Patients carrying the *BCL2-938CC* genotype showed significantly worse time to progression and overall survival than those with the *BCL2- 938AA* genotype. This genetic marker might particularly impact on treatment strategies

*c-MYC, N-MYC* and *L-MYC* are proto-oncogenes that code for proteins involved in the regulation of proliferation, differentiation, and apoptosis (Komiya et al., 2011; Paulson et al., 2009; Xion et al., 2011). Amplification of the *c-MYC* oncogene has been observed in various human malignancies (Komiya et al., 2011). Studies in small cell lung cancer have suggested that although amplification of the *MYC* family genes is seen in only about 10% of patients with newly diagnosed SCLC, this proportion increases following treatment (Komiya et al., 2011; Paulson et al., 2009; Xion et al., 2011 ; Barr et al., 1998), suggesting that MYC expression increases with resistance of SCLC to therapy. Other studies have suggested that higher levels of expression of the *MYC* gene family may play a significant role in the carcinogenesis of SCLC (Kumimoto et al., 2002). Protein overexpression is seen with *MYC*  activation by gene amplification or transcriptional dysregulation (Komiya et al., 2011). In addition, amplification of these genes may have a predictive value, since tumors with *N-MYC* amplification have been associated with poor response to chemotherapy, rapid tumor

**4.** *BCL2* **gene** 

using *BCL2* antisense approaches.

growth, and short survival (Komiya et al., 2011).

*5. MYC* **genes** 

c-Kit is a tyrosine kinase receptor that, when activated by its ligand stem cell factor (SCF), enhances the growth and survival of hematopoietic cells (Reber et al., 2006; Schneider et al., 2010). Preclinical studies have demonstrated the expression of c-KIT and SCF on almost 70% of SCLC cell lines (Reber et al., 2006), suggesting a possible role in the autocrine/paracrine stimulation of tumor growth. Almost a third of patients with ES-SCLC show evidence of ckit overexpression by immunohistochemistry (IHC) (Potti et al., 2003; 2005). In a phase II study of imatinib in SCLC, Johnson et al. (2003) enrolled 19 patients with extensive disease who were either treatment-naïve or had a chemo-sensitive relapse. No activity for imatinib was reported, with only one patient showing disease stabilization. A major drawback of this study was that 79% of the enrolled patients lacked c-KIT expression. In an attempt to improve on these findings, the Cancer and Leukemia Group B (CALGB) refined the study design and conducted a similar trial (Dy et al., 2005) in patients with c-KIT overexpression. Despite this, the results were similar to those seen in the previous study, with no observed responses and only one patient with stable disease for 31 weeks. In a third study, imatinib was used as maintenance therapy following cisplatin and irinotecan in patients with ES-SCLC and c-KIT overexpression in tumor tissue (Schneider et al., 2010). Patients who received imatinib did not have any improvement in the progression-free survival, and thus, this strategy did not warrant further investigation. One of the reasons for the inactivity of imatinib in SCLC may be that the putative cells of origin of SCLC are not developmentally dependent on c-KIT (Heinrich et al., 2002), as opposed to hematopoietic stem cells. Another reason could be the absence of activating mutations in patients with SCLC in c-*KIT* exon 11 that predict for imatinib activity in gastrointestinal stromal tumors (Burger et al., 2003).

## **7. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)**

The vascular endothelial growth factor (VEGF) family is comprised of VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E growth factors and their three VEGF receptors (VEGFR 1-3) (Tanno et al., 2004; Wójcik et. al., 2010). The VEGF signaling pathway leads to increased proliferation, migration, and invasion of endothelial cells, thus mediating tumor angiogenesis (Tanno et al., 2004; Sattler & Salgia, 2003). VEGF is a key factor in the development of new blood vessels that increases the permeability of microvessels (Thomas et al., 2003). High levels of VEGF have been reported in patients with SCLC, which are associated with tumor stage, disease progression, resistance to chemotherapy and poorer outcomes (Fischer et al., 2007). Studies affecting angiogenesis in SCLC have involved: (1) External inhibitors of angiogenesis, chiefly targeting VEGF and its receptor, (2) Endogenous inhibitors such as interferons, and, (3) Miscellaneous agents, e.g., thalidomide. SCLCs express VEGFR1-3 and VEGFR-2, which are actively involved in tumor growth and invasion (Tanno et al., 2004). The VEGF/VEGFR autocrine signaling pathway mediates proliferation and metastasis, which can be inhibited with the use of monoclonal antibodies against VEGFR-2 and VEGFR-3 in SCLC (Tanno et al., 2004). SU6668, which inhibits VEGFR, c-KIT and FGFR, blocks proliferation and angiogenesis in human lung tumor xenografts (Laird et al., 2000). In a study of 87 patients with SCLC who underwent primary resection followed by adjuvant therapy, microvessel count and expression of VEGF significantly affected survival, thereby establishing a role for angiogenesis in SCLC (Lucchi et al., 2002). Another preclinical trial demonstrated that ZD6474, a VEGFR-2 and EGFR kinase inhibitor, successfully interfered with VEGF signaling and angiogenesis, leading to decreased proliferation and increased apoptosis in SCLC xenographs. Bevacizumab is a recombinant humanized monoclonal antibody against VEGF that has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic colon cancer and NSCLC. Preliminary reports of bevacizumab following carboplatin and irinotecan treatment in patients with LS-SCLC have demonstrated response rates, 1- and 2-year survival rates and a safety profile comparable to standard therapy (Raefsky et al., 2005). However, whether the addition of bevacizumab increases progression-free and overall survival is currently unclear and needs to be studied in a randomized phase III trial. VEGF-tyrosine kinase (VEGF-TK) inhibitors have been shown to inhibit downstream signaling pathways that are activated following ligand binding to the VEGF receptor. Multiple agents with VEGF-TK inhibitory activity are being tested for their utility in therapy of various malignancies. A number of small molecules that inhibit VEGFTKs are currently in development, including ZD6474, PTK787/ ZKI222584 (vatalanib), AZD2171, BAY 43-9006 (sorafenib), SU11248 (sunitinib), and AMG706 (Herbst et al., 2005; Morabito et al., 2009). Trials incorporating SU11248 (sunitinib), a multi-targeted TK inhibitor, are being planned in SCLC.

The interferons are a family of naturally-occurring cytokines that have anticancer activity through immunomodulatory and antiangiogenic properties (Blackhall & Shepherd, 2004). Interferons affect endothelial cells by blocking production of basic fibroblast growth factor (bFGF). Patients who achieved a response to chemotherapy were randomized to either interferon maintenance or placebo in different randomized trials. In each of those trials, administration of interferon was associated with considerable toxicity, although two studies showed a trend towards improved survival with interferon treatment (Lebeau et al., 1999). Current trials are investigating the role of interferon either as a vaccine or in combination with cytotoxic chemotherapy in SCLC. Initial reports of maintenance therapy with thalidomide following induction with platinum - etoposide in patients with ES-SCLC have been promising (Conney et al., 2005; Ustuner et al., 2008). This approach needs evaluation in the future.

## **8. Epidermal growth factor receptor (EGFR)**

Although EGFR expression has been reported to be low in SCLC, gefitinib, an oral EGFR tyrosine kinase inhibitor, has been shown to inhibit EGFR signaling in SCLC cell lines (Schmid et al., 2010; Tanno et al., 2004). Anecdotal evidence has suggested tumor regression in patients with advanced stage SCLC following treatment with gefitinib (Araki et al., 2005; Okamoto et al., 2006). This suggests a potential role for gefitinib and other agents targeting the EGFR pathway in this disease. Sequist et al. (2011) performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant NSCLCs carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including, the EGFR T790M mutation or *MET* gene amplification. Furthermore, some resistant cancers showed unexpected genetic changes, such as EGFR amplification and mutations in the *PIK3CA* gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into SCLC, and were sensitive to standard SCLC treatments. Serial biopsies in three patients revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and were sensitive to a second round of treatment with EGFR

preclinical trial demonstrated that ZD6474, a VEGFR-2 and EGFR kinase inhibitor, successfully interfered with VEGF signaling and angiogenesis, leading to decreased proliferation and increased apoptosis in SCLC xenographs. Bevacizumab is a recombinant humanized monoclonal antibody against VEGF that has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic colon cancer and NSCLC. Preliminary reports of bevacizumab following carboplatin and irinotecan treatment in patients with LS-SCLC have demonstrated response rates, 1- and 2-year survival rates and a safety profile comparable to standard therapy (Raefsky et al., 2005). However, whether the addition of bevacizumab increases progression-free and overall survival is currently unclear and needs to be studied in a randomized phase III trial. VEGF-tyrosine kinase (VEGF-TK) inhibitors have been shown to inhibit downstream signaling pathways that are activated following ligand binding to the VEGF receptor. Multiple agents with VEGF-TK inhibitory activity are being tested for their utility in therapy of various malignancies. A number of small molecules that inhibit VEGFTKs are currently in development, including ZD6474, PTK787/ ZKI222584 (vatalanib), AZD2171, BAY 43-9006 (sorafenib), SU11248 (sunitinib), and AMG706 (Herbst et al., 2005; Morabito et al., 2009). Trials incorporating SU11248

The interferons are a family of naturally-occurring cytokines that have anticancer activity through immunomodulatory and antiangiogenic properties (Blackhall & Shepherd, 2004). Interferons affect endothelial cells by blocking production of basic fibroblast growth factor (bFGF). Patients who achieved a response to chemotherapy were randomized to either interferon maintenance or placebo in different randomized trials. In each of those trials, administration of interferon was associated with considerable toxicity, although two studies showed a trend towards improved survival with interferon treatment (Lebeau et al., 1999). Current trials are investigating the role of interferon either as a vaccine or in combination with cytotoxic chemotherapy in SCLC. Initial reports of maintenance therapy with thalidomide following induction with platinum - etoposide in patients with ES-SCLC have been promising

(Conney et al., 2005; Ustuner et al., 2008). This approach needs evaluation in the future.

Although EGFR expression has been reported to be low in SCLC, gefitinib, an oral EGFR tyrosine kinase inhibitor, has been shown to inhibit EGFR signaling in SCLC cell lines (Schmid et al., 2010; Tanno et al., 2004). Anecdotal evidence has suggested tumor regression in patients with advanced stage SCLC following treatment with gefitinib (Araki et al., 2005; Okamoto et al., 2006). This suggests a potential role for gefitinib and other agents targeting the EGFR pathway in this disease. Sequist et al. (2011) performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant NSCLCs carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including, the EGFR T790M mutation or *MET* gene amplification. Furthermore, some resistant cancers showed unexpected genetic changes, such as EGFR amplification and mutations in the *PIK3CA* gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into SCLC, and were sensitive to standard SCLC treatments. Serial biopsies in three patients revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and were sensitive to a second round of treatment with EGFR

(sunitinib), a multi-targeted TK inhibitor, are being planned in SCLC.

**8. Epidermal growth factor receptor (EGFR)** 

inhibitors (Sequist et al., 2011). Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

HER2/neu expression in SCLC has been less well studied. Studies have demonstrated overexpression of HER2/neu by immunohistochemistry in approximately 13–30% of patients with advanced stage SCLC (Micke et al., 2001; Potti et al., 2002, 2003). These studies also found that HER2/neu expression was associated with a poor prognosis for patients with advanced disease. Based on these findings, the anti-HER2/neu monoclonal antibody trastuzumab may be useful as a therapeutic agent in SCLC.

## **9. Matrix metalloproteinase (MMP) inhibitors**

Matrix metalloproteinases are involved in extracellular matrix (ECM) degradation (Stetler-Stevenson et al., 1993), a key process in metastasis. Use of protease inhibitors to limit extracellular matrix proteolysis by cancer cells, thereby interfering with tumor cell invasion, would thus be an attractive therapeutic target. In a retrospective analysis, elevated MMP expression was identified as a negative predictor of survival in SCLC (Michael et al., 1999). 60 to 70% of tumor cells stained positive by IHC for MMP-1 and -9, while positive signals for MMP-11, -13, and -14 were observed in 70 to 100% of tumor cells. Expression of MMP-11 and MMP-14 was determined to be an independent negative prognostic factor for Marimastat, a synthetic, orally administrated, broad spectrum MMPI with activity against collagenases, gelatinases, and stromolysins (Brown & Giavazzi 1995). The National Cancer Institute of Canada-Clinical Trials Group (NCIC-CTG) and EORTC conducted a randomized placebo-controlled trial of marimastat following induction chemotherapy in 532 patients with sensitive SCLC (Shepherd et al., 2002). The addition of marimastat after induction chemotherapy did not result in improved survival and also had a negative impact on quality of life. These disappointing results may be due to patient selection, as MMP expression was not studied in these patients. The proteinases MMP11 and MMP14 were expressed, a more selective MMPI-targeting these two proteins would arguably be more beneficial. In another study, Jumper et al. (2004) evaluated the relationship between circulating MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP-1). Thirty one male and female patients with either stage III or IV NSCLC and 17 with either stage III or IV SCLC were compared to 117 age-matched non-smoking controls of both sexes. Prior to any treatment of the patient, a baseline serum sample was obtained from each of the patients for the determination of circulating MMP-9 and TIMP-1. The results indicate that both MMP-9 and TIMP-1 were elevated in the serum of patients with SCLC or NSCLC when compared to the controls. However, the mean values for both MMP-9 and TIMP-1 in the two tumors did not differ. The natural physiological relationship between MMP-9 and the inhibitor TIMP-1 was lost in both SCLC and NSCLC, indicative of abnormal alterations by the tumor. This study suggests that advanced lung cancer alters the normal circulatory pattern of MMP-9 and TIMP-1 a finding that could aid in the understanding of tumor invasion and/or metastasis.

#### **10. Sonic hedgehog pathway**

The hedgehog (HH) signaling pathway is important during embryonic development and may be involved in development and progression of several human malignancies (Datta & Datta, 2006; Watkins & Peacock, 2004). *In vitro* studies have demonstrated extensive activation of this pathway in a subset of SCLC cell lines (Watkins et al., 2003). The Hedgehog (Hh) pathway is essential in early lung formation and development through epithelial-mesenchymal interactions (Peacock & Watkins, 2008; Watkins et al., 2003). The signaling cascade is initiated by Hh binding to the Patched-1 receptor (Ptch-1), a twelve transmembrane protein. In the absence of Hh ligand, Ptch-1 constitutively inhibits the seven transmembrane protein Smoothened (Smo), and renders the pathway inactive. However, binding of Hh ligand to Ptch-1 causes the inhibition of Smo to be relieved, which then activates a protein complex and downstream transcription of Hh targets in the nucleus, including Gli-1 and Ptch-1. Present at low levels in the basal layer of bronchial epithelium in the adult, active Hedgehog signaling results in expansion of an intraepithelial cell population during airway regeneration induced by naphthalene injury. In SCLC, there is ligand-dependent activation of the Hedgehog pathway in a juxtacrine fashion, with adjacent cells expressing Sonic hedgehog (Peacock & Watkins, 2008; Watkins et al., 2003). Further, *in vitro* and *in vivo* studies have demonstrated that SCLC can be inhibited by the steroidal alkaloid Hedgehog antagonist, cyclopamine (Watkins et al., 2003; Vestergaard et al., 2006). These data support the presence of a progenitor cell in SCLC that remains chemotherapyresistant and relies on the Hedgehog developmental pathway, which can be targeted. Gene expression analysis on members of this pathway showed that although the key transcription factor of this pathway, GL1, was only weakly expressed in cell lines, it was expressed on most SCLC tumors studied, thereby suggesting an important role for this pathway for tumor growth *in vivo* (Vestergaard et al., 2006). Thus, the data support the idea that the HH pathway may be an ideal therapeutic target in SCLC. Agents that inhibit this pathway, cyclopamine and its analog KAAD cyclopamine, will soon be in clinical trials.

## **11. mTOR pathway**

Mammalian target of rapamycin (mTOR), a downstream mediator in the PI3K/Akt signaling pathway, plays a critical role in the regulation of cell proliferation, survival, mobility and angiogenesis (Schmid et al., 2010). Inhibition of this pathway leads to inhibition of downstream signaling elements, thereby resulting in cell cycle arrest in the G1 phase (Chan 2004). Temsirolimus (CCI-779), an inhibitor of mTOR, is being investigated in a phase II trial in patients with ES-SCLC in remission following platinum-based chemotherapy. Genome-wide gene expression profiling revealed that mutant NRF2 affects diverse molecular pathways including the mTOR pathway. Mutant NRF2 upregulates RagD, a small G-protein activator of the mTOR pathway, which was also overexpressed in primary lung cancer (Shibata et al., 2010). Preliminary results show a prolongation of progression-free survival, thereby suggesting significant activity for this agent in SCLC (Pandya et al., 2005). Another agent from this class, everolimus (RAD-001), is currently being tested in a phase II trial for patients with relapsed SCLC (Schmid et al., 2010).

## **12. CD56 (NCAM)**

The neural cell adhesion molecule (NCAM, CD56) is associated with the immunoglobulin family and modulates neuroendocrine cell growth, migration, and differentiation (Jensen & Berthold, 2007; Kim and Kwon, 2010). CD56 is an isoform encoded by the NCAM gene. NCAM is found in almost 100% of SCLC and is also expressed on natural killer cells,

Datta, 2006; Watkins & Peacock, 2004). *In vitro* studies have demonstrated extensive activation of this pathway in a subset of SCLC cell lines (Watkins et al., 2003). The Hedgehog (Hh) pathway is essential in early lung formation and development through epithelial-mesenchymal interactions (Peacock & Watkins, 2008; Watkins et al., 2003). The signaling cascade is initiated by Hh binding to the Patched-1 receptor (Ptch-1), a twelve transmembrane protein. In the absence of Hh ligand, Ptch-1 constitutively inhibits the seven transmembrane protein Smoothened (Smo), and renders the pathway inactive. However, binding of Hh ligand to Ptch-1 causes the inhibition of Smo to be relieved, which then activates a protein complex and downstream transcription of Hh targets in the nucleus, including Gli-1 and Ptch-1. Present at low levels in the basal layer of bronchial epithelium in the adult, active Hedgehog signaling results in expansion of an intraepithelial cell population during airway regeneration induced by naphthalene injury. In SCLC, there is ligand-dependent activation of the Hedgehog pathway in a juxtacrine fashion, with adjacent cells expressing Sonic hedgehog (Peacock & Watkins, 2008; Watkins et al., 2003). Further, *in vitro* and *in vivo* studies have demonstrated that SCLC can be inhibited by the steroidal alkaloid Hedgehog antagonist, cyclopamine (Watkins et al., 2003; Vestergaard et al., 2006). These data support the presence of a progenitor cell in SCLC that remains chemotherapyresistant and relies on the Hedgehog developmental pathway, which can be targeted. Gene expression analysis on members of this pathway showed that although the key transcription factor of this pathway, GL1, was only weakly expressed in cell lines, it was expressed on most SCLC tumors studied, thereby suggesting an important role for this pathway for tumor growth *in vivo* (Vestergaard et al., 2006). Thus, the data support the idea that the HH pathway may be an ideal therapeutic target in SCLC. Agents that inhibit this pathway,

cyclopamine and its analog KAAD cyclopamine, will soon be in clinical trials.

Mammalian target of rapamycin (mTOR), a downstream mediator in the PI3K/Akt signaling pathway, plays a critical role in the regulation of cell proliferation, survival, mobility and angiogenesis (Schmid et al., 2010). Inhibition of this pathway leads to inhibition of downstream signaling elements, thereby resulting in cell cycle arrest in the G1 phase (Chan 2004). Temsirolimus (CCI-779), an inhibitor of mTOR, is being investigated in a phase II trial in patients with ES-SCLC in remission following platinum-based chemotherapy. Genome-wide gene expression profiling revealed that mutant NRF2 affects diverse molecular pathways including the mTOR pathway. Mutant NRF2 upregulates RagD, a small G-protein activator of the mTOR pathway, which was also overexpressed in primary lung cancer (Shibata et al., 2010). Preliminary results show a prolongation of progression-free survival, thereby suggesting significant activity for this agent in SCLC (Pandya et al., 2005). Another agent from this class, everolimus (RAD-001), is currently

being tested in a phase II trial for patients with relapsed SCLC (Schmid et al., 2010).

The neural cell adhesion molecule (NCAM, CD56) is associated with the immunoglobulin family and modulates neuroendocrine cell growth, migration, and differentiation (Jensen & Berthold, 2007; Kim and Kwon, 2010). CD56 is an isoform encoded by the NCAM gene. NCAM is found in almost 100% of SCLC and is also expressed on natural killer cells,

**11. mTOR pathway** 

**12. CD56 (NCAM)** 

neuroendocrine glands, cardiomyocytes, and in the central and peripheral nervous system (Kim and Kwon, 2010). Since malignant cells can be influenced by NCAM signaling, it has been investigated as a target for anti-cancer therapy (Jensen & Berthold, 2007). N901 is an anti-CD56 monoclonal antibody covalently linked to a blocked ricin molecule binds to SCLC tumors and cell lines (Lynch, 1993). Initial studies showed promising activity, but were hampered by the immune response that developed against the murine monoclonal antibody and the ricin molecule, leading to potentially fatal side effects (Fidias et al., 2002). In order to overcome this, a humanized version of this antibody covalently bound to the maytasinoid (microtubule- depolymerizing compound) effector molecule DM-1 has now been made available (BB-10901 or huN901-DM1). Initial studies of this compound have shown evidence of efficacy and safety (Fossella et al., 2005) and a phase II trial is currently underway.

#### **13. Chromosomal alterations**

In SCLC and other epithelial tumors, multiple chromosomal aberrations are found, reflecting genomic instability (Balsara& Testa, 2002; Sato et al., 2007.). Loss of the short arm of chromosome 3 has been consistently seen in SCLC (Balsara & Testa, 2002). The deletion of 3p leads to inactivation of three putative tumor-suppressor genes. The majority of SCLCs have deletions affecting multiple chromosomal sites, with recurrent losses at 3p, 5q, 13q and 17p, which are loci with tumor suppressor genes including p53 (Balsara& Testa, 2002). Comparative genomic hybridization analyses have revealed that a large number of SCLCs harbor gains of 1p, 2p, 3q, 5p, 8q and 19p, regions which encode well-known oncogenes, such as *MYC* and *KRAS*. SCLC cell lines found to have amplifications of 1p, 2p and 3q, and deletion of 18q display a more aggressive phenotype of the disease (Balsara & Testa, 2002). Allele loss on chromosome 3p occurs with a frequency greater than 90% in SCLC and is believed to be an early event found in lung carcinogenesis (Sato et al., 2007). The loss of the fragile histidine triad *(FHIT)* gene results in the accumulation of diadenosine tetraphosphate, stimulating DNA synthesis and proliferation (Sozzi et al., 1996). This gene has been localized to 3p14.2 and is believed to be an important tumor suppressor gene involved in the pathogenesis of lung cancer (Sozzi et al., 1996; Wistuba et al., 2001). The second tumor suppressor gene is believed to be *RASSF1A,* which is located within a 120 kilobase region of chromosome 3p21.3 which also contains the *FUS1, SEMA3B* and *SEMA3F*  loci*. RASSF1A* encodes a protein similar to RAS effector proteins and is inactivated by tumor-acquired promoter hypermethylation in 90-100% of SCLC samples (Burbee et al., 2001; Dammann et al., 2000). ASSF1 is involved in cell cycle pathways, apoptosis and microtubule stability (Agathanggelou et al., 2005).

*FUS1* is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region where allele losses and genetic alterations occur early and frequently for many human cancers. Expression of FUS1 protein is absent or reduced in the majority of lung cancers and premalignant lung lesions. Specificially, expression of the FUS1 protein is lost in 100% of SCLC cases. Interestingly, restoration of wild-type FUS1 function in 3p21.3-deficient nonsmall cell lung carcinoma cells significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle kinetics (Ji & Ross, 2008). They report that FUS1 induces apoptosis through the activation of the intrinsic mitochondrial-dependent and Apaf-1-associated pathways and inhibits the function of protein tyrosine kinases including EGFR, PDGFR, AKT, c-Abl, and c-Kit. Moreover, intravenous administration of a nanoparticle encapsulated FUS1 expression plasmid effectively delivered FUS1 to distant tumor sites and mediated an antitumor effect in orthotopic human lung cancer xenograft models (Ji & Ross, 2008). This approach is the rationale for an ongoing FUS1-nanoparticlemediated gene delivery clinical trial for the treatment of lung cancer.

Deletion of a third gene, *TGFBR2*, located at 3p21.3.22, that encodes the transforming growth factor β (TGF-β) type II receptor, has also been described in SCLC (Hougaard et al., 1999). This nonsense mutation results in the synthesis of a truncated receptor and has been linked to exposure to benzo[a]-pyrene, a component of cigarette smoke. The gene coding for Retinoic Acid Receptor Beta *(RAR-β)* is located on chromosome 3p24 (Mattei et al., 1991). Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors. Loss of heterozygosity of *RAR-B2* and *RAR-B4* isoforms is seen in almost all cases of SCLC. Methylation of the promoter region of these two isoforms may be responsible for the silencing of their expression in SCLC (Virmani et al., 2000).

## **14. Telomerase**

Telomeres are genetic elements present at the ends of linear chromosomes that play an important role in stabilizing chromosomes from degradation and cell death (Counter et al., 1992; Hyer & Silvestri, 2000). Telomerase is a ribonucleoenzyme that compensates for telomere shortening during cell division by synthesizing hexameric TTAGGG repeats at the end of the chromosomes. The functional unit of this enzyme consists of an RNA component hTR, and a catalytic component hTERT. Repression of telomerase in the somatic tissues of humans seems to have evolved as a powerful protective barrier against carcinogenesis (Newbold, 2002). Studies in patients with neuroendocrine lung tumors have demonstrated upregulation of the RNA component of telomerase in 98% of human SCLC (Sarvesvaran et al., 1999). Similar studies assessing telomerase activity in small cell lung cancer showed increased activity in all specimens analyzed (Hiyama et al., 1995). Zaffaroni et al. (2003) studied telomerase activity by the telomeric repeat amplification protocol (TRAP) assay in 38 neuroendocrine (NE) lung tumors. A positive TRAP signal was observed in 14 of 15 (93%) SCLCs, 7 of 8 (87%) large-cell NE carcinomas, and only 1 of 15 (7%) typical carcinoid tumors. When telomerase activity was correlated with the gene product-based immunophenotypic profile of individual tumors, the absence of telomerase activity was associated with a lack of BCL-2, P53, c-KIT, and CDK4 expression and presence of RB. Such a phenotype was appreciable in most of the carcinoid tumors. Conversely, telomerasepositive tumors generally showed an immunophenotype consistent with gene product alterations (including high expression of BCL-2, P53, and c-KIT, and loss of RB) and were characterized by a high proliferative index. These data support the previously reported evidence for two genetically unrelated groups of NE lung tumors that have distinct phenotypic profiles (Zaffaroni et al., 2003).

## **15. Knockout Mouse Models for SCLC**

Contrary to NSCLC, neuroendocrine carcinomas are virtually never found in spontaneous or chemically induced murine lung cancer. One reason for this could be that in these murine models, a combination of both *p53* and *Rb* mutations is almost never found, unlike most human SCLCs. To address this issue, a Cre/lox based deletion of both conditional alleles for *Rb* and *p53* was performed by intratracheal instillation with Adeno-Cre (Meuwissen et al., 2003). After 3 months, foci of neuroendocrine hyperplasia developed through the proximal

tumor sites and mediated an antitumor effect in orthotopic human lung cancer xenograft models (Ji & Ross, 2008). This approach is the rationale for an ongoing FUS1-nanoparticle-

Deletion of a third gene, *TGFBR2*, located at 3p21.3.22, that encodes the transforming growth factor β (TGF-β) type II receptor, has also been described in SCLC (Hougaard et al., 1999). This nonsense mutation results in the synthesis of a truncated receptor and has been linked to exposure to benzo[a]-pyrene, a component of cigarette smoke. The gene coding for Retinoic Acid Receptor Beta *(RAR-β)* is located on chromosome 3p24 (Mattei et al., 1991). Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors. Loss of heterozygosity of *RAR-B2* and *RAR-B4* isoforms is seen in almost all cases of SCLC. Methylation of the promoter region of these two isoforms may be responsible for the silencing of their expression in SCLC (Virmani et al., 2000).

Telomeres are genetic elements present at the ends of linear chromosomes that play an important role in stabilizing chromosomes from degradation and cell death (Counter et al., 1992; Hyer & Silvestri, 2000). Telomerase is a ribonucleoenzyme that compensates for telomere shortening during cell division by synthesizing hexameric TTAGGG repeats at the end of the chromosomes. The functional unit of this enzyme consists of an RNA component hTR, and a catalytic component hTERT. Repression of telomerase in the somatic tissues of humans seems to have evolved as a powerful protective barrier against carcinogenesis (Newbold, 2002). Studies in patients with neuroendocrine lung tumors have demonstrated upregulation of the RNA component of telomerase in 98% of human SCLC (Sarvesvaran et al., 1999). Similar studies assessing telomerase activity in small cell lung cancer showed increased activity in all specimens analyzed (Hiyama et al., 1995). Zaffaroni et al. (2003) studied telomerase activity by the telomeric repeat amplification protocol (TRAP) assay in 38 neuroendocrine (NE) lung tumors. A positive TRAP signal was observed in 14 of 15 (93%) SCLCs, 7 of 8 (87%) large-cell NE carcinomas, and only 1 of 15 (7%) typical carcinoid tumors. When telomerase activity was correlated with the gene product-based immunophenotypic profile of individual tumors, the absence of telomerase activity was associated with a lack of BCL-2, P53, c-KIT, and CDK4 expression and presence of RB. Such a phenotype was appreciable in most of the carcinoid tumors. Conversely, telomerasepositive tumors generally showed an immunophenotype consistent with gene product alterations (including high expression of BCL-2, P53, and c-KIT, and loss of RB) and were characterized by a high proliferative index. These data support the previously reported evidence for two genetically unrelated groups of NE lung tumors that have distinct

Contrary to NSCLC, neuroendocrine carcinomas are virtually never found in spontaneous or chemically induced murine lung cancer. One reason for this could be that in these murine models, a combination of both *p53* and *Rb* mutations is almost never found, unlike most human SCLCs. To address this issue, a Cre/lox based deletion of both conditional alleles for *Rb* and *p53* was performed by intratracheal instillation with Adeno-Cre (Meuwissen et al., 2003). After 3 months, foci of neuroendocrine hyperplasia developed through the proximal

mediated gene delivery clinical trial for the treatment of lung cancer.

**14. Telomerase** 

phenotypic profiles (Zaffaroni et al., 2003).

**15. Knockout Mouse Models for SCLC** 

as well as distal bronchi. After a further 3 months, these early lesions progressed into massive lung tumors with histological features typical of SCLC. Interestingly, some early type lesions remained even in the presence of extensive SCLC. Consequently, it was of importance to determine if the early neuroendocrine lesions are indeed precursors for SCLC and, if so, which additional epigenetic events are then needed for progression. Immunohistological characterization of the full-blown tumors revealed that they indeed shared neuroendocrine features with human SCLC. As in human SCLC all neuroendocrine differentiation markers, such as calcitonin gene-related protein (CGRP), neuron-specific enolase, synaptophysin, neural cell adhesion molecule, and achaete-scute homolog-1 (ASH-1) were not expressed. Furthermore, the murine SCLC readily metastasized towards similar organs as found with human SCLC (Meuwissen et al., 2003). All primary SCLC, as well as their metastases, had all *Rb* and *p53* alleles inactivated. Tumors that retained one WT *Rb* allele were all invariably adenocarcinomas without any neuroendocrine features. Therefore, the status of *Rb* most likely determines if tumors occur with mixed SCLC and NSCLC phenotypes, as has been observed in some patients (Brambilla et al., 2000). No lung tumors were found in *RbF/F* mice, which suggested that loss of *Rb* alone is not enough to initiate lung tumorigenesis (Meuwissen et al., 2003) and the additional loss of *p53* is needed.

Not only does *Rb* loss require additional genetic events to initiate lung tumorigenesis, the nature of these complementary lesions also determines which type of lung cancer will develop. For instance, *RB* inactivation and *KRas* mutations are almost never found together in the same human lung cancer. Moreover, the overall mutation rate of *RB* in human NSCLC is very low (Wistuba & Gazdar, 2003). As noted, Adeno-Cre dependent activation of *Kras* in a broad range of lung epithelial cells leads exclusively to the onset of NSCLC. However, when *LSLKrasG12D* transgenes were combined with *RbF/F* and Rb family *p130F/F* alleles for Adeno-Cre-dependent lung tumor induction, much more advanced adenocarcinomas of *KrasG12D*; *Rb-/-*; *p130-/-* genotypes resulted compared to single *KrasG12D* (Ho et al., 2009). Loss of both *Rb* and *p130*, albeit to a lesser extent, contributed to *Kras* dependent NSCLC. Clearly, in this genetic context KrasG12D overrules any effect of *Rb* loss on neuroendocrine differentiation.

Another intriguing observation came from *CC10-rtTA; tetO7-Cre; RbF/F* mice administered doxycycline during early embryogenesis, causing a complete *Rb* ablation in all bronchial Clara cells. However, only increased hypercellular neuroendocrine lesions were detected in these mice, and no effect on Clara cell homeostasis could be observed. Alternatively, when all three Rb family proteins (Rb, p107 and p130) were inactivated by a truncated SV40 large T-antigen oncoprotein (T121) in *CC10-T121* mice, severe bronchial hyperplasia with complete dedifferentiation of all Clara cells occurred. These results suggest that Rb might be specifically required for determining neuroendocrine cell fate, but only in a strict cellular and genetic context. The combined evidence from mouse models indicate it is unlikely that NSCLC and SCLC develop from similar target cells. It would be more plausible that separate, non-identical target cells can develop into different lung cancers, although each still depends on specific major genetic pathways.

Apart from the somatic *RbF/F; p53F/F* model for SCLC, two other lung cancer models have also been associated with pulmonary neuroendocrine tumors. One model made use of bitransgenic *CC10-hASH1; CC10-SV40 large T* system in which progressive neuroendocrine dysplasia and aggressive lung adenocarcinoma developed with both focal neuroendocrine differentiation (through expression of pro-neural ASH-1 transcription factor) and CC10 expression (Linnoila et al., 2000). These adenocarcinomas closely resembled human NSCLC with neuroendocrine differentiation (Linnoila et al., 1994). In the other model, the cyclin dependent kinase inhibitor *p18Ink4c* and *Men1*, a tumor suppressor gene deleted in human multiple endocrine neoplasia, were both inactivated. To determine how *p18* and *p27*  genetically interact with Men1, Pei et al. (2007) characterized *p18-Men1* and *p27-Men1* double mutant mice and showed that p18, but not p27, functionally collaborates with Men1 in suppressing lung tumorigenesis. Lung tumors developed in both *Men1+/-* and *p18-/-; Men1+/-* mice at a high penetrance and contained both neuroendocrine and nonneuroendocrine cells. The remaining wild-type *Men1* allele was lost in most lung tumors from *Men1+/-* mice, but was retained in most tumors from *p18-/-;Men1+/-* mice, showing a functional collaboration between p18 and Men1 in lung tumor suppression (Pei et al., 2007). Phosphorylation of Rb protein at both Cdk2 and Cdk4/Cdk6 sites were significantly increased in normal bronchial epithelia and tumor cells derived from *p18-/-;Men1+/-* mice compared to those from single *p18-/-* or *Men1+/-* mice. Lung tumors developed in *p18-/-;Men1+/* mice were multifocal, more heterogeneous, and highly invasive compared to those in either *p18-/-* or *Men1+/-* mice. These results revealed a previously unrecognized function of p18 in lung tumor suppression through collaboration with Men1 to control lung stem cell proliferation. To investigate the cellular origin(s) of this cancer, Sutherland et al. (2011) assessed the effect of *Trp53* and *Rb1* inactivation in distinct cell types in the adult lung using adenoviral vectors that target Cre recombinase to Clara, neuroendocrine (NE), and alveolar type 2 (SPC-expressing) cells. Using these cell type-restricted Adeno-Cre viruses, loss of *Trp53* and *Rb1* efficiently transformed NE and SPC-expressing cells, leading to SCLC, albeit SPC-expressing cells were transformed less efficiently (Sutherland et al., 2011). In contrast, Clara cells were largely resistant to transformation. Their results indicate that although NE cells serve as the predominant cell of origin of SCLC, a subset of SPC-expressing cells are also endowed with this ability.

Increasingly, it is realized that during tumorigenesis a variety of cells are recruited into the tumor to provide a range of functions that are associated with tumor progression (Calbo et al., 2011). Karnoub and colleagues (2007) showed that mesenchymal stem cells recruited into the stroma of breast cancer promote metastasis through CCL5-mediated paracrine signaling, thereby emphasizing the relevance of the interactions between tumor cells and the surrounding microenvironment. Using the mouse model for simultaneous *Rb* and *p53* inactivation, Calbo et al. (2011) established primary cultures from 21 murine NSCLCs. They found mouse SCLC primary cultures attached to the dishes in 9 of 21 cases, consistent with cell culture derived from human SCLC. They seeded single-cell suspensions from 15 mouse SCLC cultures in soft agar-containing medium, and isolated individual colonies and expanded separately. Most of the obtained clones grew as suspending aggregates of very small cells. These cells expressed neuroendocrine protein markers such as synaptophysin, achaete-scute complex homolog 1, and neural cell adhesion molecule. They also obtained clones grew as a cellular monolayer composed of larger cells with visible cytoplasm and spindle-like membrane extensions, spreading on the substrate (mesenchymal cells). This is consistent with human SCLC that are often composed of phenotypically different cells with either a neuroendocrine or a mesenchymal marker profile. Importantly, these two types of cells had a common origin because they shared specific genomic aberrations as demonstrated by SKY analysis. Calbo et al. also showed that the transition from neuroendocrine to mesenchymal phenotype could be achieved by the ectopic expression of oncogenic RasV12 in the former (Calbo et al., 2011). When engrafted as a mixed population, the mesenchymal cells endowed the neuroendocrine cells with metastatic capacity,

with neuroendocrine differentiation (Linnoila et al., 1994). In the other model, the cyclin dependent kinase inhibitor *p18Ink4c* and *Men1*, a tumor suppressor gene deleted in human multiple endocrine neoplasia, were both inactivated. To determine how *p18* and *p27*  genetically interact with Men1, Pei et al. (2007) characterized *p18-Men1* and *p27-Men1* double mutant mice and showed that p18, but not p27, functionally collaborates with Men1 in suppressing lung tumorigenesis. Lung tumors developed in both *Men1+/-* and *p18-/-; Men1+/-* mice at a high penetrance and contained both neuroendocrine and nonneuroendocrine cells. The remaining wild-type *Men1* allele was lost in most lung tumors from *Men1+/-* mice, but was retained in most tumors from *p18-/-;Men1+/-* mice, showing a functional collaboration between p18 and Men1 in lung tumor suppression (Pei et al., 2007). Phosphorylation of Rb protein at both Cdk2 and Cdk4/Cdk6 sites were significantly increased in normal bronchial epithelia and tumor cells derived from *p18-/-;Men1+/-* mice compared to those from single *p18-/-* or *Men1+/-* mice. Lung tumors developed in *p18-/-;Men1+/* mice were multifocal, more heterogeneous, and highly invasive compared to those in either *p18-/-* or *Men1+/-* mice. These results revealed a previously unrecognized function of p18 in lung tumor suppression through collaboration with Men1 to control lung stem cell proliferation. To investigate the cellular origin(s) of this cancer, Sutherland et al. (2011) assessed the effect of *Trp53* and *Rb1* inactivation in distinct cell types in the adult lung using adenoviral vectors that target Cre recombinase to Clara, neuroendocrine (NE), and alveolar type 2 (SPC-expressing) cells. Using these cell type-restricted Adeno-Cre viruses, loss of *Trp53* and *Rb1* efficiently transformed NE and SPC-expressing cells, leading to SCLC, albeit SPC-expressing cells were transformed less efficiently (Sutherland et al., 2011). In contrast, Clara cells were largely resistant to transformation. Their results indicate that although NE cells serve as the predominant cell of origin of SCLC, a subset of SPC-expressing cells are

Increasingly, it is realized that during tumorigenesis a variety of cells are recruited into the tumor to provide a range of functions that are associated with tumor progression (Calbo et al., 2011). Karnoub and colleagues (2007) showed that mesenchymal stem cells recruited into the stroma of breast cancer promote metastasis through CCL5-mediated paracrine signaling, thereby emphasizing the relevance of the interactions between tumor cells and the surrounding microenvironment. Using the mouse model for simultaneous *Rb* and *p53* inactivation, Calbo et al. (2011) established primary cultures from 21 murine NSCLCs. They found mouse SCLC primary cultures attached to the dishes in 9 of 21 cases, consistent with cell culture derived from human SCLC. They seeded single-cell suspensions from 15 mouse SCLC cultures in soft agar-containing medium, and isolated individual colonies and expanded separately. Most of the obtained clones grew as suspending aggregates of very small cells. These cells expressed neuroendocrine protein markers such as synaptophysin, achaete-scute complex homolog 1, and neural cell adhesion molecule. They also obtained clones grew as a cellular monolayer composed of larger cells with visible cytoplasm and spindle-like membrane extensions, spreading on the substrate (mesenchymal cells). This is consistent with human SCLC that are often composed of phenotypically different cells with either a neuroendocrine or a mesenchymal marker profile. Importantly, these two types of cells had a common origin because they shared specific genomic aberrations as demonstrated by SKY analysis. Calbo et al. also showed that the transition from neuroendocrine to mesenchymal phenotype could be achieved by the ectopic expression of oncogenic RasV12 in the former (Calbo et al., 2011). When engrafted as a mixed population, the mesenchymal cells endowed the neuroendocrine cells with metastatic capacity,

also endowed with this ability.

illustrating the potential relevance of tumor cell heterogeneity in dictating tumor properties (Calbo et al., 2011). In short, they showed a specific type of tumor heterogeneity of SCLC, in which the interaction between clonally derived but diversified subclones alters the behavior of the tumor as a whole (Calbo et al., 2011). One outcome was a substantially increased metastatic potential, a feature with important clinical ramifications in human SCLC. Tumor cell behavior then not only depended on the interactions with stromal cells, but was also influenced by interactions with tumor cell variants that fulfill a distinct role in the tumor tissue. This study provides a unique system, whereby the mesenchymal compartment of the tumor was generated from a separate subclone of SCLC during the tumorigenic process, providing the tumor cell population as a whole with new capabilities such as metastatic potential.

#### **16. Xenograft models for SCLC**

For the past three decades, the mainstay of preclinical cancer therapeutic research has been the use of human cancer cells lines cultured *in vitro* and of xenografts derived from these cell lines grown *in vivo* in immunodeficient mice. However, neither model consistently predicted the efficacy in clinical trials, resulting in two major barriers to the successful translation of new cancer therapeutics. First, resources are expended on drug development based on these models that ultimately fail in clinical trials. Second, many potentially useful therapies that might be beneficial in humans are discarded because the animal models fail to demonstrate efficacy in conventional cell culture and xenograft models. Emerging evidence suggests that the process of establishing conventional cell lines from human cancers results in distinct and irreversible loss of important biological properties. These include (*a*) gain or loss of gene amplification, (*b*) the ability to migrate and metastasize, (*c*) the maintenance of a distinct stem cell population, and (*d*) the preservation of dependency on embryonic signaling pathways. These properties are not restored when these conventional cell lines are grown as heterotopic or orthotopic xenografts.

Because SCLC is usually diagnosed by endobronchial biopsy or fine-needle aspiration cytology, substantial quantities of fresh or frozen tissues are typically lacking in most tumor banks. For this reason, most SCLC researches rely on conventional cell lines, which are often chemoresistant because they were derived from patients who had received cytotoxic chemotherapy (Phelps et al., 1996). In addition, all of these cell lines have experimental limitations and lack the three-dimensional tumor-stromal interactions, which seem to significantly affect the response of these cells to chemotherapy (Hodkinson et al., 2007). To establish better models for the study of SCLC, Daniel et al. (2009) generated and characterized a series of primary xenograft models derived from chemo-naive patients to more accurately model SCLC in mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then reimplanted to generate secondary xenografts. When compared with normal lung, primary tumors, xenografts, and cell lines displayed a gene expression signature specific for SCLC (Daniel et al., 2009). Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to SCLC cell lines; these genes were not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development.

Bcl-2 is a central regulator of cell survival that is overexpressed in most SCLC tumors and contributes to both malignant transformation and therapeutic resistance. Hann et al. (2008) compared primary SCLC xenografts prepared from *de novo* human tumors with standard cell line–based xenografts to evaluate a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent difference may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significantly decreased tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, they isolated resistant derivatives of an initially sensitive cell line–based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling revealed 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. These data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the testing of novel anticancer strategies in relevant preclinical models.

#### **17. Conclusions**

There are many distinct genetic pathways present in SCLC, leading to its unique biology and clinical features. A better understanding of these basic molecular and cellular changes will allow for the development of novel therapeutic strategies. Multiple molecularly targeted agents are actively being studied pre-clinically and clinically, with the hope of ultimately improving survival of patients with SCLC. Development of targeted therapy in small cell lung cancer has significantly lagged behind that of non-small cell lung cancer. Etoposide and cisplatin remain the mainstays of first-line SCLC treatment. Although the decreasing prevalence of smoking in industrialized countries will lead to decreased incidence of SCLC, the burden of disease is shifting to developing countries. Further investment in research for this disease is, therefore, warranted. Many phase 1 and 2 studies of drugs with potential activity in SCLC and phase 2 and 3 trials to improve radiotherapy are underway. Inclusion of patients with SCLC in such trials should be encouraged, especially otherwise healthy patients with relapsing or refractory SCLC, for whom treatment options are limited. A new, effective, and active combination for extensive-stage SCLC would be quickly moved up as a treatment priority.

SCLC remains a therapeutic challenge despite high initial responses to chemotherapy and radiotherapy. The fact that several promising molecularly targeted agents have not shown adequate activity in clinical trials does not mean the end of novel targeted therapies for SCLC. Nevertheless, a better understanding of SCLC biology and better preclinical models of SCLC are needed to improve available therapies.

The mouse model presented in the study by Meuwissen et al. clearly showed that somatic inactivation of both *Rb1* and *Trp53* alleles in lung epithelial cells readily leads to formation of small cell NE tumors. Their histopathologic characteristics and metastasizing capacity were strikingly similar to human SCLCs. Their mouse model will prove a valuable tool for (1) comparing genotype-phenotype similarities between human SCLC and MSCLC, (2) the identification of precursor lesions, and (3) additional factors involved in tumor progression,

Bcl-2 is a central regulator of cell survival that is overexpressed in most SCLC tumors and contributes to both malignant transformation and therapeutic resistance. Hann et al. (2008) compared primary SCLC xenografts prepared from *de novo* human tumors with standard cell line–based xenografts to evaluate a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent difference may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significantly decreased tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, they isolated resistant derivatives of an initially sensitive cell line–based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling revealed 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. These data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the

There are many distinct genetic pathways present in SCLC, leading to its unique biology and clinical features. A better understanding of these basic molecular and cellular changes will allow for the development of novel therapeutic strategies. Multiple molecularly targeted agents are actively being studied pre-clinically and clinically, with the hope of ultimately improving survival of patients with SCLC. Development of targeted therapy in small cell lung cancer has significantly lagged behind that of non-small cell lung cancer. Etoposide and cisplatin remain the mainstays of first-line SCLC treatment. Although the decreasing prevalence of smoking in industrialized countries will lead to decreased incidence of SCLC, the burden of disease is shifting to developing countries. Further investment in research for this disease is, therefore, warranted. Many phase 1 and 2 studies of drugs with potential activity in SCLC and phase 2 and 3 trials to improve radiotherapy are underway. Inclusion of patients with SCLC in such trials should be encouraged, especially otherwise healthy patients with relapsing or refractory SCLC, for whom treatment options are limited. A new, effective, and active combination for extensive-stage

SCLC remains a therapeutic challenge despite high initial responses to chemotherapy and radiotherapy. The fact that several promising molecularly targeted agents have not shown adequate activity in clinical trials does not mean the end of novel targeted therapies for SCLC. Nevertheless, a better understanding of SCLC biology and better preclinical models

The mouse model presented in the study by Meuwissen et al. clearly showed that somatic inactivation of both *Rb1* and *Trp53* alleles in lung epithelial cells readily leads to formation of small cell NE tumors. Their histopathologic characteristics and metastasizing capacity were strikingly similar to human SCLCs. Their mouse model will prove a valuable tool for (1) comparing genotype-phenotype similarities between human SCLC and MSCLC, (2) the identification of precursor lesions, and (3) additional factors involved in tumor progression,

testing of novel anticancer strategies in relevant preclinical models.

SCLC would be quickly moved up as a treatment priority.

of SCLC are needed to improve available therapies.

**17. Conclusions** 

and (4) ultimately, testing of targeted, novel tumor intervention strategies and chemoprevention.

The identification of specific cell type(s) from which SCLC originates is critical in the development of methods for early diagnosis and treatment. By using cell type-restricted Adeno-Cre vectors in directing *Trp53* and *Rb1* loss to distinct cell populations in the adult mouse lung, Sutherland et al. showed that NE cells are the predominant cells of origin of SCLC. Their study provides additional tools to address questions related to the cell of origin of lung cancer, and highlights the importance of specifically targeting NE cells for the treatment of SCLC. Their strategy to manipulate specific adult lung cell populations in a controlled manner by cell type-restricted somatic gene transfer vectors could help to answer the question of whether distinct lung pathologies have a unique cell of origin, and whether this cell of origin is a determining factor in the drug resistance profile of the various tumor subtypes.

The work of Calbo et al. showed a specific type of tumor heterogeneity, in which the interaction between clonally derived but diversified subclones alters the behavior of the tumor as a whole. One outcome was substantially increased metastasis, a feature with important clinical ramifications. Thus, tumor cell behavior not only depends on the interactions with stromal cells, but also interactions with tumor cell variants that fulfill a distinct role in the tumor tissue. Enhanced metastatic capacity serves as an illustrative example of crosstalk between specialized tumor cell clones. Disrupting the paracrine signaling involved in this interaction is worth further exploring as a strategy to mitigate tumor progression in SCLC.

## **18. Acknowledgements**

We thank K. Klein for editing. K. Inoue is supported by NIH/NCI 5R01CA106314, ACS RSG-07-207-01-MGO, a Director's Challenge pilot award (Comprehensive Cancer Center of WFU), and an intramural pilot grant award from Wake Forest University School of Medicine. P. Taneja has been supported by the Susan G. Komen Foundation postdoctoral fellowship KG080179. D. Maglic has been supported by DOD predoctoral fellowship BC100907.

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#### **19.14 Telomerase**


#### **19.15 Knockout mouse models**


#### **19.16 Mouse xenograft models**


## **Centrosome Abnormality and Human Lung Cancer**

Kazuya Shinmura and Haruhiko Sugimura *Hamamatsu University School of Medicine* 

*Japan* 

## **1. Introduction**

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**19.16 Mouse xenograft models** 

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Matthews MJ, Bunn PA Jr, Carney D, Minna JD, Mulshine JL. (1996). NCI-Navy

The centrosome, which functions as a major microtubule-organizing center (MTOC), is composed of a pair of centrioles and surrounding protein aggregates called pericentriolar material (PCM); at any given time during the cell cycle, each cell contains one or two centrosomes (Fukasawa, 2007). Centrosomes play a crucial role in the formation of bipolar mitotic spindles, which are essential for accurate chromosome segregation (Zyss & Gergely, 2009). Numerical and functional abnormalities of centrosomes result in an increase in aberrant mitotic spindle formation, merotelic kinetochore-microtubule attachment errors, lagging chromosome formation, and chromosome segregation errors, all of which are thought to be possible causes of chromosome instability (Ganem et al., 2009; Nigg & Raff, 2009). Centrosome abnormalities and chromosome instability are characteristics of human lung cancer (Masuda and Takahashi, 2002; Koutsami et al., 2006; Jung et al., 2007; Shinmura et al., 2008), and abnormalities in genes responsible for centrosome regulation have been reported in lung cancer (Fukasawa, 2007; Lee et al., 2010). In this Review, the status of centrosome abnormalities in lung cancer, the mechanisms responsible for inducing centrosome abnormalities, and the relationship between centrosome abnormalities and chromosome instability are summarized.

## **2. Centrosome abnormalities in human lung cancer: Mechanisms causing centrosome abnormalities and chromosome instability**

The presence of two centrosomes at mitosis is an important factor in the formation of bipolar mitotic spindles. Therefore, the numerical integrity of centrosomes is carefully controlled in human cells, and abrogation of this control results in centrosome amplification. First, we describe the normal centrosome duplication cycle, followed by three reports on centrosome abnormalities in lung cancer. Next, we describe investigations of the mechanism responsible for inducing centrosome amplification. Finally, we summarize the possible reasons why centrosome abnormalities cause chromosome instability.

### **2.1 Centrosome duplication cycle in human cells**

Centrioles are cylindrical structures (-0.2 m in diameter and 0.2-0.5 m in length) and are composed of nine triplet microtubule arrays organized around a central cartwheel. Centrioles contain several tubulin isoforms and non-tubulin proteins such as CETN2, CP110, SAS-6, and SAS-4 (Bettencourt-Dias & Glover, 2009). In animal cells, a pair of centrioles is embedded in a cloud of electron dense material known as PCM, and both structures constitute a larger structure named the centrosome, which serves as the main MTOC during both interphase and mitotic phase (Vorobjev & Nadezhdina, 1987). Centrosome duplication occurs once per cell cycle and is subject to strict control within cells. To organize a bipolar mitotic spindle, a centrosome is duplicated in S phase, additional PCM proteins are recruited during centrosome maturation in G2, and the two centrosomes separate at mitotic entry (Figure 1). The primary function of PCM is microtubule nucleation. The assembly of microtubules is initiated on a -tubulin ring complex (TuRC), composed by -tubulin and additional subunits known as -tubulin complex proteins (Teixidó-Travesa et al., 2010).

Fig. 1. Centrosome duplication cycle.

The centrioles duplicate once per cell cycle. The formation of the daughter centriole on each mother centriole occurs during the late G1 and S phases of the cell cycle. The daughter and mother centrioles are tightly associated in an orthogonal manner until the end of mitosis, and centriole disengagement occurs during mitotic exit. The initiation of centriole duplication requires the activity of several proteins, such as Cdk2-cyclin E and PLK4 kinases. The procentriole starts to assemble, and elongation depends on several proteins including centrin, CEP135, and -tubulin. During G2 phase, additional PCM proteins are recruited, and centrosome maturation requires the activity of Aurora A and PLK1 kinases. During late G2, the daughter centriole of the parental pair acquires subdistal appendages. Then, the two duplicated centrosomes separate and move to opposite end of the cell (centrosome separation). Finally, the two centrosomes form the poles of the bipolar mitotic spindle.

SAS-6, and SAS-4 (Bettencourt-Dias & Glover, 2009). In animal cells, a pair of centrioles is embedded in a cloud of electron dense material known as PCM, and both structures constitute a larger structure named the centrosome, which serves as the main MTOC during both interphase and mitotic phase (Vorobjev & Nadezhdina, 1987). Centrosome duplication occurs once per cell cycle and is subject to strict control within cells. To organize a bipolar mitotic spindle, a centrosome is duplicated in S phase, additional PCM proteins are recruited during centrosome maturation in G2, and the two centrosomes separate at mitotic entry (Figure 1). The primary function of PCM is microtubule nucleation. The assembly of microtubules is initiated on a -tubulin ring complex (TuRC), composed by -tubulin and additional subunits known as -tubulin complex proteins (Teixidó-Travesa et al., 2010).

**G1**

**Centriole**

**disengagement**

**Procentriole assembly**

**S**

The centrioles duplicate once per cell cycle. The formation of the daughter centriole on each mother centriole occurs during the late G1 and S phases of the cell cycle. The daughter and mother centrioles are tightly associated in an orthogonal manner until the end of mitosis, and centriole disengagement occurs during mitotic exit. The initiation of centriole duplication requires the activity of several proteins, such as Cdk2-cyclin E and PLK4 kinases. The procentriole starts to assemble, and elongation depends on several proteins including centrin, CEP135, and -tubulin. During G2 phase, additional PCM proteins are recruited, and centrosome maturation requires the activity of Aurora A and PLK1 kinases. During late G2, the daughter centriole of the parental pair acquires subdistal appendages. Then, the two duplicated centrosomes separate and move to opposite end of the cell (centrosome separation). Finally, the two centrosomes form the poles of the bipolar mitotic

**Centriole elongation**

**M**

**G2**

**Centrosome maturation Centrosome separation**

Fig. 1. Centrosome duplication cycle.

spindle.

#### **2.2 Centrosome abnormalities in lung cancer**

Centrosome amplification has been reported in a variety of human primary cancers (e.g., breast cancer, lung cancer, bladder cancer, pancreatic cancer, and prostatic cancer) (Pihan et al., 1998; Sato et al., 1999; Pihan et al., 2001; Kawamura et al., 2004; Zyss & Gergely, 2009). With regard to primary lung cancer, Koutsami et al. (2006) examined 68 primary non-small cell lung carcinomas (NSCLCs) for the presence or absence of centrosome amplification using an immunofluorescence analysis with a monoclonal antibody for -tubulin, a centrosome marker; they showed that 36 (53%) of the 68 NSCLCs exhibited centrosome amplification. Centrosome amplification was not associated with clinicopathological markers such as stage, tumor grade, and histological subtype, but was associated with aneuploidy. Jung et al. (2007) examined 175 NSCLCs for centrosome abnormalities using an immunofluorescence analysis with an anti--tubulin antibody; they showed that 50 (29%) of the 175 NSCLCs exhibited a centrosome abnormality. Aneuploidy, p16 expression, and the loss of pRB expression were significantly associated with centrosome abnormalities. Shinmura et al. (2008) examined 182 primary lung carcinomas for the presence or absence of centrosome amplification using an immunohistochemical analysis with an anti--tubulin antibody and showed that 67 (37%) of the 182 cancers exhibited centrosome amplification. Thus, centrosome amplification is a common abnormality seen in human primary lung cancers.

#### **2.3 Mechanisms inducing centrosome abnormalities**

An immunofluorescence analysis using an antibody for centrosome or centriole markers in cultured cell lines can be used to determine the status of the centrosome number in the cells. The involvement of many kinds of agents and genes in centrosome regulation has been examined using such analyses. Here, these analyses are divided into those using lung cells and those using cells derived from other organs.

#### **2.3.1 Mechanisms identified by using the lung cells**

Holmes et al. (2006) showed that chronic exposure to lead chromate causes centrosome abnormalities and aneuploidy using WTHBF-6 cells, a cell line derived from normal human bronchial fibroblasts. Hexavalent chromium compounds [Cr(VI)] are human lung carcinogens (Le´onard & Lauwerys, 1980), and "particulate" Cr(VI) compounds are one of the most potent forms. They reported centrosome amplification in interphase and mitotic cells in response to treatment with lead chromate as a model particulate Cr(VI) compound. They suggested that one possible mechanism for lead chromate–induced carcinogenesis is through centrosome dysfunction, leading to the induction of aneuploidy. The same group (Holmes et al., 2010) also showed that chronic exposure to zinc chromate, another particulate Cr(VI) compound, induces centrosome amplification and spindle checkpoint bypass using human lung fibroblasts.

Arsenic is another environmental toxicant, and the biological effects of arsenic have been studied. Liao et al. (2007) showed that arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells. They used H1355 (a lung adenocarcinoma cell line with a p53 mutation), BEAS-2B (immortalized lung epithelial cells with functional p53) and pifithrin--treated BEAS-2B (p53-inhibited cells) and reported an increase in centrosome abnormalities in both arsenite-treated p53 compromised cell lines, compared with that in arsenite-treated BEAS-2B cells. Their findings provided evidence of the carcinogenic promotional role of arsenic, especially in the presence of p53 abnormalities. The group also showed that arsenite promoted centrosome abnormalities in the presence of a p53-compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) using BEAS-2B cells (Liao et al., 2010). Their findings provided evidence of an interaction between arsenite and cigarette smoking.

Benzo[*a*]pyrene diol epoxide (B[*a*]PDE), the ultimate carcinogenic metabolite of benzo[*a*]pyrene, has been implicated in the mutagenesis of the *p53* gene, which is involved in smoking-associated lung cancer. Shinmura et al. (2008) showed that the exposure of *p53* deficient H1299 lung cancer cells to B[*a*]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. They also revealed that the centrosome amplification could be primarily attributed to excessive centrosome duplication, rather than to centriole splitting, and the forced expression of POLK DNA polymerase, which has the ability to bypass B[*a*]PDE–guanine lesions in an error-free manner, suppressing B[*a*]PDE-induced centrosome amplification. The B[*a*]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. Thus, they concluded that B[*a*]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization in addition to its mutagenic activity.

Fig. 2. Induction of centrosome amplification in *p53*-deficient H1299 lung cancer cells by exposure to benzo[*a*]pyrene diol epoxide (B[*a*]PDE).

H1299 cells were exposed to 0.6 M B[*a*]PDE for 72 hr and then immunostained with mouse anti--tubulin monoclonal antibody (GTU-88; Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 546 (red)-conjugated anti-IgG antibody (Molecular Probes, Eugene, OR, USA) was used to detect the antibody–antigen complexes. The nuclei were stained with 4',6 diamidino-2-phenylindol (DAPI, blue). An increase in the number of centrosomes, i.e., centrosome amplification, was observed in both interphase cells and mitotic phase cells. The arrows indicate the positions of centrosomes.

the carcinogenic promotional role of arsenic, especially in the presence of p53 abnormalities. The group also showed that arsenite promoted centrosome abnormalities in the presence of a p53-compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) using BEAS-2B cells (Liao et al., 2010). Their findings provided evidence of an interaction between arsenite and cigarette smoking. Benzo[*a*]pyrene diol epoxide (B[*a*]PDE), the ultimate carcinogenic metabolite of benzo[*a*]pyrene, has been implicated in the mutagenesis of the *p53* gene, which is involved in smoking-associated lung cancer. Shinmura et al. (2008) showed that the exposure of *p53* deficient H1299 lung cancer cells to B[*a*]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. They also revealed that the centrosome amplification could be primarily attributed to excessive centrosome duplication, rather than to centriole splitting, and the forced expression of POLK DNA polymerase, which has the ability to bypass B[*a*]PDE–guanine lesions in an error-free manner, suppressing B[*a*]PDE-induced centrosome amplification. The B[*a*]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. Thus, they concluded that B[*a*]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome

**Interphase Mitotic phase**

Fig. 2. Induction of centrosome amplification in *p53*-deficient H1299 lung cancer cells by

H1299 cells were exposed to 0.6 M B[*a*]PDE for 72 hr and then immunostained with mouse anti--tubulin monoclonal antibody (GTU-88; Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 546 (red)-conjugated anti-IgG antibody (Molecular Probes, Eugene, OR, USA) was used to detect the antibody–antigen complexes. The nuclei were stained with 4',6 diamidino-2-phenylindol (DAPI, blue). An increase in the number of centrosomes, i.e., centrosome amplification, was observed in both interphase cells and mitotic phase cells. The

destabilization in addition to its mutagenic activity.

**B[***a***]PDE**

exposure to benzo[*a*]pyrene diol epoxide (B[*a*]PDE).

arrows indicate the positions of centrosomes.

**untreated**

The lung is easily subjected to many kinds of environmental agents, some of which may be derived from cigarette smoking or occupational exposure. As described in the above three paragraphs, some environmental carcinogens induce centrosome amplification. Other environmental carcinogens attacking DNA may also induce centrosome amplification, since cell cycle arrest has been shown to occur during centrosome amplification. Further precise analyses of environmental agent-related centrosome amplification are needed to understand the relationship between environmental carcinogens and lung cancer more clearly.

The S-phase kinase-interacting protein-2 (SKP2) plays a key role in the progression of cells from a quiescent to proliferative state, and the SKP2 protein is overexpressed in lung cancer. Jiang et al. (2005) showed that the RNA silencing of SKP2 inhibits proliferation and centrosome amplification using the lung cancer cell lines A549 and H1792. Their results suggest that SKP2 plays an oncogenic role in lung cancer and has a centrosome regulating function.

NORE1 (RASSF5) is a member of the *RASSF* gene family, and NORE1A is the longest and major splice isoform of the *NORE1* gene (Nakamura et al., 2005). Its product, NORE1A, is a nucleocytoplasmic shuttling protein and has a growth-suppressive function (Moshnikova et al., 2006). Shinmura et al. (2011) showed that NORE1A suppresses the centrosome amplification induced by hydroxyurea using a *p53*-deficient H1299 lung cancer cell line, and NORE1A expression was down-regulated in NSCLC. Both of these findings imply that NORE1A has a key preventative role against the carcinogenesis of NSCLC.

#### **2.3.2 Mechanisms identified using cells derived from other organs**

CDK2–cyclin E, a known inducer of S-phase entry (Heichman, 1994), has an important role in the regulation of centrosome duplication (Hinchcliffe et al., 1999; Matsumoto et al., 1999). The activation of CDK2–cyclin E during late-G1 phase coordinates the initiation of centrosome and DNA duplication. Several CDK2–cyclin E targets, including nucleophosmin (NPM) (Okuda et al., 2000), have been identified. NPM binds and modulates the activities of multiple proteins including tumor suppressor proteins (e.g.*,* p53) and some oncogenic proteins (e.g., ROCK2) (Colombo et al., 2002; Ma et al., 2006b). The reduced as well as increased expression of NPM can lead to the oncogenic transformation of cells. Actually, NPM is frequently mutated, lost or overexpressed in cancers (Grisendi et al., 2006), and both the overexpression and the depletion of NPM in cultured cells can lead to neoplastic transformation (Kondo et al., 1997; Grisendi et al., 2005). NPM localizes between the paired centrioles of the unduplicated centrosome, probably functioning in centriole pairing (Shinmura et al., 2005). When NPM is phosphorylated by CDK2–cyclin E, most of the NPM dissociates from the centrosomes, leading to the centrosome duplication. In this context, NPM negatively controls centrosome duplication; indeed, the depletion of NPM leads to centrosome amplification (Grisendi et al., 2005; Wang et al., 2005). NPM was reported to have the ability to control centrosome duplication in association with ROCK2 (Ma et al., 2006b), a member of the Rho-associated, coiled-coil containing protein kinase family that is frequently overexpressed in cancer (Nishimura et al., 2003). After NPM phosphorylation by CDK2–cyclin E, the binding between NPM and ROCK2 increases and ROCK2 is activated at centrosomes, leading to centrosome duplication (Ma et al., 2006b). In ROCK2 activation, the binding of Rho small GTPase to the auto-inhibitory region is also required (Kanai et al., 2010). Among three isoforms of Rho, both RhoA and RhoC, but not RhoB, promoted centrosome duplication and centrosome amplification.

Another target of CDK2–cyclin E in centrosome regulation is MPS1, a spindle checkpoint kinase that is localized at the centrosome (Fisk et al., 2003). MPS1 is stabilized and activated by CDK2–cyclin E phosphorylation and involved in centrosome duplication. Mortalin, a member of the heat-shock protein 70 molecular chaperone family, is localized at the centrosome and physically interacts with and is phosphorylated by MPS1. The phosphorylation of mortalin activates MPS1 in a positive-feedback manner, and this phenomenon is important for MPS1-related centrosome duplication (Kanai et al., 2007). Mortalin is frequently upregulated in cancers (Wadhwa et al., 2006).

CDK2 forms a complex with cyclin A in addition to cyclin E, and CDK2–cyclin A has been implicated in the regulation of centrosome duplication (Meraldi et al., 1999). CDK2–cyclin A and CDK2–cyclin E share some substrates (Tokuyama et al., 2001). The CDK2–cyclin A complex is active in S and G2 phases during the cell cycle, and CDK2–cyclin A may have a crucial role in centrosome over-duplication and/or amplification (Hanashiro et al., 2008). As another type of CDK-cyclin complex, the overactivation of CDK4/6–cyclin D has been shown to induce centrosome amplification (Nelsen et al., 2005). The major target of CDK4/6–cyclin D is the RB tumor-suppressor protein (Duensing et al., 2000). The conditional loss of *Rb* in mice results in centrosome amplification (Balsitis et al., 2003; Iovino et al., 2006).

CDK2 activity is also negatively controlled by the CDK inhibitor p21, one of the major transactivation targets of the p53 tumor-suppressor protein (Bálint & Vousden, 2001). p53 is involved in the regulation of centrosome duplication, which was first demonstrated in cells and tissues from *p53*-deficient mice (Fukasawa et al., 1996; Fukasawa et al., 1997). When cells are exposed to DNA-synthesis inhibitors such as hydroxyurea, centrosomes undergo reduplication without DNA synthesis, resulting in centrosome amplification (Balczon et al., 1995). Centrosome reduplication occurs efficiently when *p53* is mutated or lost (Tarapore et al., 2001a). In normal cells, p53 is stabilized under cellular stresses by the inhibition of MDM2, leading to the upregulation of p21, which blocks the initiation of centrosome reduplication through the inhibition of cyclin–CDK2 complexes (Bálint & Vousden, 2001). On the other hand, p21 is not upregulated in cells lacking *p53*, allowing the activation of CDK2, which in turn triggers centrosome reduplication.

Besides the p53–p21 pathway, p53 has the ability to control centrosome duplication. p53 is localized at centrosomes (Blair Zajdel & Blair, 1988; Brown et al., 1994; Tarapore et al., 2001b; Tritarelli et al., 2004; Ma et al., 2006a; Shinmura et al., 2007) and appears to control centrosome duplication independently of its transactivation function. Even if p53 is a mutant without transactivation function, p53 retains the ability to localize to centrosomes and partially suppresses centrosome duplication (Shinmura et al., 2007). However, the mechanism underlying this role of p53 is currently unknown.

The proteins that control p53 stability are also involved in the regulation of centrosome duplication. The ectopic expression of human papilloma virus (HPV) E6 protein, which promotes the degradation of p53, induces centrosome amplification (Duensing et al., 2000). MDM2 is an E3 ubiquitin ligase that promotes the degradation of p53 and is often overexpressed in cancers (Manfredi, 2010). The forced expression of MDM2 in cells containing wild-type p53 efficiently leads to centrosome amplification (Carroll et al., 1999). Aurora A kinase (AURKA) phosphorylates p53 at Ser315, resulting in MDM2-mediated p53 destabilization (Katayama et al., 2004), and the forced expression of Aurora A induces centrosome amplification (Zhou et al., 1998).

Another target of CDK2–cyclin E in centrosome regulation is MPS1, a spindle checkpoint kinase that is localized at the centrosome (Fisk et al., 2003). MPS1 is stabilized and activated by CDK2–cyclin E phosphorylation and involved in centrosome duplication. Mortalin, a member of the heat-shock protein 70 molecular chaperone family, is localized at the centrosome and physically interacts with and is phosphorylated by MPS1. The phosphorylation of mortalin activates MPS1 in a positive-feedback manner, and this phenomenon is important for MPS1-related centrosome duplication (Kanai et al., 2007).

CDK2 forms a complex with cyclin A in addition to cyclin E, and CDK2–cyclin A has been implicated in the regulation of centrosome duplication (Meraldi et al., 1999). CDK2–cyclin A and CDK2–cyclin E share some substrates (Tokuyama et al., 2001). The CDK2–cyclin A complex is active in S and G2 phases during the cell cycle, and CDK2–cyclin A may have a crucial role in centrosome over-duplication and/or amplification (Hanashiro et al., 2008). As another type of CDK-cyclin complex, the overactivation of CDK4/6–cyclin D has been shown to induce centrosome amplification (Nelsen et al., 2005). The major target of CDK4/6–cyclin D is the RB tumor-suppressor protein (Duensing et al., 2000). The conditional loss of *Rb* in mice results in centrosome amplification (Balsitis et al., 2003; Iovino

CDK2 activity is also negatively controlled by the CDK inhibitor p21, one of the major transactivation targets of the p53 tumor-suppressor protein (Bálint & Vousden, 2001). p53 is involved in the regulation of centrosome duplication, which was first demonstrated in cells and tissues from *p53*-deficient mice (Fukasawa et al., 1996; Fukasawa et al., 1997). When cells are exposed to DNA-synthesis inhibitors such as hydroxyurea, centrosomes undergo reduplication without DNA synthesis, resulting in centrosome amplification (Balczon et al., 1995). Centrosome reduplication occurs efficiently when *p53* is mutated or lost (Tarapore et al., 2001a). In normal cells, p53 is stabilized under cellular stresses by the inhibition of MDM2, leading to the upregulation of p21, which blocks the initiation of centrosome reduplication through the inhibition of cyclin–CDK2 complexes (Bálint & Vousden, 2001). On the other hand, p21 is not upregulated in cells lacking *p53*, allowing the activation of

Besides the p53–p21 pathway, p53 has the ability to control centrosome duplication. p53 is localized at centrosomes (Blair Zajdel & Blair, 1988; Brown et al., 1994; Tarapore et al., 2001b; Tritarelli et al., 2004; Ma et al., 2006a; Shinmura et al., 2007) and appears to control centrosome duplication independently of its transactivation function. Even if p53 is a mutant without transactivation function, p53 retains the ability to localize to centrosomes and partially suppresses centrosome duplication (Shinmura et al., 2007). However, the

The proteins that control p53 stability are also involved in the regulation of centrosome duplication. The ectopic expression of human papilloma virus (HPV) E6 protein, which promotes the degradation of p53, induces centrosome amplification (Duensing et al., 2000). MDM2 is an E3 ubiquitin ligase that promotes the degradation of p53 and is often overexpressed in cancers (Manfredi, 2010). The forced expression of MDM2 in cells containing wild-type p53 efficiently leads to centrosome amplification (Carroll et al., 1999). Aurora A kinase (AURKA) phosphorylates p53 at Ser315, resulting in MDM2-mediated p53 destabilization (Katayama et al., 2004), and the forced expression of Aurora A induces

Mortalin is frequently upregulated in cancers (Wadhwa et al., 2006).

CDK2, which in turn triggers centrosome reduplication.

mechanism underlying this role of p53 is currently unknown.

centrosome amplification (Zhou et al., 1998).

et al., 2006).

Polo-like kinase 1 (PLK1) is a key regulator of centrosome maturation (Barr et al., 2004; Bettencourt-Dias and Glover, 2007). Its deregulation is linked to centrosome abnormalities and oncogenesis (Zyss and Gergely, 2009). PLK1 belongs to the mammalian PLK family, which is comprised of five members (PLK1 - PLK4 and PLK5P) (Lens et al., 2010). PLK1 is involved in a variety of mitotic events, including centrosome maturation and separation, G2/M transition, mitotic spindle formation, chromosome segregation, and cytokinesis, and several kinds of PLK1 substrates are known (Barr et al., 2004; Petronczki et al., 2008). PLK1 targets multiple centrosomal proteins (e.g., -tubulin) to fulfill the mitotic function of centrosomes. Ninein-like protein (NLP) interacts with TuRC during interphase, and participates in the establishment of the cytoplasmic microtubule network (Casenghi et al., 2003; Rapley et al., 2005). At the onset of mitosis, the cooperation of PLK1 and NLP promotes the centrosomal localization of -tubulin and other mitosis specific PCM components, resulting in a higher microtubule nucleation capacity of the mitotic centrosome (Casenghi et al., 2003; Rapley et al., 2005). The phosphorylation of NEDD1 by PLK1 is required for the targeting of TuRC to the centrosome (Zhang et al., 2009). In mitosis, centrosomes must withstand the pulling forces exerted by chromosome-attached microtubules. To withstand such forces, PLK1 also plays a role in maintaining the structural integrity of the centrosome during mitosis (Oshimori et al., 2006). Kizuna is localized at the centrosomes and is phosphorylated by PLK1 during mitosis. The reduced expression of kizuna results in centrosome fragmentation and the dispersion of PCM, leading to the formation of aberrant mitotic spindles and chromosome segregation errors.

Another PLK, PLK4, is involved in recruiting the structural components required for the formation of procentrioles at the proximal side of the older centriole, in cooperation with CDK2-cyclin E (Habedanck et al., 2004). The upregulation of PLK4 expression is a strong stimulus for centriole multiplication (Kleylein-Sohn et al., 2007). The timely degradation of PLK4 by the SCF slimb ubiquitin ligase is important for the restriction of procentriole formation (Cunha-Ferreira et al., 2009). The SCF component CUL1 also functions as a centrosomal suppressor of centriole multiplication by regulating the PLK4 protein level (Korzeniewski et al., 2009). PLK4 kinase activity also regulates its own stability (Holland et al., 2010; Guderian et al., 2010). CEP152 interacts with PLK4 and CPAP and controls centrosome duplication in human cells (Dzhindzhev et al., 2010). PLK4 is transcriptionally regulated by p53 (Li et al., 2005). Clinically, the expression of PLK4 is upregulated in colon cancer (Macmillan et al., 2001), while the expression of PLK4 is downregulated in hepatocellular carcinoma because of promoter hypermethylation and the loss of heterozygosity (LOH) (Pellegrino et al., 2010; Rosario et al., 2010).

The role of the morgana/chp-1 in centrosome regulation has been reported by Ferretti et al. (2010). Mutations in morgana result in centrosome amplification. Morgana forms a complex with Hsp90, ROCK1 and ROCK2, and directly binds to ROCK2. Morgana downregulation promotes the interaction between ROCK2 and NPM, leading to an increase in ROCK2 activity, which in turn results in centrosome amplification. Morgana is downregulated in a large fraction of lung and breast cancers. They suggested that morgana plays a role in preventing centrosome amplification and tumorigenesis.

NLP, a previously described substrate of PLK1 (Casenghi et al., 2003), is a BRCA1-associated centrosomal protein that is involved in microtubule nucleation and spindle formation (Jin et al., 2009). NLP is overexpressed as a result of *NLP* gene amplification in lung cancer, and NLP overexpression causes centrosome amplification (Shao et al., 2010).

The *BRCA1* gene is responsible for susceptibility to familial breast/ovarian cancer and participates in diverse cellular functions (Venkitaraman, 2002). The BRCA1 is localized at the centrosomes (Hsu & White, 1998; Okada & Ouchi, 2003) and is involved in the regulation of centrosome duplication (Xu et al., 1999). BRCA1 is associated with BARD1, and this association mediates the ubiquitylation of -tubulin, which is important for maintaining the numeral integrity of centrosomes. The *BRCA2* gene is another causative gene of familial breast/ovarian cancer and its protein product functions in homologous recombination (HR) repair (Venkitaraman, 2002). The loss of *BRCA2* results in centrosome amplification (Tutt et al., 1999), implying a relationship between a defect in DNA repair and the abnormal amplification of the centrosomes. HR repair is mediated by several proteins including RAD51, and the downregulation of RAD51 leads to centrosome amplification (Bertrand et al., 2003). The reduced expression or loss of XRCC2, XRCC3, and RAD51B-D, which are other HR components, induces centrosome amplification and chromosome instability (Griffin et al., 2000; Smiraldo et al., 2005; Date et al., 2006; Renglin Lindh et al., 2007; Cappelli et al., 2011).

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase (Dodson et al., 2004). A centrosome-autonomous signal that involves centriole disengagement causes centrosome amplification in G2 phase after DNA damage (Inanç et al., 2010), suggesting that genotoxic stress can decouple the centrosome cycle and chromosome cycle.

The active nucleocytoplasmic transport of proteins is mediated by the nuclear localization signal (NLS) and nuclear export signal (NES) (Turner & Sullivan, 2008). NLS-containing proteins are transported from the cytoplasm to the nucleus, whereas NES-containing proteins are exported from the nucleus to the cytoplasm by XPO1, the human homolog of yeast Crm1. The inhibition of XPO1 causes centrosome amplification via the disruption of the nucleocytoplasmic transport of NPM (Forgues et al., 2003; Shinmura et al., 2005; Wang et al., 2005). XPO1 is involved in the centrosomal localization of various proteins (Han et al., 2008). Importin and RANBP1 are other proteins involved in nucleocytoplasmic transport, and these proteins also have the ability to regulate centrosomes (Di Fiore et al., 2003; Ciciarello et al., 2004).

SGOL1 interacts with protein phosphatase 2A, is localized in the centromere, and prevents the cohesin complex from precocious cleavage at the centromere via the dephosphorylation of SA2, one of the cohesin subunits (Kitajima et al., 2006; Riedel et al., 2006). Clinically, SGOL1 expression is downregulated in colorectal cancer, and SGOL1-knockdown leads to centrosome amplification and chromosome instability in a colon cancer cell line (Iwaizumi et al., 2009; Dai et al., 2009). A SGOL1-P1 transcript containing an exon-skip of exon 3, resulting in the formation of a premature stop codon, is expressed in colorectal cancer, and the overexpression of SGOL1-P1 in a colon cancer cell line resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids, delayed mitotic progression, and centrosome amplification (Kahyo et al., 2011). Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere, suggesting that SGOL1-P1 may function as a negative factor to native SGOL1 (Kahyo et al., 2011).

#### **2.4 Relationship between centrosome abnormalities and chromosome instability**

Chromosome instability is defined as a persistently high rate of the gain and loss of whole chromosomes (Thompson et al., 2010). Chromosome instability is a major source of

The *BRCA1* gene is responsible for susceptibility to familial breast/ovarian cancer and participates in diverse cellular functions (Venkitaraman, 2002). The BRCA1 is localized at the centrosomes (Hsu & White, 1998; Okada & Ouchi, 2003) and is involved in the regulation of centrosome duplication (Xu et al., 1999). BRCA1 is associated with BARD1, and this association mediates the ubiquitylation of -tubulin, which is important for maintaining the numeral integrity of centrosomes. The *BRCA2* gene is another causative gene of familial breast/ovarian cancer and its protein product functions in homologous recombination (HR) repair (Venkitaraman, 2002). The loss of *BRCA2* results in centrosome amplification (Tutt et al., 1999), implying a relationship between a defect in DNA repair and the abnormal amplification of the centrosomes. HR repair is mediated by several proteins including RAD51, and the downregulation of RAD51 leads to centrosome amplification (Bertrand et al., 2003). The reduced expression or loss of XRCC2, XRCC3, and RAD51B-D, which are other HR components, induces centrosome amplification and chromosome instability (Griffin et al., 2000; Smiraldo et al., 2005; Date et al., 2006; Renglin Lindh et al.,

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase (Dodson et al., 2004). A centrosome-autonomous signal that involves centriole disengagement causes centrosome amplification in G2 phase after DNA damage (Inanç et al., 2010), suggesting that genotoxic stress can decouple the centrosome cycle and chromosome cycle. The active nucleocytoplasmic transport of proteins is mediated by the nuclear localization signal (NLS) and nuclear export signal (NES) (Turner & Sullivan, 2008). NLS-containing proteins are transported from the cytoplasm to the nucleus, whereas NES-containing proteins are exported from the nucleus to the cytoplasm by XPO1, the human homolog of yeast Crm1. The inhibition of XPO1 causes centrosome amplification via the disruption of the nucleocytoplasmic transport of NPM (Forgues et al., 2003; Shinmura et al., 2005; Wang et al., 2005). XPO1 is involved in the centrosomal localization of various proteins (Han et al., 2008). Importin and RANBP1 are other proteins involved in nucleocytoplasmic transport, and these proteins also have the ability to regulate centrosomes (Di Fiore et al., 2003;

SGOL1 interacts with protein phosphatase 2A, is localized in the centromere, and prevents the cohesin complex from precocious cleavage at the centromere via the dephosphorylation of SA2, one of the cohesin subunits (Kitajima et al., 2006; Riedel et al., 2006). Clinically, SGOL1 expression is downregulated in colorectal cancer, and SGOL1-knockdown leads to centrosome amplification and chromosome instability in a colon cancer cell line (Iwaizumi et al., 2009; Dai et al., 2009). A SGOL1-P1 transcript containing an exon-skip of exon 3, resulting in the formation of a premature stop codon, is expressed in colorectal cancer, and the overexpression of SGOL1-P1 in a colon cancer cell line resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids, delayed mitotic progression, and centrosome amplification (Kahyo et al., 2011). Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere, suggesting that SGOL1-P1 may function as a

**2.4 Relationship between centrosome abnormalities and chromosome instability**  Chromosome instability is defined as a persistently high rate of the gain and loss of whole chromosomes (Thompson et al., 2010). Chromosome instability is a major source of

2007; Cappelli et al., 2011).

Ciciarello et al., 2004).

negative factor to native SGOL1 (Kahyo et al., 2011).

aneuploidy (Lengauer et al., 1997; Rajagopalan and Lengauer, 2004), and chromosome instability is thought to be involved not only in cancer initiation, where aneuploidy may have a causal role, but also in cancer development, where increased rates of chromosome missegregation may enable the clonal expansion of cells with a greater malignant potential (Rajagopalan & Lengauer, 2004; Weaver et al., 2007; Gao et al., 2007; Ganem et al., 2009). Defects in chromosome cohesion, weakened spindle assembly checkpoint (SAC) signalling, impaired microtubule-kinetochore attachment, defects in cell cycle regulation, and centrosome abnormalities can cause chromosome instability (Lingle et al., 1998; Draviam et al., 2004; Thompson & Compton, 2008; Weaver & Cleveland, 2008; Thompson et al., 2010). Regarding centrosome abnormalities, two mechanisms underlying chromosome instability have been proposed. The first mechanism is that centrosome amplification generates chromosome instability by promoting multipolar anaphase, which is an abnormal division that produces more than three aneuploid daughter cells (Nigg, 2002). The other mechanism is that centrosome amplification generates chromosome instability by promoting merotelic kinetochore–microtubule attachments (Ganem et al., 2009; Silkworth et al., 2009). Merotely is a type of error in which single kinetochores attach to microtubules emanating from different poles (Salmon et al., 2005; Cimini, 2008) and is common in cells showing chromosome instability (Thompson & Compton, 2008). Cells with centrosome amplification often coalesce the extra centrosomes during mitosis to ensure that anaphase occurs with a bipolar spindle (Quintyne et al., 2005). The extra centrosomes induce transient multipolar spindle intermediates prior to the coalescence of the centrosomes into bipolar spindles; this event increases the incidence of merotelic kinetochore–microtubule attachments and elevates the chromosome missegregation rates (Ganem et al., 2009; Silkworth et al., 2009). Ganem et al. (2009) showed that the presence of extra centrosomes is correlated with an increase in lagging chromosomes (Figure 3), promoting chromosome missegregation through excessive merotely induced by transient multipolar spindle intermediates. Since merotelic attachments are poorly sensed by the SAC (Salmon et al., 2005; Cimini, 2008), the merotelic attachments arising from centrosome amplification are not fully repaired and give rise to lagging chromosomes during anaphase, possibly leading to missegregation events.

Fig. 3. Lagging chromosomes in human cancer cells.

(A, B) Lagging chromosome formation detected in a B[*a*]PDE-treated H1299 lung cancer cell line. (A) Normal segregation; (B) an anaphase cell showing lagging chromosome formation. The nuclei were stained with DAPI (blue). (C) Lagging chromosomes are shown in a hematoxylin-and-eosin-stained section of a squamous cell carcinoma of the lung. In (B) and (C), the arrows indicate lagging chromosomes.

## **3. Conclusion**

The progress in our understanding of the relationship between centrosome abnormalities and cancer during the past 15 years has been enormous. We have learned that centrosome abnormalities are common among diverse human cancers including lung cancer. Many molecules are involved in the control of the numeral and/or functional integrity of centrosomes, and the abrogation of these mechanisms results in centrosome abnormalities, which promote chromosome instability. From a therapeutic standpoint, anti-cancer drugs targeting the centrosome have now been developed (Mazzorana et al., 2011). Future studies using a genome-wide approach and new scientific technologies will further increase our knowledge of the role of the centrosome in human cells, and such knowledge will likely help to establish effective cancer therapies.

## **4. Acknowledgment**

This work was supported by grants from the MHLW (21-1), the JSPS (22590356), the MEXT (20014007 and 221S0001), and the Smoking Research Foundation.

#### **5. References**


The progress in our understanding of the relationship between centrosome abnormalities and cancer during the past 15 years has been enormous. We have learned that centrosome abnormalities are common among diverse human cancers including lung cancer. Many molecules are involved in the control of the numeral and/or functional integrity of centrosomes, and the abrogation of these mechanisms results in centrosome abnormalities, which promote chromosome instability. From a therapeutic standpoint, anti-cancer drugs targeting the centrosome have now been developed (Mazzorana et al., 2011). Future studies using a genome-wide approach and new scientific technologies will further increase our knowledge of the role of the centrosome in human cells, and such knowledge will likely

This work was supported by grants from the MHLW (21-1), the JSPS (22590356), the MEXT

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## **Defective Expression and DNA Variants of TGFBR2 in Chinese Small Cell Lung Carcinoma**

ZhenHong Zhao1, Jibin Xu2, Jun Xie2, Yang Bao2,3, Xiaotian Wang1, Lei Wang2, Junjie Wu4, Li Jin1, Zhiyun Xu2\* and Jiucun Wang1,\* *1MOE Key Laboratory of Contemporary Anthropology and State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 2Department of Cardiac and Thoracic Surgery, Changhai Hospital, Shanghai, 3Yangzhou No.1 People's Hospital, Jiangsu Province, 4Department of Pneumology, Changhai Hospital, Shanghai, China* 

#### **1. Introduction**

188 Lung Diseases – Selected State of the Art Reviews

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Lung cancer is one of the most commonly diagnosed malignant tumors, and has the highest death rate of all cancer types. Both the incidence rate and death rate of lung cancer have increased rapidly worldwide during the last 50 years. Lung cancer has now become the leading cause of cancer death in males, and the second most common cause of cancer death in females, after breast cancer. According to data provided by the International Agency for Research on Cancer, about 1.6 million new lung cancer patients were confirmed in 2008, accounting for 13% of the total cancer cases, while about 1.4 million patients died, amounting to 18% of the total deaths caused by cancer worldwide (Jemal et al., 2011).

Lung cancer can be divided according to histological subtype into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with the latter accounting for about 14% of new lung cancer cases in the USA and Europe in 2004 (Jemal et al., 2004). The clinical and histological features of SCLC were first recognized by Barnard in 1926 as being distinct from those of other types of lung cancer (Barnard, 1926). SCLC cells develop from lung Kulchitsky cells, and SCLC can be further subdivided into three different types: oat-cell type, intermediate-cell type and mixed-cell type (Travis, 1999). Smoking is the key risk factor for SCLC, and more than 95% of patients develop SCLC as a result of tobacco smoking. Smoking more cigarettes and prolonging the duration of smoking can both increase the risk of developing SCLC (Brownson et al., 1992), while stopping smoking reduces its risk, compared to persistent smokers (Khuder and Mutgi, 2001; Jackman and Johnson, 2005). SCLC is very aggressive and the median survival time without treatment is less than 4 months. Chemotherapy and radiotherapy represent the two major treatments for SCLC. According to the standards developed by the Veterans Administration Lung Cancer Study Group, SCLC can be divided into two stages: a limited stage and an extensive stage (Simon, 2003). Cancer cells in limited-stage SCLC are restricted to the ipsilateral hemithorax

<sup>\*</sup> Corresponding Author

and can be treated by both chemotherapy and radiation therapy. About 20% of patients are cured after treatment, and the median survival time is about 18 months. Patients with extensive-stage SCLC have a high response rate to chemotherapy, which is the primary treatment for this disease, but the median survival time is only about 9 months because most patients relapse and the results of salvage therapy are poor (Janne et al., 2002; Demedts et al., 2010).

Transforming growth factor-beta (TGF-β) belongs to a large superfamily of cell cytokines, and is an important component of several cellular metabolic pathways. TGF-β signaling pathways regulate many aspects of cellular function, such as cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance and survival (Jakowlew, 2006). TGF-β plays a very complex dual role in cancer development, progress and metastasis (Akhurst & Derynck, 2001; Elliott & Blobe, 2005). TGF-β inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis as a tumor suppressor during the early phase of tumorigenesis (Arteaga, et al. 1993), but also promotes tumor invasion and metastasis by inducing the epithelial-mesenchymal transition in some epithelial cells, indicating that TGF-β can also act as a tumor promoter in the late stage of cancer (Miyazono, 2009).

There are two types of TGF-β signaling pathways; Smad-dependent and Smad-independent pathways. In Smad-dependent TGF-β signaling pathways, autocrine or exogenous TGF-β binds to the TGFBR2 and TGFBR1 membrane receptors. TGFBR2 then phosphorylates TGFBR1, which activates receptor-regulated Smads (also known as R-Smads). The R-Smads usually comprise Smad2 and Smad3. Activated Smad2 and Smad3 form complexes with Smad4, the common-partner Smad (co-Smad) in mammals. The subsequent R-Smad-co-Smad complexes shuttle between the nucleus and cytoplasm, and interact with various transcription factors and transcriptional co-activators such as AP-1, Sp1, p300, and SMIF to regulate the transcription of target genes (Derynck & Zhang, 2003). The phosphorylation of R-Smads can be blocked by inhibitory Smad, which starts the ubiquitination and degradation of the R-Smad-co-Smad complexes, thus inhibiting signal transduction (Itoh & ten Dijke, 2007). This TGF-β signal transduction pathway mainly regulates cell metabolism through this network involving cell cycle capture and apoptosis. In addition to Smadmediated signaling pathways, TGF-β also activates other pathways, including Erk, JNK and p38 MAPK kinase pathways, via Smad-independent mechanisms (Moustakas & Heldin, 2007).

Both Smad-dependent and -independent TGF-β signaling pathways start by binding TGF-β to its transmembrane receptor TGFBR2, which then activates the downstream signal transduction. However, TGFBR2 expression is often reduced or even blocked in tumor cells (Levy & Hill, 2006). In bladder cancer, deficient TGFBR2 expression leads to loss of the growth inhibition function of TGF-β, and loss of expression of TGFBR2 has been shown to correlate with tumor grade (Tokunaga, et al., 1999). Other studies also found that inactivation of TGFBR2 played a central role in the development and progression of human gastric cancer, and TGFBR2 expression has shown a strong association with the degree of malignancy in gastric cancer (Chang, et al., 1997). The expression of TGFBR2 was also reduced in breast cancer (Gobbi, et al. 2000). Although the reasons for defective TGFBR2 expression are still unknown, loss of or reduced expression of TGFBR2 may be caused by histone deacetylation in lung cancer cell lines (Osada et al., 2001).

TGFBR2 mutations have also been observed in tumor cells. A DNA variant with a frameshift mutation in the poly(A)10 repeat, resulting in microsatellite instability (MSI), has been detected in the coding region of the TGFBR2 gene in several types of tumors, including colon cancer, gastric cancer, and gliomas (Markowitz et al. 1995; Pinto et al., 1997; Izumoto et al. 1997). This frameshift could affect gene function and be related to cancer development. This MSI also been detected in both NSCLC and SCLC (Kim et al., 2000; Tani et al., 1997), though the mutation rate seems to be much lower than that of deficient TGFBR2 expression rate in lung cancer. A previous study identified a novel microdeletion (c.492\_507del) in giant cell carcinoma (GCC) and large cell carcinoma (LCC) patients, compared to other NSCLC subtypes. This 16-bp microdeletion introduced a premature stop codon at positions 590–592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of a kinase domain. Although the mutated TGFBR2 played an important role in the abrogation of TGF-β signal transduction in LCC cells (Wang et al., 2007), it was not correlated with the reduced TGFBR2 expression seen in NSCLC (Xu et al., 2007).

However, TGFBR2 has rarely been studied in Chinese SCLC samples and its role in TGF-β insensitivity in this population thus remains unknown. The present study therefore examined the levels of TGFBR2 expression in 27 pairs of formalin-fixed, paraffin-embedded SCLC tumors and compared them with NSCLC samples. The entire cDNA region and promoter of the gene was then sequenced to identify the causal variants in the TGFBR2 gene that accounted for its defective expression.

## **2. Materials and methods**

#### **2.1 Specimens**

190 Lung Diseases – Selected State of the Art Reviews

and can be treated by both chemotherapy and radiation therapy. About 20% of patients are cured after treatment, and the median survival time is about 18 months. Patients with extensive-stage SCLC have a high response rate to chemotherapy, which is the primary treatment for this disease, but the median survival time is only about 9 months because most patients relapse and the results of salvage therapy are poor (Janne et al., 2002; Demedts

Transforming growth factor-beta (TGF-β) belongs to a large superfamily of cell cytokines, and is an important component of several cellular metabolic pathways. TGF-β signaling pathways regulate many aspects of cellular function, such as cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance and survival (Jakowlew, 2006). TGF-β plays a very complex dual role in cancer development, progress and metastasis (Akhurst & Derynck, 2001; Elliott & Blobe, 2005). TGF-β inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis as a tumor suppressor during the early phase of tumorigenesis (Arteaga, et al. 1993), but also promotes tumor invasion and metastasis by inducing the epithelial-mesenchymal transition in some epithelial cells, indicating that TGF-β can also act as a tumor promoter in the late

There are two types of TGF-β signaling pathways; Smad-dependent and Smad-independent pathways. In Smad-dependent TGF-β signaling pathways, autocrine or exogenous TGF-β binds to the TGFBR2 and TGFBR1 membrane receptors. TGFBR2 then phosphorylates TGFBR1, which activates receptor-regulated Smads (also known as R-Smads). The R-Smads usually comprise Smad2 and Smad3. Activated Smad2 and Smad3 form complexes with Smad4, the common-partner Smad (co-Smad) in mammals. The subsequent R-Smad-co-Smad complexes shuttle between the nucleus and cytoplasm, and interact with various transcription factors and transcriptional co-activators such as AP-1, Sp1, p300, and SMIF to regulate the transcription of target genes (Derynck & Zhang, 2003). The phosphorylation of R-Smads can be blocked by inhibitory Smad, which starts the ubiquitination and degradation of the R-Smad-co-Smad complexes, thus inhibiting signal transduction (Itoh & ten Dijke, 2007). This TGF-β signal transduction pathway mainly regulates cell metabolism through this network involving cell cycle capture and apoptosis. In addition to Smadmediated signaling pathways, TGF-β also activates other pathways, including Erk, JNK and p38 MAPK kinase pathways, via Smad-independent mechanisms (Moustakas & Heldin,

Both Smad-dependent and -independent TGF-β signaling pathways start by binding TGF-β to its transmembrane receptor TGFBR2, which then activates the downstream signal transduction. However, TGFBR2 expression is often reduced or even blocked in tumor cells (Levy & Hill, 2006). In bladder cancer, deficient TGFBR2 expression leads to loss of the growth inhibition function of TGF-β, and loss of expression of TGFBR2 has been shown to correlate with tumor grade (Tokunaga, et al., 1999). Other studies also found that inactivation of TGFBR2 played a central role in the development and progression of human gastric cancer, and TGFBR2 expression has shown a strong association with the degree of malignancy in gastric cancer (Chang, et al., 1997). The expression of TGFBR2 was also reduced in breast cancer (Gobbi, et al. 2000). Although the reasons for defective TGFBR2 expression are still unknown, loss of or reduced expression of TGFBR2 may be caused by

TGFBR2 mutations have also been observed in tumor cells. A DNA variant with a frameshift mutation in the poly(A)10 repeat, resulting in microsatellite instability (MSI), has been

histone deacetylation in lung cancer cell lines (Osada et al., 2001).

et al., 2010).

2007).

stage of cancer (Miyazono, 2009).

Twenty-seven formalin-fixed, paraffin-embedded SCLC samples and their corresponding normal tissues were collected by the Laboratory of the Department of Thoracic Surgery, Changhai Hospital between 2000 and 2007. All the patients had undergone pulmonary resection for primary SCLC at Changhai Hospital and had provided informed consent, and none had received preoperative radiotherapy or chemotherapy. The demographic and clinical features of these SCLC cases are summarized in Table 1. This research was conducted with the official approval of the academic advisory board of the Institute of Genetics, Fudan University, Shanghai, P. R. China.

An additional 65 formalin-fixed, paraffin-embedded NSCLC samples and their corresponding normal tissues were collected between 2005 and 2007, as a control group to compare with SCLC (Table 2). These tissues were also provided by the Laboratory of the Department of Thoracic Surgery, Changhai Hospital after obtaining the patients' consent. None of these patients had received radiotherapy or chemotherapy prior to surgery.

#### **2.2 Immunohistochemistry**

Expression of TGFBR2 was detected by immunohistochemistry assay using a monoclonal antibody against the extracellular domain of TGFBR2 (R & D Systems, Minneapolis, MN) via two-step immunohistochemical staining using the EnVision system (DAKO Cytomation, Denmark), as described in our previous report (26). In brief, after the paraffin sections were deparaffinized and hydrated, serial 4-μm thick sections were placed into 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. For antigen retrieval, the sections were treated with boiling 0.01 mol/L citrate buffer (pH 6.0) for 25 min and then incubated with 10% fetal calf serum for 20 min at room temperature. After the blocking serum was removed, the sections were incubated with the primary antibody (1:50) at room temperature for 1 h, followed by rinsing three times with phosphate-buffered saline (PBS).


Re: reduced TGFBR2 expression in tumor tissues, Loss: loss of TGFBR2 expression, Pr: preserved TGFBR2 expression.

The staining score of each tissue is the product of the proportion of positive staining cells and intensity scores.

Table 1. Clinical features and TGFBR2 expression of the 27 SCLC patients


Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 2. Clinical features and TGFBR2 expression in NSCLC samples

The sections were then incubated with a working solution of horseradish peroxidase-labeled goat anti-mouse immunoglobulin, as provided in the EnVision kit, for 30 min. Finally, the peroxidase activity was developed with 3,3-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Because NSCLC develops from bronchial epithelium precursors, human normal bronchial epithelium was used as a positive control. A negative control for each specimen was provided by treating the sections with PBS instead of the primary antibody.

#### **2.3 Interpretation of the staining and data evaluation**

192 Lung Diseases – Selected State of the Art Reviews

Re: reduced TGFBR2 expression in tumor tissues, Loss: loss of TGFBR2 expression, Pr: preserved

Table 1. Clinical features and TGFBR2 expression of the 27 SCLC patients

The staining score of each tissue is the product of the proportion of positive staining cells and intensity

TGFBR2 expression.

scores.

All sections were examined by standard light microscopy and scored semi-quantitatively on the basis of the percentage of immunoreactive cells and on the intensity of the staining reaction. The samples were initially classified into one of four grades, according to staining intensity: 0 (negative staining, equivalent to the negative control), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The percentages of positively-stained cells were assigned as 0 for 0–25%, 1 for 26–50%, 2 for 51–75% and 3 for 76–100%, respectively. The final score was determined as the product of the proportion and intensity scores, and ranged from 0–9. Samples were considered to be negatively stained if the final score was 0, and positively stained if the final score was 1–9. Moreover, cancer samples were classified as preserved- or reduced-type in terms of TGFBR2 expression, depending on whether the final score was the same as or less than that of its corresponding normal lung tissue.

## **2.4 DNA Extraction and mutation analysis**

Target cells from formalin-fixed, paraffin-embedded tissue sections were microdissected and scraped into microtubes. After deparaffinization with xylene and washing in ethanol, DNA was extracted by standard proteinase K digestion and phenol-chloroform extraction (Sambrook & Maniatis, 1989).

The presence of the 16-bp microdeletion in exon 4, which was previously detected in LCC and GCC, was examined in all SCLC tissues using the following forward and reverse primers to amplify the fragments of 117/101 bp, representing the wild/mutant alleles: 5' caccagcaatcctgacttgttg-3' and 5'-cggttaacgcggtagcagtag-3'. The MSI in exon 3 was detected by the STR method using an ABI 3100 Sequencer and the following forward and reverse primers were used to amplify the exon 3 fragment (normally 242 bp) of the TGFBR2 gene: 5'-tccaatgaatctcttcactc-3' and 5'-cccacacccttaagagaaga-3'. c.1167 C>T in exon 4 of TGFBR2 was detected by direct sequencing using an ABI 3100 Sequencer and the following forward and reverse primers to amplify the exon 4 fragment (242 bp) of the TGFBR2 gene: 5'-


cccaagatgcccatcgtg-3' and 5'-tcccaggctcaaggtaaagg-3'. The other primers used for promoter and exon sequencing are listed in Table 3.

Table 3. Primers used in the study

#### **2.5 Statistical analysis**

Data were analyzed using χ2 tests, corrected χ2 tests, or Fisher's exact tests. A P value of less than 0.05 was considered statistically significant.

## **3. Results**

#### **3.1 TGFBR2 expression was more often reduced in SCLC than in NSCLC**

TGFBR2 expression was assessed using immunohistochemistry. Normal human lung tissues and normal human bronchial epithelium were used as positive controls. Over 75% of cells in these tissues exhibited consistently strong staining, both showing staining scores of 3 × 3 = 9, indicating normal TGFBR2 expression (Figure 1). Immunostaining of TGFBR2 was performed in 27 SCLC tumor tissue samples and their corresponding normal tissues. All the normal tissues showed strong staining in over 75% cells with staining scores of 9. One SCLC sample showed negative TGFBR2 expression (score of 0), while the remaining 26 were TGFBR2-positive. Furthermore, 16 of the total 27 SCLC tumor samples showed reduced TGFBR2 expression (score of 1–6) and 10 showed preserved expression (Table 1).

None of the 65 NSCLC samples showed negative TGFBR2 expression (staining score of 0). In addition, only 35.4% (23/65) of all NSCLC tumor tissues showed reduced (score of 1–6) TGFBR2 expression and 64.6% (42/65) of tumors had preserved expression (score of 9) (Table 2). When adenocarcinoma and squamous cell carcinoma tissues were analyzed separately, the frequencies of preserved type were also higher (63.6% (21/33) and 66.7% (18/27) respectively) than those of reduced type (36.4% (12/33) and 33.4% (9/27) respectively). In contrast, the frequency of preserved type in SCLCs (47%, 10/27) was much lower than that of reduced type (63%, 17/27), indicating that reduced TGFBR2 expression was more frequent in SCLC cells (Table 1 & 2).

Fig. 1. Expression of TGFBR2 in lung cancer by immunohistochemical analysis (×400).


194 Lung Diseases – Selected State of the Art Reviews

cccaagatgcccatcgtg-3' and 5'-tcccaggctcaaggtaaagg-3'. The other primers used for promoter

Data were analyzed using χ2 tests, corrected χ2 tests, or Fisher's exact tests. A P value of less

TGFBR2 expression was assessed using immunohistochemistry. Normal human lung tissues and normal human bronchial epithelium were used as positive controls. Over 75% of cells in these tissues exhibited consistently strong staining, both showing staining scores of 3 × 3 = 9, indicating normal TGFBR2 expression (Figure 1). Immunostaining of TGFBR2 was performed in 27 SCLC tumor tissue samples and their corresponding normal tissues. All the normal tissues showed strong staining in over 75% cells with staining scores of 9. One SCLC

**3.1 TGFBR2 expression was more often reduced in SCLC than in NSCLC** 

Fragments Region Direction Sequence (5' - 3')

TGFBR2 promoter Promoter part1 Forward aactacaaaacatgtacaccagg TGFBR2 promoter Reverse ttctttaggtcgaagtctagagg TGFBR2 promoter Promoter part2 Forward atgcagaatctctgcctgcctc TGFBR2 promoter Reverse cgagagctttggccgacttt TGFBR2 promoter Promoter part3 Forward gtaaatacttggagcgaggaactc TGFBR2 promoter Reverse ttctgaacgtgcggtgggat TGFBR2 exon exon 1 Forward tcggtctatgacgagcag TGFBR2 exon Reverse gggaccccaggaagaccc TGFBR2 exon exon 2 Forward gggctggtatcaagttcatttg TGFBR2 exon Reverse ggagacagagatacactgactgtg TGFBR2 exon exon 3 Forward tccaatgaatctcttcactc TGFBR2 exon Reverse cccacacccttaagagaaga TGFBR2 exon exon 4-1 Forward ccaactccttctctccttgttttg TGFBR2 exon Reverse tccaagaggcatactcctcatagg TGFBR2 exon exon 4-2 Forward gtcgctttgctgaggtctataagg TGFBR2 exon Reverse ccaggctcaaggtaaaggggatctagca TGFBR2 exon exon 5 Forward ggcagctggaattaaatgatgggc TGFBR2 exon Reverse tgctcgaagcaacacatg TGFBR2 exon exon 6 Forward tttcctttgggctgcacatg TGFBR2 exon Reverse cctaagaggcaacttggttgaatc TGFBR2 exon exon 7 Forward ccaactcatggtgtccctttg TGFBR2 exon Reverse tctttggacatgcccagcctg TGFBR2 MSI Exon 3 Forward Fam-tccaatgaatctcttcactc TGFBR2 MSI Reverse cccacacccttaagagaaga TGFBR2 LOH Exon 4 Forward cccaagatgcccatcgtg TGFBR2 LOH Reverse tcccaggctcaaggtaaagg

and exon sequencing are listed in Table 3.

Table 3. Primers used in the study

than 0.05 was considered statistically significant.

**2.5 Statistical analysis** 

**3. Results** 


#### **3.2 No significant relationship was found between TGFBR2 expression and clinical features in SCLC patients**

The associations between TGFBR2 expression and other clinical features were analyzed. No significant associations were found between TGFBR2 expression and gender (P = 1.00), age (P = 0.14), tumor size (P = 1.00), nodal involvement (P = 1.00), metastasis (P = 1.00) or stage (P = 0.12) (Table 4).


Table 4. Association between TGFBR2 expression and clinical features of 27 SCLC patients

#### **3.3 TGFBR2 expression is related to tumor types**

The relationship between TGFBR2 expression and histological type was analyzed. Samples were categorized as SCLC or NSCLC subtypes because they developed from different lung cells. As shown in Table 5, a significant association between TGFBR2 expression and histological type was identified (P = 0.0151), indicating the existence of a significant difference in TGFBR2 expression levels between SCLC and NSCLC subtypes (Table 5).

For further statistical analysis, NSCLC cases were divided into AdC, SqC, Ad-SqC and other subtypes. Because of the sample sizes, comparisons were only made between SCLC and AdC, and between SCLC and SqC. The results demonstrated significant differences in TGFBR2 expression between SCLC and AdC, and between SCLC and SqC (P = 0.0402 and 0.0293, respectively) (Table 5).

#### **3.4 Mutations in exon 4 of TGFBR2**

In a previous study, we identified a microdeletion (c.492\_507del) in patients with GCC and LCC. We therefore investigated the occurrence of this microdeletion in SCLC in the present study. Genomic DNA was extracted from 21 pairs of formalin-fixed, paraffin-embedded SCLC tissues and their corresponding normal tissues. The coding and promoter regions of


Table 5. TGFBR2 expression in different subtypes of tumor

TGFBR2 were sequenced. The DNA from the other six pairs of tissues was degraded and was unsuitable for amplification. No microdeletion was observed in any of the tested SCLC samples.

However, another novel variant in exon 4 of TGFBR2 was identified in 11 of 21 SCLC tumor samples. This variant at c.1167 in the TGFBR2 coding region was T/T homozygous in eight out of 11 cases, and C/T heterozygous in the other three cases, compared with C/C homozygous in normal individuals. The corresponding normal samples for these were C/T heterozygous. In the other 10 pairs of samples, however, the site was C/C homozygous. These results suggest that loss of heterozygosity (LOH) occurred in the eight tumors whose alleles became T/T homozygous from C/T heterozygous (Figure 2 and Table 6).

Interestingly, this change was a synonymous mutation that did not alter the amino acid sequence. We investigated its effect on the expression of TGFBR2, and found that TGFBR2 expression was reduced in nearly all T carriers (81.8%), compared with that in normal tissues, while only 60% of CC carriers had reduced TGFBR2 expression (Table 6).

Fig. 2. LOH in SCLC.

196 Lung Diseases – Selected State of the Art Reviews

Age

Gender

Tumor Size

Nodal involvement

Metastasis

Stage

Cases Gender TGFBR2 expression Age P-value M F Re Pr

≤60 16(59.26%) 11 5 10 6 32-57 P=1.0000

Male 21(77.78%) 21 0 15 6 P=0.1358

T1 4 3 1 3 1 P=1.0000

N0 5 5 0 3 2 P=1.0000

M0 26 20 6 16 10 P=1.0000

Ⅰ 2 2 0 1 1 P=0.1164

Table 4. Association between TGFBR2 expression and clinical features of 27 SCLC patients

The relationship between TGFBR2 expression and histological type was analyzed. Samples were categorized as SCLC or NSCLC subtypes because they developed from different lung cells. As shown in Table 5, a significant association between TGFBR2 expression and histological type was identified (P = 0.0151), indicating the existence of a significant difference in TGFBR2 expression levels between SCLC and NSCLC subtypes (Table 5). For further statistical analysis, NSCLC cases were divided into AdC, SqC, Ad-SqC and other subtypes. Because of the sample sizes, comparisons were only made between SCLC and AdC, and between SCLC and SqC. The results demonstrated significant differences in TGFBR2 expression between SCLC and AdC, and between SCLC and SqC (P = 0.0402 and

In a previous study, we identified a microdeletion (c.492\_507del) in patients with GCC and LCC. We therefore investigated the occurrence of this microdeletion in SCLC in the present study. Genomic DNA was extracted from 21 pairs of formalin-fixed, paraffin-embedded SCLC tissues and their corresponding normal tissues. The coding and promoter regions of

>60 11(40.74%) 10 1 7 4 61-77

T2 16 11 5 14 9 T≥2

N1 10 8 2 14 8 N≥1

Female 6(22.22%) 0 6 2 4

M1 1 1 0 1 0

Ⅱ 11 8 3 5 6 Ⅲ 13 10 3 11 2 Ⅳ 1 1 0 0 1

T3 3 3 0 T4 4 4 0

N2 10 8 2 N3 2 2 0

**3.3 TGFBR2 expression is related to tumor types** 

0.0293, respectively) (Table 5).

**3.4 Mutations in exon 4 of TGFBR2** 

A. CC sequence in normal lung tissue from SCLC patients;

B. TC sequence in normal lung tissue from SCLC patients;

C. TT sequence in tumor tissue from SCLC patients.


+: tumor tissues; -: normal lung tissue of patients;

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 6. Relationship between LOH in exon 4 and TGFBR2 expression

#### **3.5 MSI in TGFBR2 in SCLC**

Poly(A)10/(A)9 heterozygosity in exon 3 of TGFBR2, representing MSI, was detected in 60% of SCLC samples (9 out of 15), as shown in Table 7. However, no association between MSI and TGFBR2 expression was found (P = 0.264).


+: tumor tissues; -: normal lung tissue of patients;

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression.

Table 7. MSI detection and relation with TGFBR2 expression

## **4. Discussion**

198 Lung Diseases – Selected State of the Art Reviews

Patient No. Tissue LOH TGFBR2 expression S1 <sup>+</sup> TT Pr - CT

S3 <sup>+</sup> CC Re - CC

S5 <sup>+</sup> CC Re - CC

S6 <sup>+</sup> TT Re - CT

S7 <sup>+</sup> CC Re - CC

S8 <sup>+</sup> TT Re - CT

S9 <sup>+</sup> CC Pr - CC

S11 <sup>+</sup> CC Pr - CC

S12 <sup>+</sup> TT Re - CT

S13 <sup>+</sup> TT Re - CT

S15 <sup>+</sup> CT Re - CT

S16 <sup>+</sup> CC Re - CC

S18 <sup>+</sup> CC Pr - CC

S19 <sup>+</sup> TT Pr - CT

S20 <sup>+</sup> CT Re - CT

S21 <sup>+</sup> CC Pr - CC

S22 <sup>+</sup> CT Re - CT

S23 <sup>+</sup> TT Re - CT

S25 <sup>+</sup> CC Re - CC

S26 <sup>+</sup> TT Re - CT

Poly(A)10/(A)9 heterozygosity in exon 3 of TGFBR2, representing MSI, was detected in 60% of SCLC samples (9 out of 15), as shown in Table 7. However, no association between MSI

Re: reduced TGFBR2 expression in tumor tissues, Pr: preserved TGFBR2 expression. Table 6. Relationship between LOH in exon 4 and TGFBR2 expression

<sup>+</sup> CC Re - CC

S27

**3.5 MSI in TGFBR2 in SCLC** 

+: tumor tissues; -: normal lung tissue of patients;

and TGFBR2 expression was found (P = 0.264).

Tumor cells are often able to escape from TGF-β-signaling-induced cell cycle capture and apoptosis. TGF-β has a dual function in tumor development (Akhurst & Derynck, 2001; Elliott & Blobe, 2005); it acts as a tumor suppressor during the initial stages of tumor development (Arteaga, et al. 1993), but promotes tumor progression during the later stages (Miyazono, 2009). High levels of TGF-β expression in tumor cells can induce tumor evolution by stimulating angiogenesis and through other potential immunosuppressive effects, as well as by directly affecting tumor cell invasion and metastasis (Pardali & Moustakas, 2007). These direct effects can be achieved via Smad-dependent pathways, or may be mediated by interference with these pathways (Derynck & Zhang, 2003). Changes in the TGF-β signaling pathway may lead to abnormal signal transduction and cause dysregulated cell growth and differentiation. The first step in any mechanism involves binding of autocrine or paracrine TGF-β to the TGFBR2 receptor on the cell membrane, before activation of various downstream receptors can occur. TGFBR2 thus plays a key role in TGF-β signaling pathways, and its expression is reduced or blocked in many tumors (Chang et al., 1997; Tokunaga et al., 1999; Gobbi et al. 2000;Levy & Hill, 2006), resulting in partial or complete disruption of the TGF-β pathway.

Previous studies demonstrated that TGFBR2 expression in NSCLC differed between LCC and AdC, SqC or non-LCC cases, but the role of defective TGFBR2 expression in the initiation and/or development of SCLC (Xu et al., 2007), and its expression status in SCLC remain largely unknown. Furthermore, SCLC is phenotypically distinct from and much more malignant than NSCLC. We therefore compared TGFBR2 expression between SCLC and NSCLC. Immunohistochemical staining with TGFBR2 antibody revealed significant differences in the incidence of reduced expression in SCLC (63.0% of cases) versus AdC (36.4% of cases, P = 0.0402) and SqC (33.3% of cases, P = 0.0293), or SCLC versus NSCLC (35% of cases, P = 0.0151). These differences in expression levels between SCLC and NSCLC were consistent with the histopathologic classification of these tumors, suggesting that defective TGFBR2 expression might contribute to the initiation and/or development of SCLC.

To determine the reason for the reduced expression of TGFBR2, we examined the mutation status of c.492 507del in exon 4, but found no changes in this sequence in SCLC tumor samples. We subsequently determined the MSI status in exon 3, and identified a DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat (which causes MSI) in the coding region of the TGFBR2 gene. A total of 60.0% of SCLC were poly(A)10/(A)9 heterozygous, but no association was found between the MSI and TGFBR2 expression. However, no MSI was identified in our previous study of NSCLC, suggesting that the MSI in SCLC is at least partly associated with its carcinogenesis. We also sequenced all seven exons and the promoter region of the TGFBR2 gene and identified a novel LOH at c.1167 in 38.1% (8/21) of SCLC tissues. Further analysis showed that most of the mutant T-allele carriers (81.8%) had reduced TGFBR2 expression in tumor tissues, compared with only 60% of C-allele carriers. These results suggest that the change from wild type to mutant type might contribute, at least in part, to the defective expression of TGFBR2 in SCLC patients, though further studies are needed to clarify the mechanisms responsible.

## **5. Conclusion**

The present study identified reduced TGFBR2 gene expression levels in formalin-fixed, paraffin-embedded sections from most SCLC tumors examined, suggesting that this might contribute to the initiation and/or development of SCLC. Sequencing analysis also indicated that change of the wild-type C-allele to the mutant T-allele at c.1167 might contribute to the defective expression of TGFBR2 in SCLC patients. Another DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat, leading to MSI, was found in the coding region of the TGFBR2 gene, but this was not associated with TGFBR2 expression.

These results suggest that defective expression of TGFBR2 might inactivate TGF-β signal transduction, leading to the loss of growth inhibition and acceleration of tumor formation, and that a C>T substitution at c.1167 might be partially responsible for this reduced expression of TGFBR2 in SCLC.

## **6. Acknowledgement**

This study was supported by grants from the National Science Foundation of China, grant number 30971594 to JC Wang and grant number 30890034 to L Jin.

## **7. References**

200 Lung Diseases – Selected State of the Art Reviews

the TGF-β signaling pathway may lead to abnormal signal transduction and cause dysregulated cell growth and differentiation. The first step in any mechanism involves binding of autocrine or paracrine TGF-β to the TGFBR2 receptor on the cell membrane, before activation of various downstream receptors can occur. TGFBR2 thus plays a key role in TGF-β signaling pathways, and its expression is reduced or blocked in many tumors (Chang et al., 1997; Tokunaga et al., 1999; Gobbi et al. 2000;Levy & Hill, 2006), resulting in

Previous studies demonstrated that TGFBR2 expression in NSCLC differed between LCC and AdC, SqC or non-LCC cases, but the role of defective TGFBR2 expression in the initiation and/or development of SCLC (Xu et al., 2007), and its expression status in SCLC remain largely unknown. Furthermore, SCLC is phenotypically distinct from and much more malignant than NSCLC. We therefore compared TGFBR2 expression between SCLC and NSCLC. Immunohistochemical staining with TGFBR2 antibody revealed significant differences in the incidence of reduced expression in SCLC (63.0% of cases) versus AdC (36.4% of cases, P = 0.0402) and SqC (33.3% of cases, P = 0.0293), or SCLC versus NSCLC (35% of cases, P = 0.0151). These differences in expression levels between SCLC and NSCLC were consistent with the histopathologic classification of these tumors, suggesting that defective TGFBR2 expression might contribute to the initiation and/or development of

To determine the reason for the reduced expression of TGFBR2, we examined the mutation status of c.492 507del in exon 4, but found no changes in this sequence in SCLC tumor samples. We subsequently determined the MSI status in exon 3, and identified a DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat (which causes MSI) in the coding region of the TGFBR2 gene. A total of 60.0% of SCLC were poly(A)10/(A)9 heterozygous, but no association was found between the MSI and TGFBR2 expression. However, no MSI was identified in our previous study of NSCLC, suggesting that the MSI in SCLC is at least partly associated with its carcinogenesis. We also sequenced all seven exons and the promoter region of the TGFBR2 gene and identified a novel LOH at c.1167 in 38.1% (8/21) of SCLC tissues. Further analysis showed that most of the mutant T-allele carriers (81.8%) had reduced TGFBR2 expression in tumor tissues, compared with only 60% of C-allele carriers. These results suggest that the change from wild type to mutant type might contribute, at least in part, to the defective expression of TGFBR2 in SCLC patients,

The present study identified reduced TGFBR2 gene expression levels in formalin-fixed, paraffin-embedded sections from most SCLC tumors examined, suggesting that this might contribute to the initiation and/or development of SCLC. Sequencing analysis also indicated that change of the wild-type C-allele to the mutant T-allele at c.1167 might contribute to the defective expression of TGFBR2 in SCLC patients. Another DNA variant with a frameshift mutation in the TGFBR2 poly(A)10 repeat, leading to MSI, was found in the coding region of

These results suggest that defective expression of TGFBR2 might inactivate TGF-β signal transduction, leading to the loss of growth inhibition and acceleration of tumor formation, and that a C>T substitution at c.1167 might be partially responsible for this reduced

though further studies are needed to clarify the mechanisms responsible.

the TGFBR2 gene, but this was not associated with TGFBR2 expression.

partial or complete disruption of the TGF-β pathway.

SCLC.

**5. Conclusion** 

expression of TGFBR2 in SCLC.


## **Neuroendocrine Tumours of the Lung**

Guadalupe Aparicio Gallego1, Vanessa Medina Villaamil1, Ana Capdevila Puerta2, Enrique Grande Pulido3 and L.M. Antón Aparicio4,5 *1Biomedical Research Institute, A Coruña University Hospital, A Coruña, 2Anatomic Pathology Service, A Coruña University Hospital, A Coruña, 3Medical Oncology Service, Ramon y Cajal University Hospital, Madrid, 4Medical Oncology Service, A Coruña University Hospital, A Coruña 5Medicine Department, University of A Coruña, A Coruña Spain* 

#### **1. Introduction**

202 Lung Diseases – Selected State of the Art Reviews

Kim, W.S., et al., Microsatellite instability(MSI) in non-small cell lung cancer(NSCLC) is

RII) frameshift mutation. Anticancer Research, 2000. 20(3A): p. 1499-1502. Kim, W.S., et al., Reduced transforming growth factor-beta type II receptor (TGF-beta RII)

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Osada, H., et al., Heterogeneous transforming growth factor (TGF)-beta unresponsiveness

Pardali K, Moustakas A: Actions of TGF-beta as tumor suppressor and pro-metastatic factor

Pinto, M., et al., Promoter methylation of TGF beta receptor I and mutation of TGF beta

Sambrook J, F.E., Maniatis T., Molecular cloning: A laboratory manual 2nd ed. Molecular cloning: A laboratory manual. 1989, New York: Cold Spring Harbor Laboratory.

Tani, M., et al., Infrequent mutations of the transforming growth factor beta-type II receptor

Tokunaga, H., et al., Decreased expression of transforming growth factor beta receptor type

Travis WD, C.T., Corrin B, Shimosato Y, Brambilla E. Epithelial tumours. In: Travis WD, CV,

Uchida, K., et al., Somatic in vivo alterations of the JV18-1 gene at 18q21 in human lung

Wang, J.-C., et al., Novel microdeletion in the transforming growth factor beta Type II

Xu, J. B., et al., Defective expression of transforming growth factor P type II receptor

carcinoma. Genes Chromosomes & Cancer, 2007. 46(2): p. 192-201.

lung cancer cell lines. Cancer Research, 2001. 61(22): p. 8331-8339.

Simon, G.R. and H. Wagner, Small cell lung cancer. Chest, 2003. 123(1): p. 259S-271S.

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highly associated with transforming growth factor-beta type II receptor(TGF-beta

expression in adenocarcinoma of the lung. Anticancer Research, 1999. 19(1A): p.

pathways in human cancer. Cytokine & Growth Factor Reviews, 2006. 17(1-2): p.

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transitions during embryogenesis and cancer progression. Cancer Science, 2007.

and loss of TGF-beta receptor type II expression caused by histone deacetylation in

receptor II are frequent events in MSI sporadic gastric carcinomas. Journal of

gene at chromosome 3p22 in human lung cancers with chromosome 3p deletions.

I is associated with poor prognosis in bladder transitional cell carcinoma patients.

Corrin B, et al., Histological typing of lung and pleural tumours, 3rd edn. Washington DC: Armed Forces Institute of Pathology. 1999, Washington DC:

receptor gene is associated with giant and large cell variants of nonsmall cell lung

(TGFBR2) in the large cell variant of non-small cell lung carcinoma. Lung Cancer,

Lung cancer arises from neoplastic changes of the epithelial cells in the lung. However, it is not known whether all or only a subset of these lung epithelial cells is susceptible to malignant transformation. Specifically, a major question is whether the changes need to take place in lung epithelial cells involving stem-cell–like properties. Lung cancer is a clinically, biologically, histologically, molecularly, and genetically heterogeneous disease. The underlying causes of this heterogeneity are unknown and could reflect changes occurring in cells with various potential for differentiation or represent different molecular changes occurring in the same lung epithelial target cells. Transformation from a normal to malignant lung cancer phenotype is thought to arise in a multistep fashion, through a series of genetic versus epigenetic alterations, ultimately evolving into an invasive cancer by clonal expansion. These progressive pathological changes in the bronchial epithelium occur primarily as one of three distinct morphological forms: squamous dysplasia, atypical adenomatous hyperplasia, and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. Bronchial squamous dysplasia and carcinoma in situ (CIS) are the recognised preneoplastic lesions for squamous cell carcinoma (SCC); atypical adenomatous hyperplasia (AAH), a putative preneoplastic lesion, for a subset of adenocarcinomas (ADC); and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) for neuroendocrine lung carcinomas. Pulmonary neuroendocrine tumours comprise approximately 2% of all lung malignancies. According to the most recent World Health Organization classification, pulmonary neuroendocrine tumours are histologically divided into a three-tier, fourcategory system including low-grade (typical carcinoid), intermediate-grade (atypical carcinoid), and high-grade (small cell carcinoma and large cell neuroendocrine carcinoma) tumours. Nearly all lung cancers exhibit the morphological and molecular features of epithelial cells and are accordingly classified as carcinomas. The cells of origin of virtually all lung cancers reside in the epithelial lining of the airways. As more is learned about the origin of neuroendocrine lung tumours, it is also increasingly clear that the biology of neuroendocrine lung tumours arising in the central airways (i.e., SCLC) is distinct from that of peripheral airway lesions. The purpose of this chapter is not so much to recapitulate the details of the neuroendocrine lung tumour classification but rather to provide an understanding of the main categories of lung carcinoma, to highlight potential pitfalls in the histopathological diagnosis of lung cancer, to summarise current information on molecular properties and cellular origins of individual neuroendocrine lung tumour subtypes, and to relate pathologies to biological behaviours.

#### **2. Neuroendocrine system of the lung**

The endocrine cells within the gut epithelium (foregut, midgut, and hindgut) constitute the largest population of hormone producing cells in the body. So far, approximately 10 different neuroendocrine lineages have been identified, and most of them show a specific rostro-caudal distribution. Pulmonary neuroendocrine cells (PNECs) are part of the diffuse neuroendocrine system (DNES) distributed throughout the body. The PNEC system (solitary PNECs and neuroepithelial bodies, or NEBs) consists of a distinct population of airway epithelial cells displaying endocrine and paracrine secretory mechanisms. Pulmonary neuroendocrine cells were readily demonstrated and uniformly distributed in normal adult human lungs. Overall, as identified by neurone specific enolase immunoreactivity, there were 10.5 neuroendocrine cells per 10 cm of epithelial length and 4 per 10,000 epithelial cells; they extended from the trachea to the alveolar ducts but none was seen in the alveoli (72% were in bronchi, 24% in bronchioles, and 4% in alveolar ducts). Of the cells identified by gastrin-releasing peptide immunoreactivity, there were 6.9 neuroendocrine cells per 10 cm of epithelial length and 2.4 per 10,000 epithelial cells. Of the cells identified by calcitonin immunoreactivity, there were 3.5 neuroendocrine cells per 10 cm of epithelial length and 1.3 per 10,000 epithelial cells. Minor cells contained serotonin (all in the terminal bronchioles), and in a small minority no peptide or amine was detected. It is currently thought that PNECs, like their counterparts in the gastrointestinal tract, are derived from multipotent epithelial progenitors, and that all epithelial cells arise from a single stem cell. All pulmonary epithelial cells including PNECs and non-NE airway epithelial cells are likely to be derived from a single stem cell. Epithelial lung stem cells, as in many organs, are often confined to discretely localised niches that are protected from environmental insults. In the lung, PNECs are associated with the stem cell niches in both the proximal and distal airways. One of the lung stem-cell niches is located in the trachea that reveals two stem-cell niches: gland ducts in the proximal compartment and select foci near the cartilage-intercartilage junction in the distal trachea. Other intrapulmonary stem cell niches include NEBs located at the airway bifurcation. Another stem cell niche is at the bronchoalveolar junctions, although PNECs may play a diminished role at this location. Among many functions assigned to them, there is a possible dual role: the regulation of lung maturation/growth and chemoreception. First, during the early stages of lung organogenesis, PNECs acting via their amine and peptide products may function as local modulators of lung growth and differentiation. Second, later in foetal life and in postnatal stages, PNECs and in particular innervated NEBs could play a role as airway chemoreceptors. The diffuse neuroendocrine system (DNES) of the lung involves neuroendocrine cells that have been shown to express a functional oxygen sensing mechanism. Aggregates of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways, preferentially located at the airway bifurcation of the lungs. Neuroendocrine cells are selectively contacted by different nerve fibres. NEBs are contacted by at least three different nerve-fibre

details of the neuroendocrine lung tumour classification but rather to provide an understanding of the main categories of lung carcinoma, to highlight potential pitfalls in the histopathological diagnosis of lung cancer, to summarise current information on molecular properties and cellular origins of individual neuroendocrine lung tumour subtypes, and to

The endocrine cells within the gut epithelium (foregut, midgut, and hindgut) constitute the largest population of hormone producing cells in the body. So far, approximately 10 different neuroendocrine lineages have been identified, and most of them show a specific rostro-caudal distribution. Pulmonary neuroendocrine cells (PNECs) are part of the diffuse neuroendocrine system (DNES) distributed throughout the body. The PNEC system (solitary PNECs and neuroepithelial bodies, or NEBs) consists of a distinct population of airway epithelial cells displaying endocrine and paracrine secretory mechanisms. Pulmonary neuroendocrine cells were readily demonstrated and uniformly distributed in normal adult human lungs. Overall, as identified by neurone specific enolase immunoreactivity, there were 10.5 neuroendocrine cells per 10 cm of epithelial length and 4 per 10,000 epithelial cells; they extended from the trachea to the alveolar ducts but none was seen in the alveoli (72% were in bronchi, 24% in bronchioles, and 4% in alveolar ducts). Of the cells identified by gastrin-releasing peptide immunoreactivity, there were 6.9 neuroendocrine cells per 10 cm of epithelial length and 2.4 per 10,000 epithelial cells. Of the cells identified by calcitonin immunoreactivity, there were 3.5 neuroendocrine cells per 10 cm of epithelial length and 1.3 per 10,000 epithelial cells. Minor cells contained serotonin (all in the terminal bronchioles), and in a small minority no peptide or amine was detected. It is currently thought that PNECs, like their counterparts in the gastrointestinal tract, are derived from multipotent epithelial progenitors, and that all epithelial cells arise from a single stem cell. All pulmonary epithelial cells including PNECs and non-NE airway epithelial cells are likely to be derived from a single stem cell. Epithelial lung stem cells, as in many organs, are often confined to discretely localised niches that are protected from environmental insults. In the lung, PNECs are associated with the stem cell niches in both the proximal and distal airways. One of the lung stem-cell niches is located in the trachea that reveals two stem-cell niches: gland ducts in the proximal compartment and select foci near the cartilage-intercartilage junction in the distal trachea. Other intrapulmonary stem cell niches include NEBs located at the airway bifurcation. Another stem cell niche is at the bronchoalveolar junctions, although PNECs may play a diminished role at this location. Among many functions assigned to them, there is a possible dual role: the regulation of lung maturation/growth and chemoreception. First, during the early stages of lung organogenesis, PNECs acting via their amine and peptide products may function as local modulators of lung growth and differentiation. Second, later in foetal life and in postnatal stages, PNECs and in particular innervated NEBs could play a role as airway chemoreceptors. The diffuse neuroendocrine system (DNES) of the lung involves neuroendocrine cells that have been shown to express a functional oxygen sensing mechanism. Aggregates of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways, preferentially located at the airway bifurcation of the lungs. Neuroendocrine cells are selectively contacted by different nerve fibres. NEBs are contacted by at least three different nerve-fibre

relate pathologies to biological behaviours.

**2. Neuroendocrine system of the lung** 

populations: vagal sensory calbidin D28k, calcitonin gene related peptide (CGRP)/substance P (SP) innervation, and intrinsic pulmonary nitrergic neurons.

#### **2.1 Neuroendocrine epithelial cells**

Neuroendocrine epithelial cells (NEC) of the respiratory system tend to occur either as single cells that are sparsely distributed throughout the epithelium of the tracheobronchial tract or in small, well-defined clusters that are supported by nonciliated bronchiolar (Clara) cells. The latter are referred to as neuroepithelial bodies (NEBs) and are located only in the epithelium of the intrapulmonary airways, often at or near a bronchiolar bifurcation. The solitary pulmonary NEC cells of most of the investigated species are fusiform or flaskshaped, resting on the basement membrane with an apical process pointing toward the airway lumen. Adult human NEC cells generally lack luminal contact. Although there are some differences between solitary NEC cells and NEBs, a large body of evidence points to their being a member of the amine precursor uptake and decarboxylation (APUD) cell series or of the paraneuron family. The endocrine system of the lung consists of at least two different cell categories. These categories exhibit similar main characteristics. They contain a biogenic amine and neuropeptide mediators, and their cytoplasm harbours neurosecretorylike granules. Regarding morphological features and location, these cells presumably have a receptor secretory function. Consequently, these classes of endocrine cells can be designated as paraneurons. Solitary NEC cells were found to be distributed over almost the entire respiratory system, while NEBs seemed to be restricted to the epithelium of the intrapulmonary airways. Neuroepithelial bodies generally consist of nonciliated, cylindrical cells with a palisade-like arrangement between the airway surface and the underlying connective tissue, although they may also appear as stratified cells. Most of the luminal side of the NEBs is covered by the supporting Clara cells. The NEBs are strategically located on the surface of the airway bifurcations. They, in fact, contain the serotonin-bioactive amine and neuropeptides, leading to the speculation of these cells as a homogeneous or heterogeneous class. They also exert control on pulmonary vessels and airway tone. The NEC cell may function as the transducer of the stimulus or the sensory nerve ending, the activity of the latter being modulated by the release of bioactive substances from NEC cells. Investigations have indicated the influence of NEC cells on epithelial cell differentiation, mucous secretion, and proliferation of local endoderm in developing airways.

#### **2.2 Pulmonary neuroendocrine cells**

Pulmonary neuroendocrine cells (PNECs) are commonly organised into innervated clusters, called NEBs, which have been proposed to serve various functions, including the regulation of embryonic lung growth and maturation through the elaboration of a variety of potent neuropeptides. Several studies have suggested that PNECs are quiescent cells with limited self-renewal capacity. However, it was recently demonstrated that PNECs have a selfrenewal capacity and can be activated to undergo multiple rounds of proliferation after TA (Clara) cell depletion. It has been suggested that PNEC-derived paracrine factors might play a role in the regulation of epithelial cell differentiation and proliferation during foetal lung development and possibly in the normal or injured adult lung. Cell proliferation has also been shown to contribute to the maintenance of PNE cells in the normal lung as well as in hyperplasia of this population in various disease states. PNE cells are known to act as a progenitor cell for the establishment of NEB hyperplasia and represent one of two proliferative populations within hyperplastic NEBs of the naphthalene-injured lung. Participation of non-PNE progenitor cells in this process has also been demonstrated and may contribute to the intermediate phase of NEB hyperplasia. These data suggest that multiple cell types contribute to the maintenance and expansion of the NEB-associated PNE population and that progenitor selection may be a dynamic feature of NEB hyperplasia. Findings from various studies have established PNE cells as a progenitor population that is sufficient for the development of both NEB hypertrophy and hyperplasia. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for the development of small cell lung carcinoma, mechanisms regulating the expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. However, when considering mechanisms that may account for PNE cell hyperplasia, the finding that multiple cell types proliferate in the NEB microenvironment raises the possibility that a non-PNE cell progenitor may yield progeny cells with the capacity to undergo PNE cell differentiation.

#### **2.3 Neuroepithelial bodies**

Neuroendocrine bodies were illustrated in 1949 in the description of neuroendocrine cells in the bronchiolar mucosa. Neuroendocrine bodies consist of a cluster of 4 to 10 neuroendocrine cells. On well-oriented sections, they can extend from the subepithelial basement membrane to the airway lumens. They are found not only in the epithelium of bronchi and bronchioles, but also in alveoli. The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of the associated PNEC. NEB-associated epithelial cells share many morphological and biochemical characteristics with cells that are distributed throughout the airway. Pulmonary NEBs are prime candidates to serve as sensory end organs in the lung. NEBs consist of highly organised clusters of specialised cells with neuroendocrine characteristics, arranged into organoids that are dispersed throughout the epithelium at all levels of the intrapulmonary airways. Structurally, NEB cells harbour cytoplasmic neurosecretory granules that are known to contain monoamine, peptide, and purine transmitters. Neuroendocrine cells are able to synthesise and release ATP, monoamine, and peptide transmitters, resulting in autocrine, paracrine, or endocrine effects. Morphologically, NEBs resemble other known chemoreceptors, such as taste buds and carotid bodies, and are thought to represent "chemosensors" among other possible functions. Hypoxic conditions appear to depolarise NEB cells via a potassium channel-mediated mechanism. In particular, the extensively innervated aggregates of the neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways but are preferentially located at the airway bifurcation points in the lungs. Proportionally, most NEBs are found in the bronchioles and in the terminal respiratory bronchioles. The NEB microenvironment may represent an analogous structure within the conducting airway epithelium for maintenance of an airway stem-cell pool. It may influence the phenotype of the CE cells, blocking the differentiation from Clara to ciliated cells and preserving a population of regenerative cells that can contribute to epithelial renewal after exposure to Clara cell toxicants. Regeneration of the chronically injured airway epithelium is associated with alterations in the number and cellularity of the NEBs as well as the enrichment of nascent epithelial cell populations of epithelial cells that are candidate stem cells. The NEB microenvironment is multifunctional, serving to maintain slow-cycling epithelial cells in the steady state epithelium and to stimulate the proliferation of TA cells either after airway injury or during airway development. Many studies have presented extensive evidence that NEBs in the lungs may be selectively contacted by at least 5 distinct nerve-fibre populations that are both sensory and motor in nature. In addition, they have different origins, indicating that NEBs should be regarded as very complex airway receptors that may be capable of accommodating various chemoand mechano-sensory modalities.

#### **2.3.1 Functions**

206 Lung Diseases – Selected State of the Art Reviews

proliferative populations within hyperplastic NEBs of the naphthalene-injured lung. Participation of non-PNE progenitor cells in this process has also been demonstrated and may contribute to the intermediate phase of NEB hyperplasia. These data suggest that multiple cell types contribute to the maintenance and expansion of the NEB-associated PNE population and that progenitor selection may be a dynamic feature of NEB hyperplasia. Findings from various studies have established PNE cells as a progenitor population that is sufficient for the development of both NEB hypertrophy and hyperplasia. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for the development of small cell lung carcinoma, mechanisms regulating the expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. However, when considering mechanisms that may account for PNE cell hyperplasia, the finding that multiple cell types proliferate in the NEB microenvironment raises the possibility that a non-PNE cell progenitor may yield progeny cells with the capacity to undergo PNE cell

Neuroendocrine bodies were illustrated in 1949 in the description of neuroendocrine cells in the bronchiolar mucosa. Neuroendocrine bodies consist of a cluster of 4 to 10 neuroendocrine cells. On well-oriented sections, they can extend from the subepithelial basement membrane to the airway lumens. They are found not only in the epithelium of bronchi and bronchioles, but also in alveoli. The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of the associated PNEC. NEB-associated epithelial cells share many morphological and biochemical characteristics with cells that are distributed throughout the airway. Pulmonary NEBs are prime candidates to serve as sensory end organs in the lung. NEBs consist of highly organised clusters of specialised cells with neuroendocrine characteristics, arranged into organoids that are dispersed throughout the epithelium at all levels of the intrapulmonary airways. Structurally, NEB cells harbour cytoplasmic neurosecretory granules that are known to contain monoamine, peptide, and purine transmitters. Neuroendocrine cells are able to synthesise and release ATP, monoamine, and peptide transmitters, resulting in autocrine, paracrine, or endocrine effects. Morphologically, NEBs resemble other known chemoreceptors, such as taste buds and carotid bodies, and are thought to represent "chemosensors" among other possible functions. Hypoxic conditions appear to depolarise NEB cells via a potassium channel-mediated mechanism. In particular, the extensively innervated aggregates of the neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium at all levels of the intrapulmonary airways but are preferentially located at the airway bifurcation points in the lungs. Proportionally, most NEBs are found in the bronchioles and in the terminal respiratory bronchioles. The NEB microenvironment may represent an analogous structure within the conducting airway epithelium for maintenance of an airway stem-cell pool. It may influence the phenotype of the CE cells, blocking the differentiation from Clara to ciliated cells and preserving a population of regenerative cells that can contribute to epithelial renewal after exposure to Clara cell toxicants. Regeneration of the chronically injured airway epithelium is associated with alterations in the number and cellularity of the NEBs

differentiation.

**2.3 Neuroepithelial bodies** 

It has been estimated that NEBs represent <1% of the epithelial cells in human lungs. Some of the supposed functions of NEB in mammalian lungs include the following: 1) the ability of NEB to function as transducers (hypoxia); 2) modulation of bronchomotor tone via targeting bronchial smooth muscle and the associated nerves located directly beneath NEB; 3) promotion and regulation of the growth of developing airways by stimulating the proliferation of local endoderm; 4) release of amine and peptide modulators; and 5) neonatal respiratory adaptation. The lung bud epithelium grows into the adjacent mesenchyme and stars branching to form the future bronchial tree. The various stages are divided into the embryonic, pseudoglandular, canalicular, saccullar and alveolar/microvascular periods. Pulmonary neuroendocrine cells are the first specialised epithelial cell type to appear in lung development. In humans, ultrastructurally distinct primitive PNECs (pre-NE cells), which contain serotonin and neuro-specific enolase (NSE), can be detected in the beginning of the pseudoglandular period. Solitary and clustered PNECs contain bombesin, the major neuropeptide in human lungs, which appears in the early weeks of gestation. As the distal segments of the developing airways elongate, a process referred to as the canalicular period, PNECs differentiate first, followed by ciliated and secretory (Clara) cells. Parallel with the increasing number of peripheral airways, the number of PNECs also increases. In the developing bronchioles, small NEBs composed of 3-5 bombesin and serotoninimmunoreactive cells appear at the airway branching points, and rare nerve endings have been demonstrated to be in contact with NEBs already in the human foetal lung. Proposed roles for PNECs in foetal and newborn lung development include the regulation of branching morphogenesis as well as cellular growth and maturation.

#### **2.3.2 Airway oxygen sensors**

Since 1930, evidence has accumulated to suggest that NEBs may function as hypoxiasensitive airway sensors. NEB cells express membrane-bound O2 sensors and are the transducers of the hypoxic stimulus. NEB cells respond to acute hypoxia, but apparently not to hypercapnia with the degranulation of dense core vesicles and release of 5 hydroxytryptamine (5-HT). Morphologic and experimental studies to support NEB functions as hypoxia-sensitive airway chemoreceptors modulated by the central nervous system include the following: a) preferential location of NEB at airway branching points; b) apical microvilli in contact with the airway lumen; c) cytoplasmic neurosecretory granules containing monoamine and neuropeptides; d) afferent sensory innervation derived from the vagus nerve; and e) proximity to blood capillaries. NEBs are predominantly innervated by sensory nerve fibres derived from cell bodies in the nodose ganglion of the vagus nerve. Morphological data support the role of NEBs as hypoxia-sensitive airway sensor systems. Studies on the effects of chronic hypoxia have shown induced cellular hyperplasia and hypertrophy in the peripheral chemoreceptors; chronic normobaric hypoxia showed a significant increase in the number of solitary pulmonary neuroendocrine cells (PNECs) as well as the enlargement of NEBs. NEB cells possess an oxygen-binding protein, cytochrome *b*, an NADPH oxidase located in the cellular membranes that acts as the O2 receptor both during normoxia and hypoxia.

## **3. Pathology of neuroendocrine tumours of the lung**

The neuroendocrine cell system is divided into cell types that form glands and diffusely distributed cells. This second group is collectively known as the diffuse neuroendocrine system (DNES), and its representatives are found in the lung, gastrointestinal tract, or urogenital tract. Neuroendocrine tumours of the lung arise from bronchial mucosal cells known as Kulchitsky cells, which are part of the DNES. The classification of lung neuroendocrine malignancies has been an evolving process (Table 1).

WHO/IASLC histological classification


WHO: World Health Organization; IASLC: International Association for the Study of Lung Cancer \* From Travis WD, Colby TV, Corrin B, et al in collaboration with pathologists from 14 countries. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer Verlag, 1999, with permission.

Table 1. Lung tumours with neuroendocrine morphology include the low-grade typical carcinoid (TC), intermediate-grade atypical carcinoid (AC), and the high-grade LCNEC and SCLC.

These classifications date back to 1972, when atypical carcinoids were initially defined according to histological criteria, including the number of mitoses per high-power field (hpf), the presence of necrosis, increased cellularity with disorganisation, nuclear pleomorphism, hyperchromatism, and an abnormal nuclear to cytoplasmic ratio (Table 2).

In 1991, a new classification proposed 4 categories of neuroendocrine lung tumours that included the following: typical carcinoid (TC), which is a low-grade malignancy; atypical carcinoid (AC), which is a medium-grade malignancy; large-cell neuroendocrine carcinoma (LCNEC), which is a high-grade malignancy; and small-cell lung cancer (SCLC), which is also a high-grade malignancy. The 2004 WHO categorisation of tumours with neuroendocrine features included the classic carcinoid low-grade TC and intermediategrade AC, as well as the high-grade malignancies LCNEC and SCLC.

Morphological data support the role of NEBs as hypoxia-sensitive airway sensor systems. Studies on the effects of chronic hypoxia have shown induced cellular hyperplasia and hypertrophy in the peripheral chemoreceptors; chronic normobaric hypoxia showed a significant increase in the number of solitary pulmonary neuroendocrine cells (PNECs) as well as the enlargement of NEBs. NEB cells possess an oxygen-binding protein, cytochrome *b*, an NADPH oxidase located in the cellular membranes that acts as the O2 receptor both

The neuroendocrine cell system is divided into cell types that form glands and diffusely distributed cells. This second group is collectively known as the diffuse neuroendocrine system (DNES), and its representatives are found in the lung, gastrointestinal tract, or urogenital tract. Neuroendocrine tumours of the lung arise from bronchial mucosal cells known as Kulchitsky cells, which are part of the DNES. The classification of lung

during normoxia and hypoxia.

WHO/IASLC histological classification

Large cell neuroendocrine carcinoma (LCNEC)

Large Cells carcinoma (Variants)

Combined small cell carcinoma

Preinvasive Lesions

Carcinoid tumour Typical carcinoid (TC) Atypical carcinoid (AC) Small cell carcinoma

\*

SCLC.

**3. Pathology of neuroendocrine tumours of the lung** 

neuroendocrine malignancies has been an evolving process (Table 1).

Combined large cell neuroendocrine carcinoma (C-LCNEC)

grade AC, as well as the high-grade malignancies LCNEC and SCLC.

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)

Non-small Cell Lung Carcinoma with Neuroendocrine Differentiation (NSCLC-NED) WHO: World Health Organization; IASLC: International Association for the Study of Lung Cancer

These classifications date back to 1972, when atypical carcinoids were initially defined according to histological criteria, including the number of mitoses per high-power field (hpf), the presence of necrosis, increased cellularity with disorganisation, nuclear pleomorphism, hyperchromatism, and an abnormal nuclear to cytoplasmic ratio (Table 2). In 1991, a new classification proposed 4 categories of neuroendocrine lung tumours that included the following: typical carcinoid (TC), which is a low-grade malignancy; atypical carcinoid (AC), which is a medium-grade malignancy; large-cell neuroendocrine carcinoma (LCNEC), which is a high-grade malignancy; and small-cell lung cancer (SCLC), which is also a high-grade malignancy. The 2004 WHO categorisation of tumours with neuroendocrine features included the classic carcinoid low-grade TC and intermediate-

From Travis WD, Colby TV, Corrin B, et al in collaboration with pathologists from 14 countries. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer Verlag, 1999, with permission. Table 1. Lung tumours with neuroendocrine morphology include the low-grade typical carcinoid (TC), intermediate-grade atypical carcinoid (AC), and the high-grade LCNEC and


N/C: nuclear/cytoplasmic; HPF: high power fields; LCNEC: large cell neuroendocrine carcinoma; SCLC: small cell lung carcinoma

Table 2. Histopathological Classification of Neuroendocrine Tumours of Lung

#### **3.1 Tumourlets**

Carcinoid tumours that grow in the peripheral lung and are smaller than 5 mm are referred to as tumourlets. By definition, tumourlets are comprised of increased numbers of individual cells, small group cells, or nodular aggregates of cells that are confined to the bronchial/bronchiolar epithelium (with larger lesions bulging into the lumen but not breaking the subepithelial basement membrane).

#### **3.2 Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia**

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare condition in which neuroendocrine cells proliferate throughout the peripheral airways in the form of neuroendocrine cell hyperplasia, tumourlets, and sometimes carcinoid tumours. In DIPNECH, neuroendocrine cell hyperplasia and tumourlets are thought to be a primary proliferation in contrast to the much more common situation where these lesions are seen as a reactive secondary lesion in the setting of airway inflammation and/or fibrosis. This condition is regarded as a precursor to carcinoid tumours because a subset of these patients experience one or more carcinoid tumours. More aggressive forms of lung carcinoma, including SCLC, have not been associated with DIPNECH.

#### **3.3 Carcinoid tumours**

Pulmonary or bronchial carcinoid tumours account for over 25% of all carcinoid tumours and for 1%-2% of all pulmonary neoplasms. Approximately 10%-20% of pulmonary carcinoids are typical carcinoids; the remaining 80%-90% are atypical carcinoids. Most of these tumours occur centrally and involve the main, lobar, or segmental airways. Sometimes they are located distal to the segmental bronchi; such tumours are the so-called peripheral carcinoids. Atypical carcinoids have been reported to be larger than typical carcinoids, with mean diameters of 3.6 cm and 2.3 cm, respectively. Moreover, atypical carcinoids are more likely to occur in the periphery of the lung than are typical carcinoids. It was generally accepted that a carcinoid tumour was a very slow-growing and benign neoplasm with no potential for invasiveness and no tendency to give rise to metastases. Carcinoid tumours have subsequently been reported in a wide range of organs, but they most commonly involve the lungs and the gastrointestinal tract. The histopathologic features that distinguish atypical carcinoids from typical carcinoids are as follows: increased mitotic activity; greater cytological pleomorphism and higher nuclear to cytoplasmic ratios; increased cellularity and architectural irregularities, and more areas of tumour necrosis. In terms of histological features, typical carcinoids show no evidence of necrosis and fewer than 2 mitoses per 10 high-power fields (or 2 mm2) of viable tumour, whereas atypical carcinoids do have areas of necrosis and 2-10 mitoses per 10 high-power fields.

## **3.4 Large-Cell Neuroendocrine Carcinoma**

Large-cell neuroendocrine carcinoma (LCNEC) was proposed as the fourth category of pulmonary neuroendocrine tumours due to its distinct clinical and pathologic findings versus the typical carcinoid, atypical carcinoid, and SCLC. LCNEC is defined as a poorly differentiated and high-grade neuroendocrine tumour that morphologically is between an atypical carcinoid and SCLC. According to the WHO suggestions, the morphologic features of LCNEC represent a spectrum between those of atypical carcinoid and those of SCLC. In 70%-80% of cases, LCNEC appears as a peripheral mass or nodule, whereas 25% manifests as a central mass. Histopathologic diagnosis criteria for LCNEC are as follows: neuroendocrine morphologic features; a high mitotic rate (>10 per 10 high-power fields); necrosis (often large zones); cytologic features different from those of SCLC; and positive immunohistochemical staining for one or more neuroendocrine markers including chromogranin A, synaptophysin , and neural cell adhesion molecular (NCAM/CD56).

## **3.5 Small-cell lung carcinoma**

SCLC accounts for approximately 20% of all bronchogenic carcinomas. Approximately 90%- 95% of SCLCs occur centrally, apparently arising in a lobar or main bronchus. In 5%-10% of cases, SCLC manifests as a peripheral nodule. These tumour cells are usually small with a round or fusiform shape and have high cellularity with a very high mitotic rate. SCLCs are highly proliferative and rarely are the mitotic rates less than 10 mitoses per 10 high-power fields. As such, virtually every high-power field contains one or more mitoses. The architecture of the tumour clusters is poorly preserved, with large areas of necrosis separating small islands of viable tumour. A distinguishing feature of SCLC is its expression of neuroendocrine markers including neuron specific enolase (NSE), synaptophysin, neural cell adhesion molecule (NCAM/CD56), and Leu-7 (CD57).

## **4. The neuroendocrine differentiation in lung tumours**

#### **4.1 Non-small cell lung cancer (NSCLC)**

The hypothesis that tumours with neuroendocrine properties should be grouped into a single category is not universally accepted for several reasons. First, a large proportion of lung carcinomas have mixed non-neuroendocrine and neuroendocrine properties. This is particularly evident in molecular profiling studies where otherwise unremarkable adenocarcinomas have been shown to express clusters of genes that are thought to reflect neuroendocrine differentiation. Second, many of the markers that are regarded as neuroendocrine markers are expressed in a variety of cells in addition to neuroendocrine cells. Third, neuroendocrine markers are expressed during the embryonic development of the lung.

#### **4.1.1 Adenocarcinoma (ACA)**

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involve the lungs and the gastrointestinal tract. The histopathologic features that distinguish atypical carcinoids from typical carcinoids are as follows: increased mitotic activity; greater cytological pleomorphism and higher nuclear to cytoplasmic ratios; increased cellularity and architectural irregularities, and more areas of tumour necrosis. In terms of histological features, typical carcinoids show no evidence of necrosis and fewer than 2 mitoses per 10 high-power fields (or 2 mm2) of viable tumour, whereas atypical carcinoids do have areas of

Large-cell neuroendocrine carcinoma (LCNEC) was proposed as the fourth category of pulmonary neuroendocrine tumours due to its distinct clinical and pathologic findings versus the typical carcinoid, atypical carcinoid, and SCLC. LCNEC is defined as a poorly differentiated and high-grade neuroendocrine tumour that morphologically is between an atypical carcinoid and SCLC. According to the WHO suggestions, the morphologic features of LCNEC represent a spectrum between those of atypical carcinoid and those of SCLC. In 70%-80% of cases, LCNEC appears as a peripheral mass or nodule, whereas 25% manifests as a central mass. Histopathologic diagnosis criteria for LCNEC are as follows: neuroendocrine morphologic features; a high mitotic rate (>10 per 10 high-power fields); necrosis (often large zones); cytologic features different from those of SCLC; and positive immunohistochemical staining for one or more neuroendocrine markers including chromogranin A, synaptophysin , and neural cell adhesion molecular (NCAM/CD56).

SCLC accounts for approximately 20% of all bronchogenic carcinomas. Approximately 90%- 95% of SCLCs occur centrally, apparently arising in a lobar or main bronchus. In 5%-10% of cases, SCLC manifests as a peripheral nodule. These tumour cells are usually small with a round or fusiform shape and have high cellularity with a very high mitotic rate. SCLCs are highly proliferative and rarely are the mitotic rates less than 10 mitoses per 10 high-power fields. As such, virtually every high-power field contains one or more mitoses. The architecture of the tumour clusters is poorly preserved, with large areas of necrosis separating small islands of viable tumour. A distinguishing feature of SCLC is its expression of neuroendocrine markers including neuron specific enolase (NSE), synaptophysin, neural

The hypothesis that tumours with neuroendocrine properties should be grouped into a single category is not universally accepted for several reasons. First, a large proportion of lung carcinomas have mixed non-neuroendocrine and neuroendocrine properties. This is particularly evident in molecular profiling studies where otherwise unremarkable adenocarcinomas have been shown to express clusters of genes that are thought to reflect neuroendocrine differentiation. Second, many of the markers that are regarded as neuroendocrine markers are expressed in a variety of cells in addition to neuroendocrine cells. Third, neuroendocrine markers are expressed during the embryonic development of

necrosis and 2-10 mitoses per 10 high-power fields.

cell adhesion molecule (NCAM/CD56), and Leu-7 (CD57).

**4.1 Non-small cell lung cancer (NSCLC)** 

the lung.

**4. The neuroendocrine differentiation in lung tumours** 

**3.4 Large-Cell Neuroendocrine Carcinoma** 

**3.5 Small-cell lung carcinoma** 

Pathology reports frequently mention the presence of NE immunophenotype or NE differentiation in NSCLC. Travis et al. have provided a new classification of the pulmonary NE proliferations and neoplasms of the lung, as part of the WHO classification of lung cancers. In this classification, NSCLC with NE differentiation (NSCLC-ND) detected only by immunostaining via electron microscopy is presented as a distinct entity in which no histological features of NE differentiation are appreciated on routine hematoxylin and eosin (HE). NSCLC represents a histologically heterogeneous group of tumours with variable clinical behaviours. Evidence for NE differentiation in non-small cell lung carcinomas (NSCLCs) is, at present, based on histochemical, ultrastructural, and immunohistochemical data. The existence of nonsmall cell lung carcinoma with neuroendocrine differentiation as a distinct entity, as well as its relevance for prognostic and treatment purpose, is controversial. A minority of NSCLCs (10-30%) show NE differentiation, and in contrast to large cell NE carcinoma, they show no evidence of this differentiation on routine light microscopic examination. Previous studies have identified NE differentiation in NSCLC in 10 to 70% of cases. Positivity for all 3 NE (Ch, SNP, and CD56) markers was not seen. The co-expression of SNP and Ch, the two most commonly used NE markers, accounted for only 0.2% (ACA) of the NSCLC. SNP staining was observed in a significant minority of NSCLC (7.5%), whereas Ch, the most specific NE marker, was very uncommon (0.4%) (Table3).


Table 3. Immunoreactivity for Neuroendocrine Markers in Different Subtypes of NSCLC

As has been suggested, the derivation of all lung tumours from a common endodermal stem cell, along with the adoption of amine precursor uptake and decarboxylation properties by this endodermal stem cell, explains divergent differentiation in NE lung tumours and the occurrence of NE subsets in NSCLC.

#### **4.1.2 Large cell carcinoma (LCC)**

Large cell carcinomas of the lung are classified into four types based on light microscopic evidence of neuroendocrine morphology. Immunohistochemical or electron microscopic assessments of neuroendocrine differentiation are categorised as follows: (1) large cell neuroendocrine carcinoma exhibits both neuroendocrine morphology and evidence of neuroendocrine differentiation; (2) large cell carcinoma with neuroendocrine differentiation exhibits neuroendocrine markers but lacks neuroendocrine morphology; (3) large cell carcinoma with neuroendocrine morphology exhibits neuroendocrine markers; and (4) classic large cell carcinoma exhibits neither neuroendocrine morphology nor differentiation. Neuroendocrine markers in NSCLC are expressed not only in large cell carcinoma but also in adenocarcinomas.

### **4.2 NETs of the lung with NE differentiation**

Lung tumours with neuroendocrine morphology include the low-grade typical carcinoid (TC), intermediate-grade atypical carcinoid (AC), and the high-grade LCNEC and SCLC. Neuroendocrine differentiation may be detected by immunohistochemical or ultrastructural studies in 10% to 20% of histologically ordinary NSCLCs such as squamous cell carcinomas, adenocarcinomas, or large cell carcinomas.

### **4.2.1 Small cell lung carcinoma (SCLC)**

SCLC tumors are considered poorly differentiated NE cancers in contrast to typical and atypical bronchial carcinoid tumors. In addition to SCLC, approximately 20-30% of NSCLC tumors express some degree of NE differentiation, predominatly in adenocarcinomas and large cell cancers. SCLC exhibits characteristic molecular abnormalities which partially overlap with those of NSCLC including frequent inactivation of the Rb-p16INK4A-related G1 checkpoint pathway, loss of p53, and frequent abnormalities in chromosome

Fig. 1. Hypothetical carcinogenetic pathway in lung cancer: The specification of distinct cell fates is often achieved through the activation of specific intercellular signaling pathways by growth factors whose expression must be spatially and temporally controlled to ensure accurate response. Typically, a target cell expresses an excess of receptors such that the availability of its ligands is rate-limiting for pathway activation. Overall, for a signal to elicit the desired response, the intensity and duration of ligand expression must be sufficient to meet its threshold concentration, and yet the signal response must be restricted to prevent excessive signaling. Excessive activation of a signaling pathway can have broad deleterious effects, thus prompting a requirement for antagonistic regulation. Abbreviations: AAA (Atypical Adenomatous Hyperplasia), ADC (Adenocarcinoma), BAC (Bronchioalveolar Carcinoma), BSD (Basal Squamous Dysplasia), DIPNECH (Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia), LCC (Large Cell Carcinoma), LCNEC (Large Cell Neuroendocrine Carcinoma), NEBs (Neuroepithelial Bodies), PNCs (Pulmonary Neuroendocrine Cells), SCC (Squamous Cell Carcinoma), SCLC (Small Cell Lung Carcinoma).

3p-assocaited tumor suppressor activity. In addition to these changes, SCLCs frequently overexpress myc genes, especially c-Myc, often via gene amplification events. Of all the genetic changes in SCLC, Rb gene mutations are utterly characteristic. Functioning RB protein is lacking in greater than 90% of SCLC and NSCLC with NE features.

## **4.2.2 Large cell neuroendocrine carcinoma (LCNEC)**

The term combined LCNEC is used for those tumours associated with other histologic types of NSCLC. Most often this represents a component of adenocarcinoma. LCNEC must be distinguished from adenocarcinoma, SCLC, large cell carcinoma, and large cell carcinoma with neuroendocrine differentiation (LCC-ND).

## **5. Embryological pathways in lung tumours**

#### **5.1 Notch**

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Lung tumours with neuroendocrine morphology include the low-grade typical carcinoid (TC), intermediate-grade atypical carcinoid (AC), and the high-grade LCNEC and SCLC. Neuroendocrine differentiation may be detected by immunohistochemical or ultrastructural studies in 10% to 20% of histologically ordinary NSCLCs such as squamous cell carcinomas,

SCLC tumors are considered poorly differentiated NE cancers in contrast to typical and atypical bronchial carcinoid tumors. In addition to SCLC, approximately 20-30% of NSCLC tumors express some degree of NE differentiation, predominatly in adenocarcinomas and large cell cancers. SCLC exhibits characteristic molecular abnormalities which partially overlap with those of NSCLC including frequent inactivation of the Rb-p16INK4A-related G1 checkpoint pathway, loss of p53, and frequent abnormalities in chromosome

Fig. 1. Hypothetical carcinogenetic pathway in lung cancer: The specification of distinct cell fates is often achieved through the activation of specific intercellular signaling pathways by growth factors whose expression must be spatially and temporally controlled to ensure accurate response. Typically, a target cell expresses an excess of receptors such that the availability of its ligands is rate-limiting for pathway activation. Overall, for a signal to elicit the desired response, the intensity and duration of ligand expression must be sufficient to meet its threshold concentration, and yet the signal response must be restricted to prevent excessive signaling. Excessive activation of a signaling pathway can have broad deleterious effects, thus prompting a requirement for antagonistic regulation. Abbreviations: AAA (Atypical Adenomatous Hyperplasia), ADC (Adenocarcinoma), BAC (Bronchioalveolar Carcinoma), BSD (Basal Squamous Dysplasia), DIPNECH (Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia), LCC (Large Cell Carcinoma), LCNEC (Large Cell Neuroendocrine Carcinoma), NEBs (Neuroepithelial Bodies), PNCs (Pulmonary Neuroendocrine Cells), SCC (Squamous Cell Carcinoma), SCLC (Small Cell Lung

**4.2 NETs of the lung with NE differentiation** 

adenocarcinomas, or large cell carcinomas.

**4.2.1 Small cell lung carcinoma (SCLC)** 

Carcinoma).

The three main functions of Notch signalling in self-renewing tissues include stem-cell maintenance, binary cell-fate decisions, and induction of differentiation. A critical aspect of Notch function in both development and post-natal life is the maintenance of stem cell viability and asymmetric cell division. Intrinsic to this process is an unequal distribution of Notch signals in the daughter cells, with the Notch-active cell maintaining its stem cell

Fig. 2. Heat map for Notch1-4 expression in a serie of 64 carcinoids studied by tissue array technology in our lab gave the next information: The mean of expression for Notch1 is 2.33 and the mode 0. 76.6% of the samples showed no expression for this marker, 4.7% showed weak expression, 1.6% moderate expression; 4.7% of the samples showed strong reactivity for Notch1 in more than 75 % of tumour cells in the core biopsy arrayed, 12.5% of the samples did not show enough tissue for testing. The average expression for Notch2 is 8.56 and the mode 12. 7. 8% of the samples showed no expression for this marker, 4.7% showed weak expression, 17.2% moderate expression, 54.7% of the samples showed intense staining for Notch2 in almost all tumour cells present in the sample, 15.6% of the samples did not show enough tissue for testing. The average Notch3 expression is 6.3, and mode of 8. 10.9% of the samples showed no expression for this marker, and 25% showed weak expression, 14% moderate expression, 31.3% of the samples showed intense staining for Notch3 in almost all tumour cells present in the sample 18.8% of the samples did not show enough tissue for testing. Finally, for Notch4 we did not find reactivity in any of the 87, 5% samples with enough tissue for its evaluation.

Fig. 3. Immunohistochemical findings for Notch1-4 in carcinoids tumors.

character and with transit-amplifying cells typically losing Notch activity. Interestingly, Notch function in lung cancer exhibits properties suggesting both tumour promotion and inhibition, depending on the tumour cell type. A prominent function of Notch signalling is to inhibit the transcriptional activities of the widely expressed E2A proteins. Notch signalling rapidly induces degradation and inactivation of E proteins and tissue-specific bHLH proteins such as hASH1. This inhibition may occur as a consequence of forming inhibitory complexes of E2A proteins with the Hes/HERP/HEY proteins, as well as the promotion of E2A protein ubiquitinylation and degradation by Notch. Notch1 and Notch2 proteins are frequently expressed in non-small-cell lung cancer (NSCLC), while Notch3 mRNA expression was detected in one-third of all NSCLC cell lines. NSCLC, which includes adenocarcinoma, squamous cell carcinoma, large cell undifferentiated carcinoma, and bronchoalveolar cell carcinoma, was initially shown to express significant levels of the Hes1 protein. In addition, there is an inverse correlation in these cell lines between the expressions of hASH1/ASCL1 and of the Hes1 protein. In contrast to NSCLC, where Notch is suspected to have a growth promoting function, SCLC appears to be growth inhibited, at least by the high level over-expression of activated Notch1 and Notch2. Notch1 is rarely detectable or inactive in SCLC, whereas a subset of SCLC exhibit Notch2. Notch3 mRNA expression was not detected in the SCLC cell lines. Expression of Notch3 has been reported to be common in NSCLC but not in SCLC. Significantly, Notch signaling has recently been shown to be induced by the ras pathway, which is active in a large fraction of NSCLC, but

Fig. 3. Immunohistochemical findings for Notch1-4 in carcinoids tumors.

character and with transit-amplifying cells typically losing Notch activity. Interestingly, Notch function in lung cancer exhibits properties suggesting both tumour promotion and inhibition, depending on the tumour cell type. A prominent function of Notch signalling is to inhibit the transcriptional activities of the widely expressed E2A proteins. Notch signalling rapidly induces degradation and inactivation of E proteins and tissue-specific bHLH proteins such as hASH1. This inhibition may occur as a consequence of forming inhibitory complexes of E2A proteins with the Hes/HERP/HEY proteins, as well as the promotion of E2A protein ubiquitinylation and degradation by Notch. Notch1 and Notch2 proteins are frequently expressed in non-small-cell lung cancer (NSCLC), while Notch3 mRNA expression was detected in one-third of all NSCLC cell lines. NSCLC, which includes adenocarcinoma, squamous cell carcinoma, large cell undifferentiated carcinoma, and bronchoalveolar cell carcinoma, was initially shown to express significant levels of the Hes1 protein. In addition, there is an inverse correlation in these cell lines between the expressions of hASH1/ASCL1 and of the Hes1 protein. In contrast to NSCLC, where Notch is suspected to have a growth promoting function, SCLC appears to be growth inhibited, at least by the high level over-expression of activated Notch1 and Notch2. Notch1 is rarely detectable or inactive in SCLC, whereas a subset of SCLC exhibit Notch2. Notch3 mRNA expression was not detected in the SCLC cell lines. Expression of Notch3 has been reported to be common in NSCLC but not in SCLC. Significantly, Notch signaling has recently been shown to be induced by the ras pathway, which is active in a large fraction of NSCLC, but

only rarely in SCLC. Notch in the SCLC cells lead to a significant increase in Hes1 and a marked down-regulation of the neurally related transcription factors hASH1 and Hes6. The loss of hASH1 may be critical in mediating the growth inhibitory effect of Notch1 in SCLC, although a role for other as yet unidentified targets cannot be excluded Activated Notch1 and Notch2, but not Hes1, caused a potent G1 arrest in the SCLC cells, accompanied by the marked up-regulation of p21wasl/cip1, overall abundance of p53, and a Rb mutant typifying the majority of SCLC

### **5.2 Hedgehog signalling in lung cancer**

The hedgehog (Hh) signalling network functions in cell-cell communication and regulates pattern formation, proliferation, cell fate, and the stem/progenitor cell maintenance and selfrenewal in many organs. A greatly simplified version of "canonical" hedgehog signalling in mammals typically involves two types of cells, a signalling cell expressing a member of the Hedgehog family of secreted ligands (*Sonic Hedgehog* (Shh), *Indian Hedgehog* (Ihh), or *Desert Hedgehog* (Dhh)) and a responding cell expressing one or more *Patched* family hedgehog receptors (*Patched-1* (PTCH2)). In the Hh pathway, increased signalling results in activation of the GLI oncogenes (GLI1, GLI2, and GLI3) that can regulate gene transcription. The Hh signalling pathway was originally shown to have persistent activation in SCLC with high expression of Shh, PTCK, and GLI1, but an important role in NSCLC, was also demonstrated.

## **5.3 BMPs and BMPRs**

The BMPs comprise a branch of the TGF- superfamily that also plays a key role in development. Several BMP ligands and BMPRs including BMP3, 4 and 7 as well as type I BMPR are expressed during embryonic lung development. BMP4 mRNA is localized at high levels in the epithelium of distal tips of terminal buds, with lower levels in the adjacent mesenchyme. These loci of BMP expression overlap with the expression domains of some other important morphogenetic signaling molecules including HNF-3, Wnt-2, Shh and FGF-10. Also, since BMPs and Shh are co-expressed in the same domains, and since Decapentaplegic, the Drosophila BMP homologue, is regulated by the Hedgehog signaling pathway, it seems possible that BMP-Shh interactions may prove to play key roles in lung morphogenesis. Recently published data on fibroblast growth factor interactions suggest that Shh, TGF-1 and BMP4 all counteract the bud-promoting effects of FGF-10.

#### **5.4 Wnt pathway in lung cancer**

Wnt signaling has many functions in animal development including its development role in empbryogenesis and in the adult lung. More specifically, studies of knockout mice demonstrated the importance of Wnt-2, Wnt-5a, and Wnt-7b in lung maturation. In addition to its role in stem cell self-renewal, tissue regeneration, and lung development, Wnt signaling is also intimately involved in tumorigenesis and cancer progression. For example, the organs where Wnt signaling influences stem cell self-renewal are the same organs where those Wnt-pathway-dependent cancers originate. Numerous reportes have demonstrated aberrant Wnt activation in lung cancer. Overexpression of Wnt-1 has been demonstrated in NSCLC cell lines and primary cancer tissues. This activation can be caused by mutations and/or deregulation of many different Wnt signaling components. Mutations in Wnt pathway components are rarely found in lung cancer. Also overexpression of Wnt-2 in NSCLC has been demonstrated. The human Wnt-2 gene, located on chromosome 7q31.3, is highly expressed in fetal lung. The link between Wnt-2 and tumorigenesis was first proposed after data indicated that Wnt-2 was amplified in human cancers. Indeed, in patients with NSCLC found that Wnt-5a expression is squamous cell carcinoma was significantly higher than that in adenocarcinoma. Recently there has been a suggested role for Wnt-7 in lung cancer. It has been reported that expression of Wnt-7a is downregulated in most lung cancer cell lines and tumor samples.

#### **5.5 bHLH**

Helix-loop-helix proteins are a diverse family of transcriptional regulators involved in fetal development and cancer. The 125 recognized human HLH proteins can be subdivided into 45 families, almost all of which have Drosophila representatives as well. These families include achaete-scute homologs, E proteins, Atonal, NeuroD, neurogenin, ID proteins, HES, and Hesrelated proteins, and others. bHLH genes control cell differentiation in various tissues and are categorised into two distinct groups, activator genes and repressor genes. In mammals, bHLH genes such as mammalian achaete-scute complex homolog-1 (MASH1) and mammalian atonal homolog (MATH)-1 are expressed in neural precursor cells, and they up-regulate lateexpressing bHLH genes such as NeuroD to direct terminal differentiation. On the other hand, HES1, one of the hairy and enhancer of split (HES) homologues, represses neuronal differentiation by the suppression of proneural bHLH factors. Repressive bHLH factors such as HES1 are regulated by the Notch pathway. The Notch ligands activate the Notch receptors, and the activated intracellular domain of the Notch receptors interacts with the DNA-binding protein RBP-Jk to activate the expression of repressive bHLHs such as HES1 and HES5, which, in turn, suppress the expression of activator bHLHs such as MASH1 and NeuroD. Immunohistochemical studies have revealed that Notch1, Notch3, Jagged1, and Jagged2 were expressed in neuroendocrine cells of the airway epithelium, while Dll1 was detected in the pulmonary neuroendocrine cells. Thus, the differentiation of the lung epithelial cells depends on a bHLH factor network, and the Notch pathway may be involved in determining the cell differentiation fate in the airway epithelium.

#### **5.5.1 Achaete-scute homolog (ASH-1)**

Mash1 (termed Hash1 in humans) plays a critical role in development of the central and autonomic nervous systems and in tissues of the so-called diffuse NE system including the adrenal medullary chromaffin cells, thyroid parafollicular C-cells, and pulmonary NE cells. MASH1 is important in the development of the diffuse neuroendocrine system, including pulmonary neuroendocrine cells. During neurogenesis, MASH1 expression is confined to mitotically active precursors where it is involved in the early stages of lineage commitment; in more mature neurons the expression is extinguished. MASH1 and mammalian atonal homolog-1 (Math1) up-regulate NeuroD in neural precursor cells to direct terminal differentiation, whereas HES1 represses neuronal differentiation by the suppression of proneural factors such as MASH1. In the developing mouse lung, Mash1 first becomes detectable at approximately E13.5 in neuroepitelial bodies (NEB´S), clusters of NE cells frequently located at branchpoints of large and medium-sized airways. Mash 1 expression in mouse lung peaks near birth and the declines in adulthood, following the peak and decline of lung NE cells. One target of achaete-scute proteins is the cell surface ligand delta, which leads to activation of the Notch pathway in adjoining cells and repression of the neuronal fate. In human lung tumours, the expression of hASH1 mRNA was significantly higher in

highly expressed in fetal lung. The link between Wnt-2 and tumorigenesis was first proposed after data indicated that Wnt-2 was amplified in human cancers. Indeed, in patients with NSCLC found that Wnt-5a expression is squamous cell carcinoma was significantly higher than that in adenocarcinoma. Recently there has been a suggested role for Wnt-7 in lung cancer. It has been reported that expression of Wnt-7a is downregulated in

Helix-loop-helix proteins are a diverse family of transcriptional regulators involved in fetal development and cancer. The 125 recognized human HLH proteins can be subdivided into 45 families, almost all of which have Drosophila representatives as well. These families include achaete-scute homologs, E proteins, Atonal, NeuroD, neurogenin, ID proteins, HES, and Hesrelated proteins, and others. bHLH genes control cell differentiation in various tissues and are categorised into two distinct groups, activator genes and repressor genes. In mammals, bHLH genes such as mammalian achaete-scute complex homolog-1 (MASH1) and mammalian atonal homolog (MATH)-1 are expressed in neural precursor cells, and they up-regulate lateexpressing bHLH genes such as NeuroD to direct terminal differentiation. On the other hand, HES1, one of the hairy and enhancer of split (HES) homologues, represses neuronal differentiation by the suppression of proneural bHLH factors. Repressive bHLH factors such as HES1 are regulated by the Notch pathway. The Notch ligands activate the Notch receptors, and the activated intracellular domain of the Notch receptors interacts with the DNA-binding protein RBP-Jk to activate the expression of repressive bHLHs such as HES1 and HES5, which, in turn, suppress the expression of activator bHLHs such as MASH1 and NeuroD. Immunohistochemical studies have revealed that Notch1, Notch3, Jagged1, and Jagged2 were expressed in neuroendocrine cells of the airway epithelium, while Dll1 was detected in the pulmonary neuroendocrine cells. Thus, the differentiation of the lung epithelial cells depends on a bHLH factor network, and the Notch pathway may be involved in determining the cell

Mash1 (termed Hash1 in humans) plays a critical role in development of the central and autonomic nervous systems and in tissues of the so-called diffuse NE system including the adrenal medullary chromaffin cells, thyroid parafollicular C-cells, and pulmonary NE cells. MASH1 is important in the development of the diffuse neuroendocrine system, including pulmonary neuroendocrine cells. During neurogenesis, MASH1 expression is confined to mitotically active precursors where it is involved in the early stages of lineage commitment; in more mature neurons the expression is extinguished. MASH1 and mammalian atonal homolog-1 (Math1) up-regulate NeuroD in neural precursor cells to direct terminal differentiation, whereas HES1 represses neuronal differentiation by the suppression of proneural factors such as MASH1. In the developing mouse lung, Mash1 first becomes detectable at approximately E13.5 in neuroepitelial bodies (NEB´S), clusters of NE cells frequently located at branchpoints of large and medium-sized airways. Mash 1 expression in mouse lung peaks near birth and the declines in adulthood, following the peak and decline of lung NE cells. One target of achaete-scute proteins is the cell surface ligand delta, which leads to activation of the Notch pathway in adjoining cells and repression of the neuronal fate. In human lung tumours, the expression of hASH1 mRNA was significantly higher in

most lung cancer cell lines and tumor samples.

differentiation fate in the airway epithelium.

**5.5.1 Achaete-scute homolog (ASH-1)** 

**5.5 bHLH** 

SCLC (75%) than in LCNEC (50%); conversely, HES1 mRNA was lower in SCLC (59%) than in LCNEC (87%). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics that are more similar to the epithelium phenotype, suggesting that the biological characteristics of these two tumours are different. On the contrary, non-neuroendocrine carcinoma cells do not express hASH1 but show high HES1 expression. In NSCLC (squamous cell carcinoma vs. adenocarcinoma), the expression of hASH1 mRNA was lower, (0% vs. 15%, respectively), whereas HES1 mRNA was higher (10% vs. 100%, respectively). Neuroendocrine pulmonary carcinomas express MASH1 but not HES1, whereas adenocarcinoma and squamous cell carcinoma express HES1. Surprisingly, Merkel cell carcinoma, the cutaneous counterpart of small cell carcinoma MASH1, was completely negative in 100% of the cases.

#### **5.5.2 Hairy and Enhancer-of-split (HES)**

Hes1, a key effector of the Notch signalling pathway, is expressed broadly in nonneuroendocrine cells in the airway epithelium. In the developing lung, Notch1 and HES1 are strongly expressed in the non-neuroendocrine airway epithelial cells, whereas MASH1 is restricted to the clustered pulmonary neuroendocrine cells. HES1 directly represses hASH1 expression by binding to a class C site in the ASH1 promoter. Today, the published results suggest that the differentiation of neuroendocrine cells in normal lungs is affected by the absence of the MASH1 gene. Elements of the Notch signalling pathway, especially that of HES1, appear to be critical negative regulators of achaete-scute homolog 1 expression in normal lungs and in lung cancer. For example, HES1 transgenic knockout mice exhibit substantial hyperplasia and premature differentiation of lung NE cells associated with an increase in MASH1-expressing pulmonary epithelium. It has been shown that the overexpression of HES1 in SCLC cells leads to the repression of hASH1 expression via a transcriptional mechanism.

#### **5.5.3 Retinoblastoma (Rb)**

The RB gene is a prototypical tumour suppressor gene, and the loss of RB function is believed to be a key event in the initiation or progression of several human malignancies. Most RB gene alterations result in the loss of RB protein expression or in a truncated RB protein, which does not enter the nucleus. Thus, heterogeneous positive nuclear RB immunostaining is, in general, indicative of normal RB function, whereas negative intranuclear RB immunostaining in all tumour cells reflects aberrant RB protein expression. Typical and atypical carcinoids manifest a heterogeneous RB-positive staining pattern. Atypical carcinoids in general show an increase in the number of tumour cells with nuclear staining compared to typical carcinoids. In contrast, small-cell and large-cell neuroendocrine carcinomas fail to show RB staining in any tumour nuclei, indicating the loss of RB function. From these results, it can be concluded that a progressively higher degree of malignancy from typical carcinoids to atypical carcinoids to small-cell carcinomas is paralleled by the loss of neuroendocrine markers, increased proliferative markers, increased frequency of p53 immunostaining, and decreased frequency of RB immunostaining.

#### **5.5.4 p53**

Although p53 alterations have been previously studied in pulmonary neuroendocrine tumours, either these studies have used immunochemistry alone rather than genotypic analysis or they have examined a limited spectrum of pulmonary neuroendocrine neoplasias. The distribution of p53 immunohistochemical staining has 4 patterns: negative in typical carcinoids (TCs), 50% of ACs, 20% LCNECs, and 12% SCLCs; less than 10% but more than 5-10 HPF (focal) in a subset (30%) of aggressive adenocarcinomas; and 50-100% of tumour cells (diffuse), exclusively seen in LCNECs and SCLCs. Three patterns of immunohistochemical staining intensities of the p53 protein were seen: negative; weak or mild; and moderate to marked staining. Similar to other cancers, multiple genetic events contribute to the development of neuroendocrine lung tumours. This has already been demonstrated in SCLCs, which are known to exhibit alterations in their oncogenes such as *cmyc* and in tumour suppressor genes such as p53 and Rb. In addition, it has been shown that alterations in oncogenes such as H-ras, c-myc, and c-raf-1 can modulate the expression of neuroendocrine antigens in lung cancer cell lines. Thus, evidence is accumulating that the expression of neuroendocrine differentiation in pulmonary neuroendocrine tumours is fundamentally controlled by multiple genetic determinants.

## **6. Conclusions**

Although incidence of newly diagnosed patients with carcinoid tumors of the lung is low, the long survival for those with low and intermediate differentiation grade, and the deeper knowledge we now have on molecular processes that governs tumors growth make these tumors a challenging field in Oncology. Systemic treatment for metastatic carcinoid tumors of the lung has not change significantly in the last two decades, and this fact leads to a poor improvement in overall survival, contrary to what has happened in other solid tumors. Nowadays, most of researchers in neuroendocrine field consider that every single neuroendocrine tumors has its own features depending on the organ where it seats, the capacity to produce and secrete active hormones to blood stream, and the proliferation rate. Novel agents like antiangiogenic tyroisine kinase inhibitors, mTOR inhibitors or oral chemotherapeutic agents like temozolomide and capecitabine have been used to treat metastatic neuroendocrine tumors of the lung without a clear activity. Unfortunately, these clinical trials with new agents were not driven to lung tumors but to other neuroendocrine tumors of the gastrointestinal tract. Therefore, other pathways are needed to be investigated. A non insignificant number of recent publications are correlating embryological pathways with carcinoid tumors of the lung development. In this sense, some elements of the Notch signalling pathway, especially HES1, appear to be critical negative regulators of hASH-1 expression in normal lungs and in lung cancer. This fact may influence in carcinoid tumor development at this place. New compounds under clinical development targeting embryological pathways like Notch, Hedgehog or Wnt pathways may have a future impact in the treatment of disseminated carcinoids of the lung. The more we are able to select patients molecularly the greater the chance of success in future clinical trials conducted in this setting. However, none of this would be meaningless if the histological diagnosis is not accurate. There is a need to leverage the knowledge in the scientific community of the variety of neuroendocrine-derived tumors that may arise in the lung. The teamwork between pulmonologists, thoracic surgeons, pathologists, molecular biologists, oncologists, and radiotherapists is mandatory to offer to our patients the best treatment approach at the right time for their diseases.

## **7. References**

218 Lung Diseases – Selected State of the Art Reviews

analysis or they have examined a limited spectrum of pulmonary neuroendocrine neoplasias. The distribution of p53 immunohistochemical staining has 4 patterns: negative in typical carcinoids (TCs), 50% of ACs, 20% LCNECs, and 12% SCLCs; less than 10% but more than 5-10 HPF (focal) in a subset (30%) of aggressive adenocarcinomas; and 50-100% of tumour cells (diffuse), exclusively seen in LCNECs and SCLCs. Three patterns of immunohistochemical staining intensities of the p53 protein were seen: negative; weak or mild; and moderate to marked staining. Similar to other cancers, multiple genetic events contribute to the development of neuroendocrine lung tumours. This has already been demonstrated in SCLCs, which are known to exhibit alterations in their oncogenes such as *cmyc* and in tumour suppressor genes such as p53 and Rb. In addition, it has been shown that alterations in oncogenes such as H-ras, c-myc, and c-raf-1 can modulate the expression of neuroendocrine antigens in lung cancer cell lines. Thus, evidence is accumulating that the expression of neuroendocrine differentiation in pulmonary neuroendocrine tumours is

Although incidence of newly diagnosed patients with carcinoid tumors of the lung is low, the long survival for those with low and intermediate differentiation grade, and the deeper knowledge we now have on molecular processes that governs tumors growth make these tumors a challenging field in Oncology. Systemic treatment for metastatic carcinoid tumors of the lung has not change significantly in the last two decades, and this fact leads to a poor improvement in overall survival, contrary to what has happened in other solid tumors. Nowadays, most of researchers in neuroendocrine field consider that every single neuroendocrine tumors has its own features depending on the organ where it seats, the capacity to produce and secrete active hormones to blood stream, and the proliferation rate. Novel agents like antiangiogenic tyroisine kinase inhibitors, mTOR inhibitors or oral chemotherapeutic agents like temozolomide and capecitabine have been used to treat metastatic neuroendocrine tumors of the lung without a clear activity. Unfortunately, these clinical trials with new agents were not driven to lung tumors but to other neuroendocrine tumors of the gastrointestinal tract. Therefore, other pathways are needed to be investigated. A non insignificant number of recent publications are correlating embryological pathways with carcinoid tumors of the lung development. In this sense, some elements of the Notch signalling pathway, especially HES1, appear to be critical negative regulators of hASH-1 expression in normal lungs and in lung cancer. This fact may influence in carcinoid tumor development at this place. New compounds under clinical development targeting embryological pathways like Notch, Hedgehog or Wnt pathways may have a future impact in the treatment of disseminated carcinoids of the lung. The more we are able to select patients molecularly the greater the chance of success in future clinical trials conducted in this setting. However, none of this would be meaningless if the histological diagnosis is not accurate. There is a need to leverage the knowledge in the scientific community of the variety of neuroendocrine-derived tumors that may arise in the lung. The teamwork between pulmonologists, thoracic surgeons, pathologists, molecular biologists, oncologists, and radiotherapists is mandatory to offer to our patients the best treatment approach at the

fundamentally controlled by multiple genetic determinants.

**6. Conclusions** 

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#### **7.10 The neuroendocrine pathway of differentiation**


## **Chemotherapy for Large Cell Neuroendocrine Carcinoma of the Lung: Should It Be Treated with the Same Strategy as Small Cell Lung Carcinoma?**

Katsuhiko Naoki, Kenzo Soejima, Takashi Sato, Shinnosuke Ikemura, Hideki Terai, Ryosuke Satomi, Sohei Nakayama, Satoshi Yoda and Koichiro Asano *Keio Cancer Center / Division of Respiratory Medicine, Department of Internal Medicine, School of Medicine, Keio University, Tokyo / Yuai Clinic, Yokohama Japan* 

### **1. Introduction**

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signaling induces cell cycle arrest in small cell lung cancer cells. Cancer Res 2001;

Notch signaling induces rapid degradation of achaete-scute homolog.1. Mol Cell

Lung cancer is leading cause of cancer death in many advanced countries and one of the challenging malignancies because of poor prognosis. Lung cancer is traditionally divided into two major categories, so called small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) because of distinctive prognostic and treatment strategies between them. On the other hand, there is a spectrum of tumors called pulmonary neuroendocrine (NE) tumors that are thought to originate from neuroendocrine cells in the pulmonary and bronchial epithelium. Until recently, pulmonary NE tumors were classified into three categories, i.e., typical carcinoid (TC), atypical carcinoid (AC), and SCLC. Large cell neuroendocrine carcinoma (LCNEC) of the lung was officially identified by Travis et al. in 1991 as a fourth category, a unique higher grade NSCLC existing between TC and SCLC (Travis et al., 1991). It is often difficult to diagnose LCNEC with small biopsy specimens because accurate diagnosis needs morphological and immunohistochemical information. Although earlier reports mainly focused on prognosis after surgical procedures, several recent studies reported on the efficacy of chemotherapy for advanced LCNEC. Because of the limited numbers of cases (in surgical series, LCNEC represents ~3% of lung cancers), large scale prospective studies have not been reported. Standard treatment for LCNEC, especially if advanced, is not established although LCNEC is included in NSCLC in the treatment algorithm in many guidelines. However, accumulating data including recent retrospective studies have suggested that there is similarity in the prognosis and treatment response between LCNEC and SCLC.

In this review, we will focus on the treatment of advanced LCNEC for the better selection of chemotherapeutic regimens for the patients with this relatively rare lung cancer.

## **2. Large cell neuroendocrine carcinoma of the lung**

LCNEC is classified as a variant of large cell carcinoma in NSCLC whilst LCNEC has neuroendocrine characteristics similar to SCLC such as morphology and the immunohistochemical staining pattern. This discrepancy raises the question as to what the best therapeutic modality is, that is, should we treat LCNEC as NSCLC or SCLC?


Abbreviations: LCNEC, large cell neuroendocrine carcinoma; HPF, high power field; NSCLC, non-small cell lung carcinoma; N/C, nuclear-cytoplasmic ratio; NE, neuroendocrine

Table 1. Tumors with neuroendocrine morphology (Travis 2010, Gollard et al., 2010)

Recently, Varlotto et al. reported survival analysis of resected cases with LCNEC and SCLC (Varlotto et al., 2011). They compared overall survival (OS) and lung cancer-specific survival (LCSS) of patients with LCNEC and SCLC or other large cell lung carcinomas (OLCs) using the US National Cancer Institute database (SEER program). Although, the survival rates tended to be better in LCNEC and OLCs compared to SCLC, multivariate analysis showed no statistical differences (4-year OS rates are 41 % in LCNEC, 42% in OLC, and 32% in SCLC; 4-year LCSS rates are 57 % in LCNEC, 54% in OLC, and 42% in SCLC). The SEER database does not include chemotherapy information, so that we do not know the impact of chemotherapy on survival. Other reports also noted that survival in the early stage LCNEC is similar to SCLC (Asamura et al., 2006, Sun et al., 2009) and not better than NSCLC (Iyoda et al., 2007).

## **3. Chemotherapy for advanced LCNEC**

LCNEC is classified in the category of NSCLC pathologically (Brambilla et al., 2001), so that the guideline recommended treatment of advanced LCNEC as NSCLC (NCCN guidelineTM 2011), and many trials have included this disease as a NSCLC. However, recent accumulating data have brought new insights regarding possibly better results with SCLC regimens.

From the published literature, we found four major studies showing the treatment results with chemotherapy for advanced LCNEC (Igawa et al., 2010, Fujiwara et al., 2007, Yamazaki et al., 2005, Rossi et al., 2005) (Table 2). All studies were retrospective and a total of 83 patients were treated with first line systemic chemotherapy. Chemotherapy regimens can be classified into two groups: SCLC-based regimens (total n=44; platinum and etoposide n=27, platinum and irinotecan (CPT-11) n=16, CPT-11 only n=1) and NSCLC-based regimens (total n=39; platinum and paclitaxel (PTX) n=11, platinum and gemcitabine n=10, cisplatin with vindesine and mitomycin n=6, cisplatin and vindesine n=4, other platinum doublet n=2, other single agent n=6).

LCNEC is classified as a variant of large cell carcinoma in NSCLC whilst LCNEC has neuroendocrine characteristics similar to SCLC such as morphology and the immunohistochemical staining pattern. This discrepancy raises the question as to what the

Morphology carcinoid carcinoid neuroendocrine neuroendocrine

Necrosis abscent present (focal punctate) present (extensive) present (extensive) Cytologic features NSCLC (large cell, low N/C) small cell, scant cytoplasm Immunohistochemistry positive for NE markers positive for NE markers

Typical Carcinoid Atypical Carcinoid LCNEC Small cell carcinoma

(10HPF) high: >11 / 2mm2 (10HPF) high: >11 / 2mm2 (10HPF)

best therapeutic modality is, that is, should we treat LCNEC as NSCLC or SCLC?

Table 1. Tumors with neuroendocrine morphology (Travis 2010, Gollard et al., 2010)

Recently, Varlotto et al. reported survival analysis of resected cases with LCNEC and SCLC (Varlotto et al., 2011). They compared overall survival (OS) and lung cancer-specific survival (LCSS) of patients with LCNEC and SCLC or other large cell lung carcinomas (OLCs) using the US National Cancer Institute database (SEER program). Although, the survival rates tended to be better in LCNEC and OLCs compared to SCLC, multivariate analysis showed no statistical differences (4-year OS rates are 41 % in LCNEC, 42% in OLC, and 32% in SCLC; 4-year LCSS rates are 57 % in LCNEC, 54% in OLC, and 42% in SCLC). The SEER database does not include chemotherapy information, so that we do not know the impact of chemotherapy on survival. Other reports also noted that survival in the early stage LCNEC is similar to SCLC (Asamura et al., 2006, Sun et al., 2009) and not better than NSCLC (Iyoda

LCNEC is classified in the category of NSCLC pathologically (Brambilla et al., 2001), so that the guideline recommended treatment of advanced LCNEC as NSCLC (NCCN guidelineTM 2011), and many trials have included this disease as a NSCLC. However, recent accumulating data have brought new insights regarding possibly better results with SCLC

From the published literature, we found four major studies showing the treatment results with chemotherapy for advanced LCNEC (Igawa et al., 2010, Fujiwara et al., 2007, Yamazaki et al., 2005, Rossi et al., 2005) (Table 2). All studies were retrospective and a total of 83 patients were treated with first line systemic chemotherapy. Chemotherapy regimens can be classified into two groups: SCLC-based regimens (total n=44; platinum and etoposide n=27, platinum and irinotecan (CPT-11) n=16, CPT-11 only n=1) and NSCLC-based regimens (total n=39; platinum and paclitaxel (PTX) n=11, platinum and gemcitabine n=10, cisplatin with vindesine and mitomycin n=6, cisplatin and vindesine n=4, other platinum doublet

(10HPF) <2-10 / 2mm<sup>2</sup>

**2. Large cell neuroendocrine carcinoma of the lung** 

Abbreviations: LCNEC, large cell neuroendocrine carcinoma;

**3. Chemotherapy for advanced LCNEC** 

Mitosis <2 / 2mm2

NSCLC, non-small cell lung carcinoma; N/C, nuclear-cytoplasmic ratio;

HPF, high power field;

NE, neuroendocrine

et al., 2007).

regimens.

n=2, other single agent n=6).


PTX, paclitaxel; VNR, vinorelbine; DTX, docetaxel; MVP, mitomycin + vindesine + cisplatin; VDS, vindesine; GEM, gemcitabine.

Table 2. Previous report regarding 1st line chemotherapy for advanced large cell neuroendocrine cell carcinoma (LCNEC)

The response rate (RR) was 47.7% (21/44) for SCLC-based regimens and 35.9% (14/39) for NSCLC-based regimens (Table 3). In particular, a platinum doublet yielded a RR of 56.3% (9/16) with platinum and CPT-11, 44.4% (12/27) with platinum and etoposide, 54.5% (6/11) with platinum and PTX, 16.7% (2/12) with platinum and other third generation agents. Rossi et al. (2005) showed a significant survival benefit with a SCLC-based regimen compared with a NSCLC regimen (OS of 51 months (M) vs 21M). This result is far better than other reports (OS 7.9M-10.3M, 1-year survival rate 35-47.6%), suggesting that the result was from combined effects of surgery and chemotherapy.


(Abbreviations are the same as in Table 1)

Table 3. Summary of previous reports regarding 1st line chemotherapy for advanced large cell neuroendocrine cell carcinoma (LCNEC)

As for second line treatment, there is no report other than a recent publication with amrubicin treatment (Yoshida et al., 2011). Amrubicin has efficacy for both SCLC and NSCLC, and has been used commonly in Japan in a second line setting with SCLC. Promising results for SCLC have also been recently reported from the USA (Ettinger et al., 2010, Jotte et al., 2011).

Currently there are a few prospective clinical trials for LCNEC in the 1st line settings (ClinicalTrials.gov and UMIN-CTR Clinical Trial, accessed 2nd Aug, 2011). One is not yet open but is an interesting phase II study with RAD001 + carboplatin/paclitaxel for advanced LCNEC. The other is an ongoing phase II study with cisplatin + irinotecan for advanced LCNEC.

The former study with RAD001 is an interesting study utilizing an mTOR inhibitor which inhibits one of the signaling pathways, i.e. the PI3K-mTOR pathway, that lies downstream of receptor tyrosine kinases (RTKs) such as EGFR and c-MET. EGFR is one of the most


Table 4. Efficacy of Amrubicin in the 2nd line treatment for LCNEC and SCLC

important RTKs in NSCLC (Paez et al., 2004), and moreover c-MET is reported to be an important RTK in SCLC as well as NSCLC (Nakachi et al., 2010, Rossi et al., 2005, Schmid et al., 2010). RAD001 has limited but apparent antitumor activity against pretreated SCLC as a single agent (Tarhini et al., 2010). According to these results, targeting signaling pathways with cytotoxic agent might be the next challenge for SCLC and LCNEC. Because many of the current effort in NSCLC is searching for driver mutations (Paez et al., 2004, Naoki et al., 2002), such an effort is also important in SCLC and LCNEC.

## **4. Conclusion**

234 Lung Diseases – Selected State of the Art Reviews

Rossi et al. (2005) showed a significant survival benefit with a SCLC-based regimen compared with a NSCLC regimen (OS of 51 months (M) vs 21M). This result is far better than other reports (OS 7.9M-10.3M, 1-year survival rate 35-47.6%), suggesting that the result

SCLC based total n=44 48%

NSCLC based total n=39 36%

Table 3. Summary of previous reports regarding 1st line chemotherapy for advanced large

As for second line treatment, there is no report other than a recent publication with amrubicin treatment (Yoshida et al., 2011). Amrubicin has efficacy for both SCLC and NSCLC, and has been used commonly in Japan in a second line setting with SCLC. Promising results for SCLC have also been recently reported from the USA (Ettinger et al.,

Currently there are a few prospective clinical trials for LCNEC in the 1st line settings (ClinicalTrials.gov and UMIN-CTR Clinical Trial, accessed 2nd Aug, 2011). One is not yet open but is an interesting phase II study with RAD001 + carboplatin/paclitaxel for advanced LCNEC. The other is an ongoing phase II study with cisplatin + irinotecan for

The former study with RAD001 is an interesting study utilizing an mTOR inhibitor which inhibits one of the signaling pathways, i.e. the PI3K-mTOR pathway, that lies downstream of receptor tyrosine kinases (RTKs) such as EGFR and c-MET. EGFR is one of the most

Chemotherapy Pt RR

Platinum+VP16 n=27 44% Platinum+CPT11 n=16 56% CPT11 n=1 0%

Platinum+PTX n=11 55%

PTX n=1 100% CDDP+VNR n=1 100% VNR n=1 0% CDDP+DTX n=1 100% DTX n=1 0% CDDP+GEM n=10 0% GEM n=2 0% MVP n=6 33% CDDP+VDS n=4 75% CDDP n=1 0%

was from combined effects of surgery and chemotherapy.

(Abbreviations are the same as in Table 1)

2010, Jotte et al., 2011).

advanced LCNEC.

cell neuroendocrine cell carcinoma (LCNEC)

Although there is an issue regarding accurate diagnosis with small biopsy specimens, accumulating retrospective data suggest that patients with advanced LCNEC will benefit from systemic chemotherapy.

The current recommendation for the treatment of advanced LCNEC is similar to that of SCLC, i.e. platinum based combination chemotherapy, mainly with etoposide or CPT-11 and possibly with PTX. Further prospective data is needed to elucidate the best combination therapy.

## **5. References**


Clinical Trials.gov, 2011 Aug,

http://clinicaltrials.gov/


Ettinger DS, Jotte R, Lorigan P, Gupta V, Garbo L, Alemany C, Conkling P,

Fujiwara Y, Sekine I, Tsuta K, Ohe Y, Kunitoh H, Yamamoto N, Nokihara H, Yamada K, &

Gollard R, Jhatakia S, Elliott M, & Kosty M. Large cell/neuroendocrine carcinoma. *Lung* 

Igawa S, Watanabe R, Ito I, Murakami H, Takahashi T, Nakamura Y, Tsuya A, Kaira K,

small-cell lung cancer. *Lung Cancer* 2010 Jun;68(3):438-45, ISSN 0169-5002 Inoue A, Sugawara S, Yamazaki K, Maemondo M, Suzuki T, Gomi K, Takanashi S,

Iyoda A, Hiroshima K, Nakatani Y, & Fujisawa T. Pulmonary large cell neuroendocrine

Jotte R, Conkling P, Reynolds C, Galsky MD, Klein L, Fitzgibbons JF, McNally R, Renschler

Nakachi I, Naoki K, Soejima K, Kawada I, Watanabe H, Yasuda H, Nakayama S, Yoda S,

Naoki K, Chen TH, Richards WG, Sugarbaker DJ, Meyerson M. Missense mutations of the

Onoda S, Masuda N, Seto T, Eguchi K, Takiguchi Y, Isobe H, Okamoto H, Ogura T,

*Mol Cancer Res* 2010 Aug;8(8):1142-51, ISSN:1541-7786

http://www.nccn.org/professionals/physician\_gls/pdf/nscl.pdf

Non-Small Cell Lung Cancer, *NCCN Guidelines™*, 2011 Aug,

Spigel DR, Dudek AZ, Shah C, Salgia R, McNally R, Renschler MF, & Oliver JW. Phase II study of amrubicin as second-line therapy in patients with platinumrefractory small-cell lung cancer. *J Clin Oncol* 2010 May 20;28(15):2598-603, ISSN

Tamura T. Effect of platinum combined with irinotecan or paclitaxel against large cell neuroendocrine carcinoma of the lung. *Jpn J Clin Oncol* 2007 Jul;37(7):482-6,

Naito T, Endo M, Yamamoto N, & Kameya T. Comparison of chemotherapy for unresectable pulmonary high-grade non-small cell neuroendocrine carcinoma and

Inoue C, Inage M, Yokouchi H, Watanabe H, Tsukamoto T, Saijo Y, Ishimoto O, Hommura F, & Nukiwa T. Randomized phase II trial comparing amrubicin with topotecan in patients with previously treated small-cell lung cancer: North Japan Lung Cancer Study Group Trial 0402. *J Clin Oncol* 2008 Nov 20;26(33):5401-6, ISSN

carcinoma: its place in the spectrum of pulmonary carcinoma. *Ann Thorac Surg* 2007

MF, & Oliver JW. Randomized phase II trial of single-agent amrubicin or topotecan as second-line treatment in patients with small-cell lung cancer sensitive to firstline platinum-based chemotherapy. *J Clin Oncol*. 2011 Jan 20;29(3):287-93, ISSN

Satomi R, Ikemura S, Terai H, Sato T, & Ishizaka A. The combination of multiple receptor tyrosine kinase inhibitor and mammalian target of rapamycin inhibitor overcomes erlotinib resistance in lung cancer cell lines through c-Met inhibition.

BRAF gene in human lung adenocarcinoma. *Cancer Res* 2002 Dec 1;62(23):7001-3,

Yokoyama A, Seki N, Asaka-Amano Y, Harada M, Tagawa A, Kunikane H, Yokoba M, Uematsu K, Kuriyama T, Kuroiwa Y, & Watanabe K; Thoracic Oncology

Clinical Trials.gov, 2011 Aug,

0732-183X

0732-183X

0732-183X

ISSN:0008-5472

ISSN 0368-2811

*Cancer* 2010 Jul;69(1):13-8, ISSN 0169-5002

Aug;84(2):702-7, ISSN 0003-4975

http://clinicaltrials.gov/

Research Group Study 0301. Phase II trial of amrubicin for treatment of refractory or relapsed small-cell lung cancer: Thoracic Oncology Research Group Study 0301. *J Clin Oncol* 2006 Dec 1;24(34):5448-53, ISSN 0732-183X


Yoshida H, Sekine I, Tsuta K, Horinouchi H, Nokihara H, Yamamoto N, Kubota K, & Tamura T. Amrubicin Monotherapy for Patients with Previously Treated Advanced Large-cell Neuroendocrine Carcinoma of the Lung. *Jpn J Clin Oncol* 2011 Jul;41(7):897-901, ISSN 0368-2811

## **Radiation Therapy in Management of Small-Cell Lung Cancer**

#### Erkan Topkan and Cem Parlak

*Baskent University Adana Medical Faculty, Department of Radiation Oncology Turkey* 

#### **1. Introduction**

238 Lung Diseases – Selected State of the Art Reviews

Yoshida H, Sekine I, Tsuta K, Horinouchi H, Nokihara H, Yamamoto N, Kubota K, &

Jul;41(7):897-901, ISSN 0368-2811

Tamura T. Amrubicin Monotherapy for Patients with Previously Treated Advanced Large-cell Neuroendocrine Carcinoma of the Lung. *Jpn J Clin Oncol* 2011

> Worldwide, lung carcinoma (LC) is the commonest and deadliest form of cancer in men and women, exceeding the mortality of prostate, breast, and colorectal cancers combined (Jemal et al., 2009). Irrespective of histologic subtype, more than 90% of all LC is strongly associated with cigarette smoking (Alavanja et al., 2002), and the risk significantly increases with the number of cigarettes smoked per day, degree of inhalation, age at initiation, and life-long cumulative exposure (Tyczynski et al., 2003). Although survival at 5-year is less than 15%, yet, there exists no single proven chemopreventive measure reducing the risk of LC development, except for cessation of cigarette smoking.

> Histologically, non-small cell LC (NSCLC) and small-cell LC (SCLC) are the two commonest types of LC, constituting 85-90% and 10-15% of all cases, respectively (Rosenzweig et al., 2010). SCLC (previously called oat cell carcinoma) is relatively less common than NSCLC; however, because of a more aggressive growth pattern and clinical course, its treatment is more challenging. Although there is no significant difference in outcome by histologic subtype, the World Health Organization classification subdivides SCLC into three cell types; pure or classic, variant cell, and mixed (Brambilla et al., 2001). SCLC displays a high propensity to metastasize, and usually a remarkable but temporary responsiveness to chemotherapy and radiotherapy (RT). Although some may be cured, most patients succumb to disease because of rapid development of drug resistance and resultant disease progression. Median survival for metastatic SCLC is only 10 months, which is interestingly very similar to patients with relatively much more drug resistant NSCLC of similar stage (Chute et al., 1997). Despite great improvements in imaging, pathology, genetics, chemotherapy, and RT techniques, this did not translate into the clinical outcomes, thus, the current overall survival rate for SCLC patients is not different than that was 20 years before (Chute et al., 1997).

> This chapter has been designated to focus specifically on treatment of SCLC patients, with specific emphasises on the technical basis and outcomes of current RT and concurrent chemoradiotherapy (C-CRT). Therefore, readers interested in more comprehensive information about the epidemiology, etiology, preventive measures, pathologic and genetic basis and surgical treatment of SCLC are referred to excellent reviews available in this book.

## **2. Staging**

Management of SCLC begins with the accurate staging of the disease. Historically, using the Veteran's Administration Lung Study Group (VALG) criteria (Zelen, 1973), staging of SCLC was simplified to include two stages: limited stage SCLC (LS-SCLC), and extensive stage SCLC (ES-SCLC). LS-SCLC is defined as the disease confined to the ipsilateral hemithorax which can be safely encompassed within a tolerable single radiation port. Involvement of ipsilateral supraclavicular lymph node region is also included in limited-stage disease. In contrast; patients with ES-SCLC have disease that is beyond the ipsilateral hemithorax. Besides the hematogenous spread, involvement of contralateral supraclavicular lymph node region and/or presence of malignant pleural- and/or pericardial effusion are included in extensive-stage disease. Only less than one third of SCLC patients present with limited-stage disease, while remaining two thirds have extensive-stage disease, and are treated with palliative chemotherapy.

The revised American Joint Committee on Cancer (AJCC) staging system implemented the TNM staging for NSCLC, but its use for SCLC was also recommended in sixth and seventh editions (Edge, 2010; Greene, 2002). Recent study by Vallieres et al., constituting of 349 patients with resected SCLC confirmed the utility of TNM-based pathologic staging in terms of survival outcomes. But, because of being restricted to only 5% SCLC patients presenting with an operable disease at presentation, TNM-based staging has not been routinely adopted two tired VALG staging system (LS-SCLC and ES-SCLC) in this group of patients (Vallieres et al., 2009).

## **3. Prognostic factors**

Despite paramount improvements in imaging and treatment modalities, prognosis of patients with SCLC is still unacceptably poor with median survival ranges of only 15-20 months for LS-SCLC and 8-13 months for ES-SCLC (Lally et al, 2007). Furthermore, 5-year survivors are reported to be only in the respective ranges of 10-13% and 1-2%, emphasizing the futility of the condition (Lassen, 1995; Tai, 2003). A number of factors have been assigned to carry prognostic importance for patients with SCLC but the most important tumor related factor is the VALG stage (LS-SCLC versus ES-SCLC). In LS-SCLC, early stage disease that corresponds to TNM stage 1 carries the best prognosis specifically in the absence of elevated serum lactate dehydrogenase levels (Byhardt, 1986; Lassen, 1995). Similar to other tumor sites, weight loss and poor performance status are significant predictors of unfavorable outcome (Paesmans et al., 2000). Likewise, men fare poorer than women (Lally et al, 2007). In ES-SCLC, the number of organ sites and site of involvement are also strongly associated with prognosis (Albain et al, 1991). Compared to other sites, involvement of bone marrow, liver, or central nervous system signify unfavorable disease course.

Compared to NSCLC, SCLC is more frequently associated with paraneoplastic syndromes either via antibody-mediated tissue destruction or via ectopic hormone production (Lally et al, 2007). Although, some debate exists, unlike antibody-mediated paraneoplastic syndromes, ectopic hormone production is generally accepted as a predictor of poor outcome. Favorable prognosis linked to antibody-mediated paraneoplastic syndromes may be related with presence of a fully competent immune system, indicating the need for exploration of immunotherapy adjunct to standard treatment approaches in this patients group.

## **4. Treatment for limited-stage small cell lung carcinoma**

#### **4.1 Chemotherapy**

240 Lung Diseases – Selected State of the Art Reviews

Management of SCLC begins with the accurate staging of the disease. Historically, using the Veteran's Administration Lung Study Group (VALG) criteria (Zelen, 1973), staging of SCLC was simplified to include two stages: limited stage SCLC (LS-SCLC), and extensive stage SCLC (ES-SCLC). LS-SCLC is defined as the disease confined to the ipsilateral hemithorax which can be safely encompassed within a tolerable single radiation port. Involvement of ipsilateral supraclavicular lymph node region is also included in limited-stage disease. In contrast; patients with ES-SCLC have disease that is beyond the ipsilateral hemithorax. Besides the hematogenous spread, involvement of contralateral supraclavicular lymph node region and/or presence of malignant pleural- and/or pericardial effusion are included in extensive-stage disease. Only less than one third of SCLC patients present with limited-stage disease, while remaining two thirds have extensive-stage disease, and are treated with

The revised American Joint Committee on Cancer (AJCC) staging system implemented the TNM staging for NSCLC, but its use for SCLC was also recommended in sixth and seventh editions (Edge, 2010; Greene, 2002). Recent study by Vallieres et al., constituting of 349 patients with resected SCLC confirmed the utility of TNM-based pathologic staging in terms of survival outcomes. But, because of being restricted to only 5% SCLC patients presenting with an operable disease at presentation, TNM-based staging has not been routinely adopted two tired VALG staging system (LS-SCLC and ES-SCLC) in this group of patients

Despite paramount improvements in imaging and treatment modalities, prognosis of patients with SCLC is still unacceptably poor with median survival ranges of only 15-20 months for LS-SCLC and 8-13 months for ES-SCLC (Lally et al, 2007). Furthermore, 5-year survivors are reported to be only in the respective ranges of 10-13% and 1-2%, emphasizing the futility of the condition (Lassen, 1995; Tai, 2003). A number of factors have been assigned to carry prognostic importance for patients with SCLC but the most important tumor related factor is the VALG stage (LS-SCLC versus ES-SCLC). In LS-SCLC, early stage disease that corresponds to TNM stage 1 carries the best prognosis specifically in the absence of elevated serum lactate dehydrogenase levels (Byhardt, 1986; Lassen, 1995). Similar to other tumor sites, weight loss and poor performance status are significant predictors of unfavorable outcome (Paesmans et al., 2000). Likewise, men fare poorer than women (Lally et al, 2007). In ES-SCLC, the number of organ sites and site of involvement are also strongly associated with prognosis (Albain et al, 1991). Compared to other sites, involvement of bone marrow,

Compared to NSCLC, SCLC is more frequently associated with paraneoplastic syndromes either via antibody-mediated tissue destruction or via ectopic hormone production (Lally et al, 2007). Although, some debate exists, unlike antibody-mediated paraneoplastic syndromes, ectopic hormone production is generally accepted as a predictor of poor outcome. Favorable prognosis linked to antibody-mediated paraneoplastic syndromes may be related with presence of a fully competent immune system, indicating the need for exploration of immunotherapy adjunct to standard treatment approaches in this patients

liver, or central nervous system signify unfavorable disease course.

**2. Staging** 

palliative chemotherapy.

(Vallieres et al., 2009).

group.

**3. Prognostic factors** 

In LS-NSCLC, chemotherapy trials conducted in the 1970s improved survival from weeks to months. Over the following three decades, several studies have shown that combination chemotherapy regimens were clearly more efficacious than single agent regimens. Response rates of 70%-85%, with complete response of 20%-30%, are encouraging but virtually almost every patient relapses (Lally et al, 2007). Results of randomized investigations and metaanalysis for the most active regimen indicated the superiority of etoposide plus cisplatin (EP) combination over the other tested combinations (Fukuoka, 1991; Pujol, 2000; Roth, 1992; Sundstrom, 2002). Therefore, the EP combination has become the standard care chemotherapy combination in United States and Europe since 1980s. Although cisplatin is the backbone of chemotherapy, carboplatin may be substituted for cisplatin in older patients or in those with renal insufficiency without an apparent efficacy loss (Okamoto et al., 2005).

Chemotherapy combinations constituting a variety of newer agents, like irinotecan, have been tested in an effort to improve current outcomes in LS-SCLC. However, these agents do not appear to be more active than older counterparts. Irinotecan, which was the most promising of them, has been tested in three randomized phase 3 trials (Hanna, 2006; Lara, 2009; Noda, 2002). The first trial by Noda et al. demonstrated the superiority of cisplatin plus irinotecan (IP) over the standard EP combination in a Japanese Clinical Oncology Group (JCOG) trial (Noda et al., 2002). However, two subsequent trials launched in United States could not validate these results (Hanna, 2006; Lara, 2009). In both trials response and survival rates in patients treated with investigational IP were found to be equivalent to standard EP. The potential benefit of adding a third agent to standard EP has also been extensively investigated. Higher response rates at a cost of significantly increased toxicity were achieved, with no notable improvement in median survival duration over EP alone (Loehrer, 1995; Mavroudis, 2001; Niell, 2005; Pujol, 2001).

Based on these results, the current standard for the first line chemotherapy in this group of patients is 4 to 6 cycles of EP combination, and further treatment with either maintenance therapy or four cycles of topotecan following standard EP regimen has not been proved to improve outcomes (Schiller, 2001; Sculier, 1998).

#### **4.2 Thoracic radiotherapy**

Before the introduction of chemotherapy in the 1970s, thoracic radiotherapy (TRT) was the mainstay of treatment for LS-SCLC. However, management of LS-SCLC with chemotherapy alone results in unacceptable rates of intrathoracic failures, ranging from 75 to90% (Faivre-Finn et al. 2005). In this setting, integration of TRT to chemotherapy reduces these failures up to 30 to 60%. Impact of such a decrease in intrathoracic failures has been extensively investigated by two meta-analyses (Pignon, 1992; Warde & Payne, 1992). In the first one, Warde and Payne (Warde & Payne, 1992) analyzed 11 randomized studies including 1911 patients, and reported a significantly longer overall survival with the combination of TRT and chemotherapy than with chemotherapy alone, with an absolute benefit of 5.4% at 2-year (p<0.001). In the other meta-analysis, Pignon et al. (Pignon et al., 1992) included 13 trials consisting of 2103 LS-SCLC patients. Combination of TRT and chemotherapy again resulted in an absolute survival advantage of 5.4% at 3-year compared to chemotherapy alone (p=0.001). Based on the results of these two meta-analyses combination of TRT and chemotherapy became the established standard of care in LS-SCLC.

TRT delivered both sequentially and concurrently with chemotherapy has been intensively assessed. Although sequential treatment approach has the theoretical benefits by chance of irradiating smaller target volumes with resultant reduced toxicity rates, the associated longer overall treatment time potentially increases the risk of accelerated tumor repopulation and development of treatment-resistant clones. In this context, concurrent use of chemotherapy and TRT does not only reduce the risk for accelerated repopulation but also offers a chance for better locoregional control by utilizing the radiosensitizing efficacy of chemotherapeutic agents. Nevertheless, despite such potential benefits, because of increased risk for higher rates of acute toxicity with concurrent chemoradiotherapy, sequential use of chemotherapy and TRT may be more feasible in elderly patients or those with larger tumors.

Data on the optimum radiotherapy dose and fractionation come mostly from retrospective and phase 2 prospective studies. The results from non-randomized studies indicate a notable increase in local control when the dose of TRT is increased from 35 to 40 Gy, and a slightly further gain with 50 Gy. Laboratory studies have suggested that typical SCLC cell lines have radiation survival curves with little shoulders indicating that accelerated fractionation schemes would, therefore, be advantageous (van Meerbeeck et al., 2011). In 1999, two different cooperative groups randomized patients to once-a-day versus twice-aday TRT with concurrent chemotherapy, as depicted in Table 1 (Bonner, 1999; Turrisi, 1999). In the study by Bonner et al., authors reported the North Central Cancer Treatment Group (NCCTG) experience, and concluded that there was no difference in survival with twicedaily TRT versus once daily counterpart (Bonner et al., 1999). However, this study has been criticized because of using split-course RT schedule which is currently an established factor to increase the chance for accelerated repopulation, and therefore, affect treatment outcomes in an unfavorable fashion. In the landmark study by Turissi et al. (Int-0096), authors reported the long-term outcomes of 358 patients enrolled onto the cooperative group study of Eastern Cooperative Oncology Group/ Radiation Therapy Oncology Group (ECOG/RTOG) with the diagnosis of LS-SCLC. Results of this study demonstrated that twice-daily 45 Gy (1.5 Gy BID) and concurrent CRT was significantly superior over conventionally fractionated TRT scheme (Turrisi et al., 1999). Based on the results of this latter study, the current standard of care for medically fit LS-SCLC became the 45 Gy (1.5 Gy BID) TRT and concurrent EP. Nonetheless, because of the higher frequency of dose limiting ≥ Grade 3 esophagitis in twice-daily TRT scheme, 54 Gy (1.8 Gy per fraction) in 30 days and concurrent EP is also a common and acceptable treatment scheme.

Carcinoma and Leukemia Group B (CALGB) conducted two phase 2 trials to investigate the potential benefit of a higher dose of 70 Gy given in 35 fractions within 7 weeks (Bogart, 2004; Miller, 2007). In the first study, median survival, 2-year survival rate, and ≥ Grade 3 esophagitis rate were 22 months, 48%, and 21%, respectively (Bogart et al., 2004). In the second study respective rates were 20 months, 35%, and 30% (Miller et al., 2007). Based on the promising results of these two trials, two ongoing randomized Phase 3 trials were conducted to compare standard Turissi protocol with escalated doses of conventionally fractionated TRT. Results of these two landmark trials summarized in Table 2, will address the question whether the higher doses delivered by once daily scheme in 7-week, could compensate for the longer interval between the initiation of treatment and the end of TRT, in the expense of increased risk for accelerated tumor cell repopulation.

In treatment of LS-SCLC, another issue of interest is whether TRT should be administered early or late during the chemotherapy course. This question has been addressed by a number of trials with no firm conclusions (Gregor, 1997; Jeremic, 1997; Murray, 1993; Perry,

TRT delivered both sequentially and concurrently with chemotherapy has been intensively assessed. Although sequential treatment approach has the theoretical benefits by chance of irradiating smaller target volumes with resultant reduced toxicity rates, the associated longer overall treatment time potentially increases the risk of accelerated tumor repopulation and development of treatment-resistant clones. In this context, concurrent use of chemotherapy and TRT does not only reduce the risk for accelerated repopulation but also offers a chance for better locoregional control by utilizing the radiosensitizing efficacy of chemotherapeutic agents. Nevertheless, despite such potential benefits, because of increased risk for higher rates of acute toxicity with concurrent chemoradiotherapy, sequential use of chemotherapy and TRT

Data on the optimum radiotherapy dose and fractionation come mostly from retrospective and phase 2 prospective studies. The results from non-randomized studies indicate a notable increase in local control when the dose of TRT is increased from 35 to 40 Gy, and a slightly further gain with 50 Gy. Laboratory studies have suggested that typical SCLC cell lines have radiation survival curves with little shoulders indicating that accelerated fractionation schemes would, therefore, be advantageous (van Meerbeeck et al., 2011). In 1999, two different cooperative groups randomized patients to once-a-day versus twice-aday TRT with concurrent chemotherapy, as depicted in Table 1 (Bonner, 1999; Turrisi, 1999). In the study by Bonner et al., authors reported the North Central Cancer Treatment Group (NCCTG) experience, and concluded that there was no difference in survival with twicedaily TRT versus once daily counterpart (Bonner et al., 1999). However, this study has been criticized because of using split-course RT schedule which is currently an established factor to increase the chance for accelerated repopulation, and therefore, affect treatment outcomes in an unfavorable fashion. In the landmark study by Turissi et al. (Int-0096), authors reported the long-term outcomes of 358 patients enrolled onto the cooperative group study of Eastern Cooperative Oncology Group/ Radiation Therapy Oncology Group (ECOG/RTOG) with the diagnosis of LS-SCLC. Results of this study demonstrated that twice-daily 45 Gy (1.5 Gy BID) and concurrent CRT was significantly superior over conventionally fractionated TRT scheme (Turrisi et al., 1999). Based on the results of this latter study, the current standard of care for medically fit LS-SCLC became the 45 Gy (1.5 Gy BID) TRT and concurrent EP. Nonetheless, because of the higher frequency of dose limiting ≥ Grade 3 esophagitis in twice-daily TRT scheme, 54 Gy (1.8 Gy per fraction) in 30 days and

Carcinoma and Leukemia Group B (CALGB) conducted two phase 2 trials to investigate the potential benefit of a higher dose of 70 Gy given in 35 fractions within 7 weeks (Bogart, 2004; Miller, 2007). In the first study, median survival, 2-year survival rate, and ≥ Grade 3 esophagitis rate were 22 months, 48%, and 21%, respectively (Bogart et al., 2004). In the second study respective rates were 20 months, 35%, and 30% (Miller et al., 2007). Based on the promising results of these two trials, two ongoing randomized Phase 3 trials were conducted to compare standard Turissi protocol with escalated doses of conventionally fractionated TRT. Results of these two landmark trials summarized in Table 2, will address the question whether the higher doses delivered by once daily scheme in 7-week, could compensate for the longer interval between the initiation of treatment and the end of TRT,

In treatment of LS-SCLC, another issue of interest is whether TRT should be administered early or late during the chemotherapy course. This question has been addressed by a number of trials with no firm conclusions (Gregor, 1997; Jeremic, 1997; Murray, 1993; Perry,

may be more feasible in elderly patients or those with larger tumors.

concurrent EP is also a common and acceptable treatment scheme.

in the expense of increased risk for accelerated tumor cell repopulation.


Table 1. Limited-stage small cell lung cancer with daily versus twice-daily TRT


Table 2. Benchmark ongoing trials of chemoradiotherapy for limited-stage small-cell lung carcinoma

1987; Skarlos, 2001; Takada, 2002; Work, 1997). In the landmark phase 3 ECOG/RTOG trial reported by Turissi et al., the shortening of total irradiation period from 5 weeks to 3 weeks was associated with an absolute 10% (16% versus 26%) increase in 5-year survival (Turrisi et al., 1999). Results of three other trials revealed a significantly superior survival advantage for early TRT over late TRT, confirming the findings of intergroup trial (Jeremic, 1997; Murray, 1993; Takada, 2002). The impact of timing of TRT relative to chemotherapy has also been addressed by various meta-analyses. De Ruysscher and colleagues conducted a metaanalysis of phase III trials combining TRT and platinum-based chemotherapy, and concluded that the most important predictor of 5-year survival was the interim between the start of any treatment until the end of RT (SER), with shorter SERs (<30 days) being associated with the highest 5-year survival rates (>20%) (De Ruysscher et al., 2006). With a subsequent meta-analysis, Pijls-Johannesma and colleagues evaluated the impact of timing of TRT by comparing early versus late TRT, by defining early TRT as within 30 days of beginning chemotherapy. In presence of platinum-based chemotherapy, the 2- and 5-year survival rates were favoring early TRT, and this difference was significant only if the TRT was administered in a treatment period of less than 30 days. In this study, patient compliance was found to be of paramount importance, indicating the importance of patient selection in clinical trials (Pijls-Johannesma et al., 2007). In a relatively older meta-analysis by Fried et al., late TRT was defined as beginning 9 weeks after the initiation of chemotherapy or after the completion of third cycle of chemotherapy. Similar to other subsequent meta-analyses, this meta-analysis also demonstrated a statistically significant benefit of early TRT over late TRT in terms of 2 year overall survival. On subset analysis of studies that used hyperfractionated TRT, treatment with early versus late TRT revealed a survival benefit, but not when once-daily TRT was employed. Likewise, the survival benefit for early versus late TRT was observed uniquely in studies using platinum-based chemotherapy, which was not notable in studies using nonplatinum-based chemotherapy (Fried et al., 2004). Results of the available studies and metaanalyses suggested an interaction between TRT and chemotherapy and, accelerated tumor cell repopulation was postulated to be triggered by the first dose of any effective cytotoxic agent (De Ruysscher et al., 2006). Therefore, to obtain the highest chance for local/regional control, the last clonogenic tumor cell should be killed by the end of TRT (van Meerbeeck et al., 2011). Hence, long-term survival decreases with increasing time between the initiations of any treatment and the completion of TRT.

In summary, current evidence recommends the early administration of 45 Gy (1.5 Gy, BID) with concurrent EP at systemic doses in medically fit LS-SCLC patients.

#### **4.4 Radiotherapy techniques and treatment fields**

Treatment for lung tumors, including SCLC, is complex. In order to ensure safe and effective RT, several issues must be considered: (a) accurate target volume delineation; (b) proximity of dose limiting normal structures (lung, spinal cord, esophagus, heart, brachial plexus, and liver); (c) anatomic slope of the chest surface; (d) inhomogenities resulting from the presence of nonuniform tissues on the way of RT; (e) frequent need for irregular field dose calculations; (f) respiratory motion of the targeted tumor and normal tissues such as lung, heart and liver, depending on the location of the primary tumor and involved lymphatic region(s).

The ultimate goal of any RT application is to deliver the prescribed dose homogenously (not cooler than 95% and not hotter than 107%) to the planned target volume and keep the dose to non-tumorous normal tissues as minimum as possible respecting their tissue architecture (serial versus parallel) and their radiation tolerance limits. In this setting, with the aid of imaging with anatomic computerized tomography (CT), functional 18-F-fluorodeoxyglucose positron emission tomography (PET), preferably fusion of both) and the use of 3–dimensional conformal RT, and novel 4-dimensional image-guided RT (IGRT), it is easier than before to achieve these goals. Additionally, the dose-volume histograms (DVH) created for each patient makes it possible to anticipate the potential early and late toxicity risks based on the organ of interest measures and, therefore, modify the treatment plans as necessitated.

There is considerable debate on the size of the RT portals of SCLC. Historically, RT portals were large, encompassing the primary tumor as well as both hilar, entire mediastinal and both supraclavicular lymph node regions with generous margins. This was believed to be necessary to ensure adequate coverage of gross disease prior to the routine use of CT- based RT planning. Although such large field plans may guarantee the irradiation of target volumes, they are also associated with increased acute and late toxicity rates and unplanned

chemotherapy. In presence of platinum-based chemotherapy, the 2- and 5-year survival rates were favoring early TRT, and this difference was significant only if the TRT was administered in a treatment period of less than 30 days. In this study, patient compliance was found to be of paramount importance, indicating the importance of patient selection in clinical trials (Pijls-Johannesma et al., 2007). In a relatively older meta-analysis by Fried et al., late TRT was defined as beginning 9 weeks after the initiation of chemotherapy or after the completion of third cycle of chemotherapy. Similar to other subsequent meta-analyses, this meta-analysis also demonstrated a statistically significant benefit of early TRT over late TRT in terms of 2 year overall survival. On subset analysis of studies that used hyperfractionated TRT, treatment with early versus late TRT revealed a survival benefit, but not when once-daily TRT was employed. Likewise, the survival benefit for early versus late TRT was observed uniquely in studies using platinum-based chemotherapy, which was not notable in studies using nonplatinum-based chemotherapy (Fried et al., 2004). Results of the available studies and metaanalyses suggested an interaction between TRT and chemotherapy and, accelerated tumor cell repopulation was postulated to be triggered by the first dose of any effective cytotoxic agent (De Ruysscher et al., 2006). Therefore, to obtain the highest chance for local/regional control, the last clonogenic tumor cell should be killed by the end of TRT (van Meerbeeck et al., 2011). Hence, long-term survival decreases with increasing time between the initiations of any

In summary, current evidence recommends the early administration of 45 Gy (1.5 Gy, BID)

Treatment for lung tumors, including SCLC, is complex. In order to ensure safe and effective RT, several issues must be considered: (a) accurate target volume delineation; (b) proximity of dose limiting normal structures (lung, spinal cord, esophagus, heart, brachial plexus, and liver); (c) anatomic slope of the chest surface; (d) inhomogenities resulting from the presence of nonuniform tissues on the way of RT; (e) frequent need for irregular field dose calculations; (f) respiratory motion of the targeted tumor and normal tissues such as lung, heart and liver, depending on the location of the primary tumor and involved lymphatic

The ultimate goal of any RT application is to deliver the prescribed dose homogenously (not cooler than 95% and not hotter than 107%) to the planned target volume and keep the dose to non-tumorous normal tissues as minimum as possible respecting their tissue architecture (serial versus parallel) and their radiation tolerance limits. In this setting, with the aid of imaging with anatomic computerized tomography (CT), functional 18-F-fluorodeoxyglucose positron emission tomography (PET), preferably fusion of both) and the use of 3–dimensional conformal RT, and novel 4-dimensional image-guided RT (IGRT), it is easier than before to achieve these goals. Additionally, the dose-volume histograms (DVH) created for each patient makes it possible to anticipate the potential early and late toxicity risks based on the organ of

There is considerable debate on the size of the RT portals of SCLC. Historically, RT portals were large, encompassing the primary tumor as well as both hilar, entire mediastinal and both supraclavicular lymph node regions with generous margins. This was believed to be necessary to ensure adequate coverage of gross disease prior to the routine use of CT- based RT planning. Although such large field plans may guarantee the irradiation of target volumes, they are also associated with increased acute and late toxicity rates and unplanned

with concurrent EP at systemic doses in medically fit LS-SCLC patients.

interest measures and, therefore, modify the treatment plans as necessitated.

treatment and the completion of TRT.

region(s).

**4.4 Radiotherapy techniques and treatment fields** 

treatment delays, which may negatively impact both quality of life measures and local/regional control rates and related survival outcomes. This issue is specifically argued when TRT is delayed after the completion of induction chemotherapy. Although some authors advocate generous portals encompassing the pre-chemotherapy volumes as stated above, others argue that only limited portals encompassing the pre-chemotherapy primary tumor and high-risk nodal areas with a 1-cm margin are adequate, since effective chemotherapy has the theoretical chance to cope with subclinical or microscopic disease eliminating the need for generous portals. This latter approach has the additional potential for decreased treatment related toxicity specifically when TRT is administered concurrently with chemotherapy. Treatment directed at pre- versus post-chemotherapy volumes is also an ongoing issue of conflict. The unique randomized trial that addressed this issue is the one conducted by the South West Oncology Group (SWOG). In this study, patients achieving a partial response after chemotherapy were randomized to pre- versus post-chemotherapy volume irradiation arms. Outcomes of this benchmark study did not indicate any superiority for pre-chemotherapy volume irradiation arm over post-chemotherapy irradiation counterpart, in terms of neither local/regional nor survival rates (Kies et al., 1987). This issue has latter been investigated by Liengswangwong et al. in a retrospective analysis. The authors were unable to find a benefit favoring pre-chemotherapy large-field TRT over post-chemotherapy limited-field TRT (Liengswangwong et al., 1994).

In NSCLC, elective irradiation of hilar and/or mediastinal lymphatic regions has gradually been replaced by treatment limited to nodes identified by CT or FDG –PET as being involved. For SCLC, evidence is scarce to support this approach. In a prospective study by De Ruysscher et al., authors limited the RT fields to only CT-positive mediastinal lymph nodes in a cohort of 27 patients with LS-SCLC. The authors reported an isolated regional recurrence rate of 11%, which was higher than similar studies using elective mediastinal irradiation. However, because of small sample size, no definitive conclusions can be drawn from this study (De Ruysscher et al., 2006). In a larger phase 2 study including 60 LS-SCLC patients, van Loon et al., irradiated only the lymph nodes that appeared to be involved on FDG-PET, and reported an isolated nodal failure rate of 3%, which awaits to be confirmed by further studies with larger cohorts (van Loon et al., 2010). A typical 3-D conformal RT plan used in our institution and associated DVH is shown in Figure 1.

#### **4.5 Prophylactic cranial irradiation**

Approximately 10-14% of SCLC patients have detectable brain metastases (BM) at the time of initial diagnosis (Hardy et al., 1990). During the course of disease, the incidence of BM increases up to more than 50%, which is far beyond in postmortem examinations (Hirsch, 1983; Nicholson, 2002). The incidence of BM is directly proportional with the survival time, indicating a potential for further increase with implementation of more effective treatment protocols (Komaki, 1981; van Oosterhout, 1996). Compared to patients with LS-SCLC, the risk for BM occurrence is higher for patients with ES-SCLC reaching 69% at 2- years of diagnosis (van Oosterhout, 1996; Yang GY & Matthews, 2000).

Impact of BM on socioeconomic issues and quality of life is significantly worse than the impact of failure at other metastatic sites. Patients with BM are often obliged to spend significant time hospitalized, and suffer loss of independence (Felletti et al., 1985). Despite cranial irradiation and/or chemotherapy, the treatment of clinically established BM is partially satisfactory with intracranial disease control rates of about 50% and overall survival of 4 to 6 months (Carmichael, 1988; Lucas, 1986; Postmus, 1989).

Fig. 1. An example of typical 3-D conformal RT plan and associated DVH.

Several randomized trials have been conducted to investigate the utility of prophylactic cranial irradiation (PCI) in prevention of BM development in patients with LS-SCLC. Logically, in patients with extracranial disease under control, PCI may eliminate the intracranial microscopic tumor cell deposits with relatively low radiation doses and, therefore, may increase the long-term survival. Results of two randomized controlled trials from France (PCI85) and United Kingdom (UK02) demonstrated a trend for better survival with PCI but neither could reach statistical significance (Arriagada, 1995; Gregor, 1997). Up till now, no individual randomized trial has conclusively demonstrated a survival benefit for PCI, which may be related with their deficiency in provision of sufficient power to detect moderate differences in survival.

To conduct a meta-analysis of trials using PCI and to make recommendations for clinical practice, the PCI Overview Collaborative Group was established. The meta-analysis reported by Auperin et al. in 1999 included individual data from patients enrolled on seven prospective randomized PCI trials. Trials eligible in the meta-analysis were limited to those, in which patients had been treated with systemic chemotherapy with/without TRT to a complete clinical response, and no known BM. PCI treatments were generally between 24 to 40 Gy, administered in 2-3 Gy per day. Results of this meta-analysis, for the first time, demonstrated a statistically significant survival advantage favoring PCI over non-PCI arm. The relative risk for death in the treatment group, as compared to control group, was 0.84 (95 CI, 0.73-0.97; P= 0.01), which corresponds to a 5.4% higher rate of survival at 3 years (20.7% versus 15.3%), and the survival advantage persisted over time. As a percent gain over control, this represents a 35% increase in the proportion of surviving patients. There was also significant difference in disease-free survival at 3 years from 13.3% in the non-PCI group to 22.1% in the PCI group (p<0.0001). PCI was additionally associated with a 25.3% absolute decrease in the cumulative incidence of BM at 3 years, from 58.6% to 33.3% (p<0.0001) (Auperin et al., 1999). Results of this comprehensive meta-analysis have recently been confirmed by the review of data from Surveillance Epidemiology and End Results (SEER) reported by Patel et al. Of 7995 LS-SCLC patients included, 670 received PCI. Better overall and cause-specific survival were observed in patients treated with PCI, and corresponding 2- and 5-year survival rates were 23% and 11% without PCI and 425% and 19% with PCI (Patel et al., 2009).

Based on the results of meta-analysis by Auperin et al., PCI became the standard of care in patients with LS-SCLC demonstrating complete response following systemic and/or local/regional treatment (Auperin et al., 1999). However, an important concern about the use of PCI is the need for determination of an established non-toxic but effective fractionation scheme and total dose. Available data have shown that lower doses of PCI may be less effective in preventing CNS failures (Auperin, 1999; Gregor, 1997). Recently, Le Pechoux et al. published the results of benchmark international PCI trial evaluating radiation dose for PCI in LS-SCLC. The study randomized 720 LS-SCLC patients from 157 centers to one of two PCI arms: Arm-1 included patients receiving standard-dose PCI to 25 Gy in 2.5 Gy per fraction, and Arm 2 included patients receiving higher dose PCI to 36 Gy delivered in 2 Gy once daily or 1.5 Gy twice daily. No significant difference of BM incidence was reported between two study arms, but there was a significantly higher rate of cancer-related mortality in the higher dose arm as a result of unexplained finding of more deaths from extracranial disease progression (Le Pechoux et al., 2009). Based on the results of this study, 25 Gy delivered at 2.5 Gy per fraction per day remains the standard of care for PCI in LS-SCLC patients.

## **5. Treatment for extensive-stage small cell lung carcinoma**

#### **5.1 Radiotherapy**

246 Lung Diseases – Selected State of the Art Reviews

Fig. 1. An example of typical 3-D conformal RT plan and associated DVH.

moderate differences in survival.

Several randomized trials have been conducted to investigate the utility of prophylactic cranial irradiation (PCI) in prevention of BM development in patients with LS-SCLC. Logically, in patients with extracranial disease under control, PCI may eliminate the intracranial microscopic tumor cell deposits with relatively low radiation doses and, therefore, may increase the long-term survival. Results of two randomized controlled trials from France (PCI85) and United Kingdom (UK02) demonstrated a trend for better survival with PCI but neither could reach statistical significance (Arriagada, 1995; Gregor, 1997). Up till now, no individual randomized trial has conclusively demonstrated a survival benefit for PCI, which may be related with their deficiency in provision of sufficient power to detect

To conduct a meta-analysis of trials using PCI and to make recommendations for clinical practice, the PCI Overview Collaborative Group was established. The meta-analysis reported by Auperin et al. in 1999 included individual data from patients enrolled on seven prospective randomized PCI trials. Trials eligible in the meta-analysis were limited to those, in which patients had been treated with systemic chemotherapy with/without TRT to a complete clinical response, and no known BM. PCI treatments were generally between 24 to 40 Gy, administered in 2-3 Gy per day. Results of this meta-analysis, for the first time, demonstrated a statistically significant survival advantage favoring PCI over non-PCI arm. The relative risk for death in the treatment group, as compared to control group, was 0.84 (95 CI, 0.73-0.97; P= 0.01), which corresponds to a 5.4% higher rate of survival at 3 years (20.7% versus 15.3%), and the survival advantage persisted over time. As a percent gain over control, this represents a 35% increase in the proportion of surviving patients. There was also significant difference in Combination chemotherapy is the mainstay in the management of ES-SCLC, but as intrathoracic disease control may be a significant challenge to overcome in a significant proportion of patients, role of consolidative TRT has been addressed in several trials. Jeremic et al. randomized patients with ES-SCLC, who responded completely at extrathoracic sites and at least partially at thorax, to consolidation TRT versus observation arms after 3 cycles of systemic chemotherapy. In experimental arm, TRT was administered in an accelerated hyperfractionated scheme of 54 Gy given in 1.5 Gy twice daily fractions concurrently with EP chemotherapy. The median and a 5-year overall survival in the TRT arm and no TRT arm were 17 versus 11 month and 9.1% versus 3.7%, respectively (Jeremic et al, 1999). However, the results of ongoing studies addressing the question of TRT both in Netherlands and in Canada should be awaited before its routine recommendation for patients with ES-SCLC.

#### **5.2 Prophylactic cranial irradiation**

Although the beneficial effects of PCI on prevention of BM occurrence and on augmentation of overall and disease-free survival have been well established in LS-SCLC patients, this issue had remained to be answered in ES-SCLC until the publication of the results of recent EORTC trial. In this benchmark study, patients with ES-SCLC who had a response to chemotherapy were randomized to PCI versus observation arms. The cumulative risk of symptomatic BM at 1-year and the 1-year survival rate were 14.6% versus 40.4% (p<0.001), and 27.1% versus 13.3% (p=0.003), both favoring the PCI arm (Slotman et al., 2007). Following this study, similar to LS-SCLC, PCI became the standard of care in ES-SCLC patients except for those experience disease progression during chemotherapy.

#### **6. Treatment related toxicity**

Acute side effects of CRT often begin during the second or third weeks of treatment. Cessation of the tobacco abuse should be the first step in the management of SCLC patients to increase the efficacy of intended CRT as well as to decrease the incidence and severity of the treatment related side effects. Dermatitis may be seen but severe cases are rare. Prevention of trauma is the key for prevention of severe and difficult to treat dermatitis development. Aloe vera gel and perfume-free ointments can be safely used in mild to moderate dermatitis. Acute esophagitis is usually the dose limiting toxicity of mediastinal irradiation, which rarely progress to severe late esophagitis. In patients with mild to moderate swallowing difficulty, semisolid nutrition and liquid form analgesics may be beneficial. Although confirmation with randomized controlled Phase 3 trials are needed, based on the results of two recent retrospective series by Algara et al. and Topkan et al., prophylactic use of glutamine may be beneficial in preventing and reducing the severity of acute esophagitis (Algara, 2007; Topkan, 2009). Nonproductive dry cough may be seen if trachea and/or major bronchi is/are involved in the high dose radiation portals.

After the completion of TRT, radiation pneumonitis (RP) may be seen at 2 to 4 weeks. RP is a form of radiation-induced lung disease, mimicking bacterial pneumonia. Symptoms usually include non-productive dry cough, dyspnea, chest pain, palpitations, malaise, and occasionally fever. Rales can be noted on auscultation. Plain X-rays and CT are beneficial in demonstrating the extent of perivascular haziness and alveolar filling densities primarily within the radiation portal. Treatment of RP includes use of 60 mg/day prednisone for two weeks followed by gradual tapering at following 3 to 12 weeks. There is no evidence supporting the use of prophylactic use of glucocorticoids or antibiotics in preventing or reducing the severity of RP.

Late toxicities involve chronic esophagitis, pericarditis, myocarditis, pancarditis, spinal cord injury, radiation induced lung fibrosis, and secondary cancers. Incidence and severity of all these late toxicities depend on the total dose and dose per fraction, fractionation scheme, interim between subsequent fractions, volume of non-target organ exposed to specified doses of RT, and concurrent use of chemotherapy. Currently, excluding the symptomatic management measures, the best treatment method is prevention of toxicity in the presence of agents with at best limited healing efficacy. To achieve this, tolerance doses must be strictly respected. Specific for radiation-induced lung fibrosis, which may potentially be fatal, a recent study demonstrated promising efficacy of pentoxyfilline and alpha-tocopherol combination in reduction of fibrotic lung area up to 50% at median 24 months of drug use.

At long-term, potential neurocognitive toxicity of PCI is of great concern, since sequalae like severe memory loss, intellectual impairment or even dementia, ataxia, or seizures have been reported in retrospective studies with small size and questionable methodology. For example, neurocognitive assessments prior to chemotherapy and/or PCI are lacking despite the fact that almost 50% of SCLC patients have neurologic and neurocognitive impairments prior to onset of PCI (Arriagada, 1995; Gregor, 1997; Grosshans, 2008; Komaki, 1995). Neurocognitive impairment risk has been reported to strongly associate with daily fraction sizes of >3 Gy (Paumier & A Le Péchoux, 2010). In one study, Shaw et al. found that the risk for neurocognitive impairment following PCI was 2% and 10% at 2- and 5-year follow-ups,

and 27.1% versus 13.3% (p=0.003), both favoring the PCI arm (Slotman et al., 2007). Following this study, similar to LS-SCLC, PCI became the standard of care in ES-SCLC

Acute side effects of CRT often begin during the second or third weeks of treatment. Cessation of the tobacco abuse should be the first step in the management of SCLC patients to increase the efficacy of intended CRT as well as to decrease the incidence and severity of the treatment related side effects. Dermatitis may be seen but severe cases are rare. Prevention of trauma is the key for prevention of severe and difficult to treat dermatitis development. Aloe vera gel and perfume-free ointments can be safely used in mild to moderate dermatitis. Acute esophagitis is usually the dose limiting toxicity of mediastinal irradiation, which rarely progress to severe late esophagitis. In patients with mild to moderate swallowing difficulty, semisolid nutrition and liquid form analgesics may be beneficial. Although confirmation with randomized controlled Phase 3 trials are needed, based on the results of two recent retrospective series by Algara et al. and Topkan et al., prophylactic use of glutamine may be beneficial in preventing and reducing the severity of acute esophagitis (Algara, 2007; Topkan, 2009). Nonproductive dry cough may be seen if

patients except for those experience disease progression during chemotherapy.

trachea and/or major bronchi is/are involved in the high dose radiation portals.

After the completion of TRT, radiation pneumonitis (RP) may be seen at 2 to 4 weeks. RP is a form of radiation-induced lung disease, mimicking bacterial pneumonia. Symptoms usually include non-productive dry cough, dyspnea, chest pain, palpitations, malaise, and occasionally fever. Rales can be noted on auscultation. Plain X-rays and CT are beneficial in demonstrating the extent of perivascular haziness and alveolar filling densities primarily within the radiation portal. Treatment of RP includes use of 60 mg/day prednisone for two weeks followed by gradual tapering at following 3 to 12 weeks. There is no evidence supporting the use of prophylactic use of glucocorticoids or antibiotics in preventing or reducing the severity of RP. Late toxicities involve chronic esophagitis, pericarditis, myocarditis, pancarditis, spinal cord injury, radiation induced lung fibrosis, and secondary cancers. Incidence and severity of all these late toxicities depend on the total dose and dose per fraction, fractionation scheme, interim between subsequent fractions, volume of non-target organ exposed to specified doses of RT, and concurrent use of chemotherapy. Currently, excluding the symptomatic management measures, the best treatment method is prevention of toxicity in the presence of agents with at best limited healing efficacy. To achieve this, tolerance doses must be strictly respected. Specific for radiation-induced lung fibrosis, which may potentially be fatal, a recent study demonstrated promising efficacy of pentoxyfilline and alpha-tocopherol combination in reduction of fibrotic lung area up to 50% at median 24 months of drug use. At long-term, potential neurocognitive toxicity of PCI is of great concern, since sequalae like severe memory loss, intellectual impairment or even dementia, ataxia, or seizures have been reported in retrospective studies with small size and questionable methodology. For example, neurocognitive assessments prior to chemotherapy and/or PCI are lacking despite the fact that almost 50% of SCLC patients have neurologic and neurocognitive impairments prior to onset of PCI (Arriagada, 1995; Gregor, 1997; Grosshans, 2008; Komaki, 1995). Neurocognitive impairment risk has been reported to strongly associate with daily fraction sizes of >3 Gy (Paumier & A Le Péchoux, 2010). In one study, Shaw et al. found that the risk for neurocognitive impairment following PCI was 2% and 10% at 2- and 5-year follow-ups,

**6. Treatment related toxicity** 

respectively. Furthermore, the authors reported that all toxicities were seen with regimens using daily fraction sizes of >3 Gy (Shaw et al., 1994). Notably, two randomized studies with neurocognitive assessments in patients randomized to PCI versus non-PCI did not demonstrate any deterioration in neurologic functions at 30 months, and quality of life measures at baseline, at 6 and 12 months (Arriagada, 1995; Gregor, 1997). However, these findings do not mean that PCI has no potential toxicity and should be administered to every patient with the diagnosis SCLC, rather they impact the importance of patient selection based on neurocognitive tests for safer PCI applications.

## **7. Treatment algorithm for LS-SCLC and ES-SCLC**

Our current instutitional treatment algorithm for LS-SCLC and ES-SCLC patients is as depicted in Figure 2.

Fig. 2. Management Algorithm for LS-SCLC and ES-SCLC

## **8. Conclusion**

Significant progress has been made in diagnosis and treatment of SCLC in the last 25 years, but, with an overall median survival time of 18 to 20 months, even in patients with limitedstage disease, it is still not possible to consider SCLC in the category of curable cancers. Despite this disappointing figure, mandating further research on this highly fatal disease, all improvements in LS-SCLC have been achieved by concurrent use chemotherapy and TRT (as early as possible), chemotherapy and PCI. For medically fit patients with ES-SCLC, combination chemotherapy followed by PCI (in non-progressive cases) is the standard of care, and further consolidation with TRT is currently under investigation. It is of paramount importance that patients with ES-SCLC be given the chance to participate in future trials for identification of a new and effective treatment combination, which may potentially offer a longer survival.

## **9. References**


Significant progress has been made in diagnosis and treatment of SCLC in the last 25 years, but, with an overall median survival time of 18 to 20 months, even in patients with limitedstage disease, it is still not possible to consider SCLC in the category of curable cancers. Despite this disappointing figure, mandating further research on this highly fatal disease, all improvements in LS-SCLC have been achieved by concurrent use chemotherapy and TRT (as early as possible), chemotherapy and PCI. For medically fit patients with ES-SCLC, combination chemotherapy followed by PCI (in non-progressive cases) is the standard of care, and further consolidation with TRT is currently under investigation. It is of paramount importance that patients with ES-SCLC be given the chance to participate in future trials for identification of a new and effective treatment combination, which may potentially offer a

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## **Lung Parenchyma Sparing Resection for Pulmonary Malignancies**

Arpad Pereszlenyi

*Department of Thoracic Surgery, Vivantes Klinikum Neukölln, Berlin, Germany* 

## **1. Introduction**

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It is well-known that the incidence of lung malignancies increases. The increase of primary lung cancer is especially alarming. But the lung is also a target organ for other secondary malignancies, such as metastases of different origins. For the primary lung cancer, the therapy of choice is its radical resection (together with systematic lymphadenectomy). For pulmonary metastases, less radical resections are necessary. Both could be challenging for thoracic surgeons in case of limited lung function and in the case of multiple bilateral lung nodules – metastases. Furthermore, the newly detected lung tumor is in most cases diagnosed in elderly, active smokers with a limited lung function and significant comorbidity. Therefore, the above-mentioned planned radical resection in these "limited" patients is not possible. Thoracic surgeons face apparently a dead-lock situation having to operate radically and sparing enough functional lung parenchyma at the same time.

This paper is dedicated to the topic of lung parenchyma sparing resection. It's first part describes a laser resection of multiple lung lesions – metastases. The laser segmental lung resection and the sleeve bronchoplastic (angioplastic) resection are introduced in it's second and third part, respectively.

#### **2. Laser resection for lung metastases**

#### **2.1 History**

Already in 1786, John Hunter reported the first case report in history of pulmonary metastases. The primary cancer was a malignant tumor of the femur and the patient died of widespread pulmonary metastazing only 7 weeks after the leg was amputated (Allen et al., 1993; Van Schil et al. 2008). In 1927, the first surgeon who performed a lung resection of pulmonary metastasis was Jan Divis from Prague (Divis, 1927). Barney and Churchill could note the real success after the surgery, removing a lung metastasis by a lobectomy. The renal-cell carcinoma was removed by nephrectomy, subsequently. The patient survived for over 20 years without any signs of recurrence (Barney & Churchill, 1939).

Finally, a retrospective analysis of 205 patients after resection of pulmonary metastases should be mentioned. This report was published by Thomford in 1965 with a 5 Years-Survival-Rate (Y-S) of 30.3% (Thomford 1965).

### **2.2 Introducing the laser in thoracic surgery**

In 1985, after establishing a 1064 nm Nd:YAG laser for standard endobronchial interventions (Häusinger et al, 1984), LoCicero reopened the debate on the use of lasers in open thoracic surgery as well (LoCicero, 1985, 1989). However, since his CO2 laser was a pure absorption or cutting laser, it proved inadequate for lung surgery and thus could not establish itself in this medical discipline. As a result, a number of medical centers in the United States, Japan and Europe began experimenting with 1064 nm Nd:YAG lasers, using bare fibers and sapphire tips to perform superficial resections (Branscheid, 1992; Kodama 1991, 1992; Lo Cicero 1985, 1989; Mineo, 1998; Moghissi, 1988; Rolle, 1988). As it follows, all of these teams achieved only low patient-loads and published no further results, mainly because the technical difficulties posed by the available 1064 nm lasers could not be overcome without further basic research. Table 1.

The new era began by introducing the 1318 nm wavelength Nd:YAG laser system of 40 W power output. This high performance Nd: YAG laser system consisted of the thin flexible quartz fibres (400 μm) with low water content and of a four lens focusing handpiece. This new laser system was exclusively used in all patients undergoing a lung parenchyma sparing resection in our study. The next Section presents the description of this laser system.


Table 1. Literature Survey: Nd:YAG/CO2 Laser resection in thoracic surgery.

In 1985, after establishing a 1064 nm Nd:YAG laser for standard endobronchial interventions (Häusinger et al, 1984), LoCicero reopened the debate on the use of lasers in open thoracic surgery as well (LoCicero, 1985, 1989). However, since his CO2 laser was a pure absorption or cutting laser, it proved inadequate for lung surgery and thus could not establish itself in this medical discipline. As a result, a number of medical centers in the United States, Japan and Europe began experimenting with 1064 nm Nd:YAG lasers, using bare fibers and sapphire tips to perform superficial resections (Branscheid, 1992; Kodama 1991, 1992; Lo Cicero 1985, 1989; Mineo, 1998; Moghissi, 1988; Rolle, 1988). As it follows, all of these teams achieved only low patient-loads and published no further results, mainly because the technical difficulties posed by the available 1064 nm lasers could not be overcome without

The new era began by introducing the 1318 nm wavelength Nd:YAG laser system of 40 W power output. This high performance Nd: YAG laser system consisted of the thin flexible quartz fibres (400 μm) with low water content and of a four lens focusing handpiece. This new laser system was exclusively used in all patients undergoing a lung parenchyma sparing resection in our study. The next Section presents the description of this laser system.

**Length (nm) Laser Application** 

Sealing of Air Leaks

n=47 Wedge and Segmental Resections

"Coin Lesions"

Metastases n=25

resections

Metastases n=14 Laser only n=51 comb. with with

Lobectomy

Metastases n=25 Segmental Resection (NSCLC) n=25

Metastases n=23

of Multiple Pulmonary Metastases n=328

CO2 Hemostasis,

Nd:YAG 1064 Local Excision,

Nd:YAG 1064 Resection of Lung

Nd:YAG 1064 Resection of Lung

Nd:YAG 1064 Resection of Lung

Nd:YAG 1064 Resection of Lung

Nd:YAG 1318 Lobe-Sparing Resection

Nd:YAG 1064/1318 Experimental/clinical

Nd:YAG 1064 Laser assisted pulmonary

**Author Article Laser Wave** 

**2.2 Introducing the laser in thoracic surgery** 

further basic research. Table 1.

Ann Thorac Surg

Laser in Med and Surg

J Thoracic Cardiovasc Surg

J Thoracic Cardiovasc Surg

J Thoracic Cardiovasc Surg

Eur J Cardiothorac Surg

J Thorac Cardiovasc Surg

Table 1. Literature Survey: Nd:YAG/CO2 Laser resection in thoracic surgery.

Kyobu Geka

Chest

LoCicero 1985

Rolle 1988

Moghissi 1988

LoCicero 1989

Kodama 1991

Branscheid 1992

Kodama 1992

Mineo 1988

Rolle 2006

## **2.2.1 Scientific background, description of 1318 Nd:YAG Laser**

Due to its parenchymal tissue having a typical water content of 80% but a very low tissue density (just a fifth of the liver parenchyma), a very low heat capacity and a variable air content, the lung is an ideal organ for phototermal laser applications. Therefore, resecting lung parenchyma requires a laser with a powerful coagulation capability in addition to excellent cutting properties, given the high vessel density. After all, the surgeon must always expect fistulae and increasing bronchopulmonary leaks, particularly when dissecting lung parenchyma, the more so the deeper one works down in central direction. The absorption behavior of different lasers in water differs a lot (Bayly, 1963; Bramson, 1968; Dinstl, 1981).

The 1318 nm wavelength Nd:YAG laser significantly differs from the standard (1064 nm) wavelength by its ten times higher absorption in water but still offers sufficient laser light scatter, due its proximity to the beginning infrared spectrum, to satisfy the vital coagulation requirement as well. In fact the 1318 nm wavelength provided the intended combination effect - cutting capability plus coagulation capability - so perfectly as it could not be achieved with the 1064 nm wavelength (Rolle, 1988, 1989). As a welcome side-effect, we also found strong lung tissue shrinkage, which provides two additional advantages: mechanical reinforcement of the coagulation effect, and fistula sealing far into the central lobe region. In fact, the surfaces coagulated and sealed off through defocused irradiation with the 1318 nm laser withstand artificial ventilation pressures of up to 25 cm H20.

As for the founding and developing of the above mentioned laser system, the name of Professor Axel Rolle has to be mentioned in this place (Rolle, 1988, 1989, 1999).

The following design features were incorporated to develop a 1318 nm commercial design Trumph (formerly Hüttinger Medizintechnik, Umkirch, Germany) and Martin companies. The second wavelength is first generated by adapted reflection of the laser mirrors. High beam quality allows coupling into thin (less than 0.6 mm) optical quartz fibers with minimum losses. For flexible transmission to the area of application, special water-free quartz fibers are required as laser light absorption in water is 10 times higher at the 1318-nm wavelength (Bayly, et al., 1963; Stokes et al., 1981).

A four-lens focusing handpiece was developed to concentrate the laser light and allow manual manipulation of the beam onto lung tissue to keep the working-point focus in the tissue at 4 mm while avoiding heat generation in the focusing handpiece. The extremely high laser power density of 24kW/cm2 allows fast and precise cutting with simultaneous coagulation and sealing of lung tissue. A high performance smoke evacuation system eliminates the vaporization fumes, which are unavoidable during parenchyma dissection with this laser (Fig.1).

#### **2.3 Surgical technique**

Laser metastasectomy is performed via an anterolateral thoracotomy (staged 3 to 4 weeks, if bilateral) after fulfilling the standard indication criteria for pulmonary metastasectomy (histologically confirmed primary tumor after its radical resection or its fully controlled stage). Preoperative evaluations are the same as a for routine thoracic intervention; including a history and physical examination, chest computed tomography (CT), pulmonary function tests, and a bone scan. If the signs or symptoms are suggestive, a head CT is also obtained. Patients with identified extrapulmonary metastases are excluded from surgery.

The technique, indication and possibility to save lobes are demonstrated on a case report. A 59-years-old female patient with a history of radically resected colorectal carcinoma

Fig. 1. Components of modern Laser equipment for the application on lung tissue (1318 nm wavelength, 40 W power output, beam quality, energy efficiency, high performance smoke evacuation system, 0.4 mm diameter of fibre, focusing handpiece, flexible quartz fibres /low water content/).

(Adenocarcinoma of rectosigmoid pT4 pN1 pM1 (Liver), G3, Status post hemihepatectomy, chemotherapy and radiation) was referred to our Institute. A significant progress of (isolated) lung metastases was reported. The chest CT demonstrates the situation after the successful laser resection of pulmonary metastases on the right side and just before the procedure on left (Fig. 2).

Fig. 2. Case Report 1: The Chest CT demonstrates the situation after the successful laser resection of pulmonary metastases on the right side and just before the procedure on left.

Fig. 1. Components of modern Laser equipment for the application on lung tissue (1318 nm wavelength, 40 W power output, beam quality, energy efficiency, high performance smoke evacuation system, 0.4 mm diameter of fibre, focusing handpiece, flexible quartz fibres /low

(Adenocarcinoma of rectosigmoid pT4 pN1 pM1 (Liver), G3, Status post hemihepatectomy, chemotherapy and radiation) was referred to our Institute. A significant progress of (isolated) lung metastases was reported. The chest CT demonstrates the situation after the successful laser resection of pulmonary metastases on the right side and just before the

Fig. 2. Case Report 1: The Chest CT demonstrates the situation after the successful laser resection of pulmonary metastases on the right side and just before the procedure on left.

water content/).

procedure on left (Fig. 2).

The new 1318 nm Nd:YAG laser system offers a unique opportunity to perform the procedure in a parenchyma-saving and lobe-sparing way. Therefore, the bilateral laser procedure was performed. The intraoperative situation can be seen on the series of photographs: the pulmonary artery is mobilized on a vessel loop; the upper vein lies next to the central, 30 mm great metastasis (Fig. 3). The laser resection of this metastasis was than performed. The next figure (Fig. 4) shows the situation immediately after the laser resection. The intraoperative situation – its close relation to segmental pulmonary vein - can be easily recognized. Exposed bronchial branches and segmental vessels at the segmental level were over-sewn and ligated with absorbable suture (4-0/ 3-0). The lung architecture and orientation was reconstructed following each nodular resection by reapproximating the visceral pleura with a running absorbable suture (4-0 Vicryl) (Fig. 5). This technique avoided a distortion of the lung tissue to allow consistent orientation and palpation of the initially noted lung nodules. At the end of the procedure, the resected lung was re-insuflated by a standard way in accordance with the routine thoracic surgical practice.

By performing the metastasectomy the above-described way, it was possible to save the patient's lobes and to operate on both lungs by a laser parenchyma sparing manner. The patient, now one year after the procedure, is in good condition with a full physical activity, living free of metastases.

Fig. 3. Intraoperative view: Laser resection of 30 mm central metastasis localized in left upper lobe centrally to pulmonary artery. Intraoperative situation: pulmonary artery and the upper vein are mobilized on vessel loop (blue).

Fig. 4. The intraoperative situation – its close relation to segmental pulmonary vein - can be easily recognized. Exposed bronchial branches and segmental vessels at the segmental level were over-sewn and ligated with absorbable suture (4-0/ 3-0).

Fig. 5. Intraoperative view: Reconfiguration of the left upper lobe with continuous suture of the pleura visceralis.

#### **2.4 Results**

262 Lung Diseases – Selected State of the Art Reviews

Fig. 4. The intraoperative situation – its close relation to segmental pulmonary vein - can be easily recognized. Exposed bronchial branches and segmental vessels at the segmental level

Fig. 5. Intraoperative view: Reconfiguration of the left upper lobe with continuous suture of

were over-sewn and ligated with absorbable suture (4-0/ 3-0).

the pleura visceralis.

From January 1996 to May 2004, lung laser resections were performed in 328 patients. There were 164 males and 164 females and the main indications for laser lung resections included lung metastases of the following primaries: renal carcinoma in 112 cases, colorectal in 91 and breast cancer in 35 cases. In the remaining 90 cases laser resection was performed for metastases of lung cancer (n=12), malignant melanoma (n=1), sarcomas (n=15), head and neck carcinoma (n=12) and for metastases of other less frequent ones (n=27). These results were already reported and published elsewhere (Rolle et al., 2006).

This retrospective study analyzes the second largest indication group of colorectal carcinoma lung metastases (Pereszlenyi et al., 2006a, 2006b). 46 females and 45 males with median age of 64 yrs, ranged from 43 to 80 years were included. The number of complete removed metastases was 629, ranged 1-56; median 7 per patient. All laser resections were performed by the Nd:YAG laser system of 1318 nm wavelength with its lung parenchyma saving effect. The complete resection (R0) was achieved in 78 patients, incomplete (R1/2) in 13 patients.

There was no perioperative mortality. Follow-up was completed for all patients and ranged from 1-30 Mo with a median of 20 months. 1 Year-Survival (Y-S) for complete (R0) resection was 82%, 2 Y-S was 68%, 3 Y-S 42% and 5 Y-S was 22% (Fig. 6). For incomplete (R1/2) resection (n=13): 1 Year-Survival was 85%, 2 Y-S was 54%, 3 Y-S 46%, 4 Y-S 9% and 5 Y-S was 0 (Fig. 6).

Despite of that the 7 Metastases pro patient was removed and 19% of lymphatic-nodes involvement, the radical resection (R0) could be achieved. In 13 patients was the resection incomplete (R1/R2). For the R0 versus R1/2 see the Figure 6. The survival with and without lymphatic-node-involvement (N1-hilum, N2-mediastinum) after the radical resection (R0N0 versus R0N1/2) is demonstrated in Figure 7.

Fig. 6. Kaplan-Meier curve showing survival according to resection (Bullets = complete R0, squares = incomplete R1/R2 resections).

Fig. 7. Kaplan-Meier curve showing survival according to lymphnode-involvement (Bullets = complete without LN-involvement R0N0, squares = complete with LN1/N2-involvement R0 N1/N2).

#### **3. Laser segmental lung resection**

The technique of conventional lung segment resection, so called segmentectomy, is well known from the pioneer age of the thoracic surgery (pneumoftiseology) when the apical sublobar lung resections were performed for lung tuberculosis. Nowadays, those resections are not so widely spread due to their "not enough radicalism" for lung cancer cases.

However, as it is already stated in the Introduction, these resections will gain more and more importance due to their lung parenchyma-sparing effect and the improving results on early postoperative morbidity and mortality (Keenan et al., 2004; Harada et al., 2005).

In this place, it should be emphasized that the segmental resection is also an anatomical lung resection as the lobectomy. It respects the anatomical structure of the lung with its bronchial and vascular composites together with its lymphatic flows.

A significant role in the technique of the segmental resection belongs to the laser system. Its cutting effect enables the thoracic surgeon to perform this kind of resection exactly within the anatomical boarders of the pulmonary segment. Therefore it is feasible also for segments where the "classical" segment resections can only hardly be obtained, e.g. segment III, IX, X etc.

#### **3.1 Surgical technique**

Laser segmentectomy is performed via an anterolateral muscle-sparing thoracotomy after fulfilling the standard indication criteria for a lung cancer resection (histologically confirmed lung tumor in its functional and oncologic operable stage). Preoperative evaluations are the same as for a routine thoracic intervention; including a history and physical examination, chest computed tomography (CT), pulmonary function tests, and a PET scan. If there are positive mediastinal lymphatic nodes proved by the test, a videomediastinoscopy in order to clarify the N2/N3 status is added prior to the resection.

Fig. 7. Kaplan-Meier curve showing survival according to lymphnode-involvement (Bullets = complete without LN-involvement R0N0, squares = complete with LN1/N2-involvement

The technique of conventional lung segment resection, so called segmentectomy, is well known from the pioneer age of the thoracic surgery (pneumoftiseology) when the apical sublobar lung resections were performed for lung tuberculosis. Nowadays, those resections

However, as it is already stated in the Introduction, these resections will gain more and more importance due to their lung parenchyma-sparing effect and the improving results on early postoperative morbidity and mortality (Keenan et al., 2004; Harada et al., 2005). In this place, it should be emphasized that the segmental resection is also an anatomical lung resection as the lobectomy. It respects the anatomical structure of the lung with its

A significant role in the technique of the segmental resection belongs to the laser system. Its cutting effect enables the thoracic surgeon to perform this kind of resection exactly within the anatomical boarders of the pulmonary segment. Therefore it is feasible also for segments where the "classical" segment resections can only hardly be obtained, e.g. segment III, IX, X etc.

Laser segmentectomy is performed via an anterolateral muscle-sparing thoracotomy after fulfilling the standard indication criteria for a lung cancer resection (histologically confirmed lung tumor in its functional and oncologic operable stage). Preoperative evaluations are the same as for a routine thoracic intervention; including a history and physical examination, chest computed tomography (CT), pulmonary function tests, and a PET scan. If there are positive mediastinal lymphatic nodes proved by the test, a video-

mediastinoscopy in order to clarify the N2/N3 status is added prior to the resection.

are not so widely spread due to their "not enough radicalism" for lung cancer cases.

bronchial and vascular composites together with its lymphatic flows.

R0 N1/N2).

**3.1 Surgical technique** 

**3. Laser segmental lung resection** 

Next, a case report presents the technique of laser segmental lung resection. A 66-years-old male patient with histological confirmed Adenocarcinoma of the right upper lobe was evaluated for a lung resection. Because of the patient's significant comorbidity (ischemic heart disease, arterial hypertension, diabetes mellitus) and poor lung function (COPD, active smoker with history of 30 Pack/Years, FEV1 55%), the limited lung resection – Laser resection of the Segment 1 - was performed.

For the patient's preoperative images see Figure 8.

Fig. 8. Chest CT and X-ray pictures of Case 2. The perioperative images are on the left and the postoperative images are on the right side.

After the mobilization of the lung hilus, the segmental vessels and bronchus were mobilized on vessel loops. Fig.9. After this step, the lung parenchyma resection was performed by the laser system within the anatomical boarders of the Segment 1. Fig.10. The visceralisation (reapproximating the visceral pleura with a running absorbable suture /4-0 Vicryl/) in order to restore the architecture of the upper lobe followed. As it was already described above, this technique avoided a distortion of the lung tissue to allow consistent orientation and palpation of the lung parenchyma. At the end of the procedure, the resected lung was re-insuflated by a standard way in accordance with the routine thoracic surgical practice. Radical lymphadenectomy is routinely added to this procedure (Pereszlenyi et al., 2006).

The patient is now 3 years after the procedure, without any signs of tumor recurrence. The histological examination of the resected specimen has proved an Adenocarcinoma of the lung 3 cm of diameter, radically (R0) removed via the laser resection of the Segment 1. For postoperative CT scans see Figure 8.

Fig. 9. Right lung hilus is mobilized; truncus anterior on the blue vessel loop with its segmental artery branches A1, A3.

Fig. 10. Laser segmental resection within the anatomical boarders of the Segment 1.

#### **3.2 Results**

From January 1996 to December 2001, laser segmental lung resections were performed in 53 patients (Pereszlenyi et al., 2006). The results after these resections were compared to standard lobectomies /n=154/ for non-small cell lung cancer (NSCLC). The data of this comparison are presented in the Table 2.

As it follows, the 1, 3, 5 Year-Survival (Y-S) is comparable in both groups, there is no statistical significance /p=0.696/ in terms of tumor recurrence rate (26.4% versus 29.2%). The postoperative mortality rate (11.3%) after laser lung segmental resection is explained by the significant comorbidity, limited lung function in elderly patients in whom this kind of lung parenchyma sparing resection was performed. Tab.2.

Fig. 9. Right lung hilus is mobilized; truncus anterior on the blue vessel loop with its

Fig. 10. Laser segmental resection within the anatomical boarders of the Segment 1.

From January 1996 to December 2001, laser segmental lung resections were performed in 53 patients (Pereszlenyi et al., 2006). The results after these resections were compared to standard lobectomies /n=154/ for non-small cell lung cancer (NSCLC). The data of this

As it follows, the 1, 3, 5 Year-Survival (Y-S) is comparable in both groups, there is no statistical significance /p=0.696/ in terms of tumor recurrence rate (26.4% versus 29.2%). The postoperative mortality rate (11.3%) after laser lung segmental resection is explained by the significant comorbidity, limited lung function in elderly patients in whom this kind of

segmental artery branches A1, A3.

**3.2 Results** 

comparison are presented in the Table 2.

lung parenchyma sparing resection was performed. Tab.2.

To conclude, we are convinced that radical surgical resection is still the therapy of choice in NSCLC treatment. However, laser segmental lung resection represents an optimal treatment eventuality especially for those high risk patients in whom the standard resection – lobectomy is not feasible or performable.

Our study demonstrates the possibility and justification of this treatment modality with comparable results after the standard ones (Harada et al., 2005).


Table 2. Laser segmental lung resection versus standard lobectomy for primary lung cancer (n=207). Selection criteria for laser segmental resection: high risk patients (elderly patient, poor performance status, FEV1<65%, significant comorbidity) and peripheral tumors of ≤ 4cm diameter.

## **4. Sleeve bronchoplastic lung resection**

Last but not least, the sleeve lobectomy should be mentioned. Centrally localized lung tumors can be resected by a lobectomy extended into a resection of central part of the invaded bronchus. The shape of such resected bronchus has a form of a sleeve. That's where the term "sleeve" lobectomy originates. There are numerous published studies showing the clear benefit for this type of lung resections. The most important advantage includes the avoidance of a major lung resection, e.g. pneumonectomy by performing a sleeve lobectomy. Its technique is well known, but in case of constructing so called "Neo-carina" followed by its re-implantation to the main stem bronchus can be very challenging also for experienced surgeons. Fig.11.

Fig. 11. Sleeve middle and lower (bi-)lobectomy: Re-implantation the upper lobe bronchus into main-stem bronchus right (or into distal trachea with construction of the "neo-carina") after resection the tumor within the bilobectomy. Scheme.

In case of multiple lung lesions, the laser system plays a significant role. The centrally located endobronchial tumor is removed by a sleeve lobectomy, and any smaller peripheral lesions (satellites) are resected by laser. Fig.12. The technique of the laser resection is presented in detail in the first part of this chapter. Fig. 13.

Fig. 12. Case Report 3: Chest CT shows the central lung tumor in the right lower lobe and two small satellites in the periphery of the lung.

Fig. 13. Scheme of laser resection of small peripheral nodule located in the middle lobe after sleeve lower lobe resection for a large central located endobronchial tumor.

### **4.1 Surgical technique, patients and methods**

Between January 2005 and January 2011, 58 patients (42 males, 16 females, mean age 61 yrs range 24 – 83) underwent sleeve lobectomy (SL) in our Institute. The indications for SL were: non-small cell lung carcinoma (NSCLC) in 47 /Fig.14/, and pulmonary metastases in 11 patients. Fig.15. As the metastatic pulmonary lesions were multiple, the Laser Resection (LR)

In case of multiple lung lesions, the laser system plays a significant role. The centrally located endobronchial tumor is removed by a sleeve lobectomy, and any smaller peripheral lesions (satellites) are resected by laser. Fig.12. The technique of the laser resection is

Fig. 12. Case Report 3: Chest CT shows the central lung tumor in the right lower lobe and

Fig. 13. Scheme of laser resection of small peripheral nodule located in the middle lobe after

Between January 2005 and January 2011, 58 patients (42 males, 16 females, mean age 61 yrs range 24 – 83) underwent sleeve lobectomy (SL) in our Institute. The indications for SL were: non-small cell lung carcinoma (NSCLC) in 47 /Fig.14/, and pulmonary metastases in 11 patients. Fig.15. As the metastatic pulmonary lesions were multiple, the Laser Resection (LR)

sleeve lower lobe resection for a large central located endobronchial tumor.

presented in detail in the first part of this chapter. Fig. 13.

two small satellites in the periphery of the lung.

**4.1 Surgical technique, patients and methods** 

of these satellites was added to the SL procedure. The main indication for laser resection were metastases of renal-cell (n=4) and colorectal-carcinoma (n=3). Fig.15. The most detected histologic type of NSCLC was squamous-cell carcinoma (n=22), followed by adenocarcinoma (n=14). The laser resection (combined by SL) was performed after fulfilling the standard criteria for metastasectomy: primary tumors were radically removed and there were no evidence of any distant extrathoracic metastases. In four NSCLC patients the arterial sleeve was added to the left upper bronchial SL. Lymphadenectomy was routinely added to the both parenchyma-saving procedures.

The Tumor Stage is shown in the Figure 16 according to the newest, revised TNM Classification from 2009 (Goldstraw et al., 2007).

Fig. 14. Tumor histology distribution (NSCLC; n=47).

Fig. 15. Tumor histology distribution (Lung Metastases; n=11).

Fig. 16. Tumor Stage according to TNM Classification 2009 (n=47). yT0N0 - As the result of the successful neoadjuvant treatment, the tumor is not detectable in the resected lung specimen.

The location of the tumors and types of sleeve resections are shown in Table 3. Almost 40% of the patients underwent right sleeve upper lobe resection.


Table 3. The location of tumors and types of sleeve resections (n=58).

Following the double-lumen tube intubation and the standard anterolateral, muscle-sparing thoracotomy, we proceed with the resection. A bronchial anastomosis is performed by interrupted monofilament absorbable suture of PDS 3-0 or 4-0. The vascular anastomosis was performed by running suture of the non-absorbable material of Prolene 5-0 (or 4-0) in four NSCLC patients after the left upper sleeve lobectomy was finished.

Mediastinal (hilar) lymphadenectomy is routinely performed as well as an intraoperative frozen section analysis of the resected bronchus (and vessel). After the reconstruction of the bronchus, a routine fiber bronchoscopy is always performed by the first surgeon. In our series, we didn't cover the bronchial anastomosis by any autologeous flap or any other materials.

#### **4.2 Results**

270 Lung Diseases – Selected State of the Art Reviews

Fig. 16. Tumor Stage according to TNM Classification 2009 (n=47). yT0N0 - As the result of the successful neoadjuvant treatment, the tumor is not detectable in the resected lung

The location of the tumors and types of sleeve resections are shown in Table 3. Almost 40%

Side and Type of Procedure **Number of Patients (%)** 

 Upper Lobe 23 (39) Middle Lobe 1 (2) Middle and Lower Lobe 1 (2) Lower Lobe 14 (24)

 Upper Lobe 12 (21) Lower Lobe 7 (12)

Following the double-lumen tube intubation and the standard anterolateral, muscle-sparing thoracotomy, we proceed with the resection. A bronchial anastomosis is performed by

Table 3. The location of tumors and types of sleeve resections (n=58).

of the patients underwent right sleeve upper lobe resection.

specimen.

Right Lung

Left Lung

A negative bronchial (vascular) margin was achieved in all. No 30-days postoperative mortality occurred. Follow-up (completed for all patients with median of 12 Mo) showed no anastomotic complications, no local recurrence on the bronchial (arterial) anastomosis. Survival was analyzed according to Kaplan-Meier method with the estimated 1, 2, 4 years overall survival. Fig. 17.

Fig. 17. Kaplan-Meier curve showing Survival at 20, 40, 60 Months.

In accordance with the reported results from the literature we can also conclude, the bronchial and vascular sleeve lobectomy can be performed safely, and is a good alternative solution to avoid pneumonectomy (Konstantinou et al., 2009; Ludwig et al., 2005).

As shown in our study, selected patients with central lung metastases can be also included for this procedure after fulfilling the standard criteria for pulmonary metastasectomy. The central role for these kinds of procedures belongs to the laser lung-parenchyma-saving resection. In our study all laser resections were performed by a 1318-nm Nd:YAG laser system of 40 W power output.

Laser resection may expand the scope of surgical treatment for pulmonary metastases, allowing a more complete resection. The indications for laser resection may expand to include patients who are not considered ideal candidates for lung metastasectomy because of poor residual lung function or multifocal pulmonary disease. The 1318-nm Nd:YAG laser for the resection of pulmonary metastases demonstrates a significant influence on the conservation of tissue during metastasectomy and appears to minimize complications.

#### **5. References**


resection. In our study all laser resections were performed by a 1318-nm Nd:YAG laser

Laser resection may expand the scope of surgical treatment for pulmonary metastases, allowing a more complete resection. The indications for laser resection may expand to include patients who are not considered ideal candidates for lung metastasectomy because of poor residual lung function or multifocal pulmonary disease. The 1318-nm Nd:YAG laser for the resection of pulmonary metastases demonstrates a significant influence on the con-

Allen E, Turk JL, Murley R. The case book of John Hunter FRS. London: Royal Society of

Barney JD, Churchill ET. Adenocarcinoma of the kidney with metastasis to the lung cured

Bayly IG, Kartha VB, Stevens WH. The absorption spectra of liquid phase H2O, HDO, and

Bramson M. Infrared Radiation. A handbook for applications. Plenum Press, New York,

Branscheid D, Krysa S, Wollkopf G, Bulzebruck H, Probst G, Horn M, Schirren J, Vogt-

Dinstl K, Fischer PL. Der Laser. Grundlagen und klinische Anwendung. Springer, Berlin,

Divis J. Ein Beitrag zur operativen Behandlung der Lungengeschwülste. Acta Chir Scand

Goldstraw P, Crowley J, Chansky K, et al. The IASLC lung cancer staging project: proposals

Keenan RJ, Landreneau RJ, Maley RH Jr et al. Segmental resection spares pulmonary function in patients with stage I lung cancer. Ann Thorac Surg 2004;78:228-233. Kodama K, Doi O, Higashiyama M, Tatsuta M, Iwanaga T. Surgical management of lung

Konstantinou M, Potaris K, Sakellaridis T, Chamalakis G. Sleeve lobectomy for patients with

Moykopf I. Does ND-YAG laser extend the indicatons for resection of pulmonary

for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant tumours. J Thorac Oncol 2007;2:706 –714. Harada H, Okada M, Sakamoto T et al. Functional advantage after radical segmentectomy versus lobectomy for lung cancer. Ann Thorac Surg 2005;80:2041-2045. Häussinger K, Held E, Huber R: Endobronchial laser therapy, differential therapeutic use,

metastases. Usefulness of resection with the neodymium:yttrium-aluminiumgarnet laser with median sternotomy. J Thorac Cardiovasc Surg 1991;101:901-8. Kodama K, Doi O, Higashiyama M, Yokoouchi H. Usefulness of Nd:YAG laser for the

excision of multiple lung metastases and segmentectomy for primary lung cancer.

non small-cell lung cancer: a simplified approach. Eur J Cardiothorac Surg

servation of tissue during metastasectomy and appears to minimize complications.

D20 from 0.7 micron to 10 micron. Infrared Physics 1963;3:211-23.

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metastases? Eur J Cardiothorac Surg 1992;6:590-7.

and clinical value. Klin Wochenschr 1984;62:74-80.

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Van Schil PE, Hendriks JM, Van Putte BP, Stockman BA, Lauwers PR, Ten Broecke PW, Grootenboers MJ, Schramel FM: Isolated lung perfusion and realted techniques for the treatment of pulmonary metastases. Eur J Cardiothorac Surg 2008;33: 486-495.

## **Surgery in Small-Cell Lung Cancer: Past, Present and Future**

Cristian Rapicetta1, Sara Tenconi1, Tommaso Ricchetti1, Sally Maramotti2 and Massimiliano Paci1 *1Thoracic Surgery Unit, Cardio-Thoracic-Vascular and Critical Care Department 2Laboratory of Molecular Biology, Department of Oncology* 

*Arcispedale Santa Maria Nuova - IRCCS, Reggio nell'Emilia Italy* 

#### **1. Introduction**

274 Lung Diseases – Selected State of the Art Reviews

Van Schil PE, Hendriks JM, Van Putte BP, Stockman BA, Lauwers PR, Ten Broecke PW,

Grootenboers MJ, Schramel FM: Isolated lung perfusion and realted techniques for the treatment of pulmonary metastases. Eur J Cardiothorac Surg 2008;33: 486-495.

> Small-Cell Lung Carcinoma (SCLC) represents about 15% of all lung cancers diagnosed worldwide. Although its incidence is diminished in the last decades, SCLC continues to represent an almost fatal disease due to its propensity to local relapse and distant metastasis, despite initial responsiveness to therapies. Biological behaviour of SCLC has therefore lead to consider it as a systemic disease *per se* not amenable of surgical resection: the Veterans Administration Lung Study Group (VALSG) two-stage classification was in fact based on field irradiation criteria and has been applied to SCLC for long-time.

> The introduction of TNM staging system, the common recurrences of local disease despite initial complete response after chemo-radiation therapy, the lack of a valid maintenance therapy after remission or a second-line therapy after relapse renewed interest in surgery in a multimodal treatment setting. However, the second prospective randomized trial in 1994, did not confirm any significant advantage of surgery compared to chemo-radiation therapy and several retrospective studies published in the same years failed to provide strong evidences of surgery's benefits. Lack of homogeneity in design of clinical trials, which are mostly dated, patients selection and other confounding factors made results of metaanalysis too much inconsistent to be added to guidelines; for these reasons, nowadays, surgery is recommended only in small peripheral nodules without nodal involvement (proven by invasive preoperative staging).

> Advances in comprehension of biological pathways underlying carcinogenesis in SCLC are the next steps that could deeply modify the approach to disease (patients selection and prognostic stratification, chemosensitivity and treatment modality) beyond the mere histology. Molecular profile should lead to identify subsets of tumours with more favourable prognosis, especially in terms of systemic control of disease, which is actually a major issue in SCLC; these subsets could be overlapped to NSCLC regarding to natural history of disease, making their treatment similar, including indication to surgery.

> The aim of this review is to analyze literature to deduce which has been, is actually and could be the role of surgery on overall survival and pattern of recurrence of patients affected by SCLC.

## **2. Clinical background**

## **2.1 Epidemiology**

Small-cell Lung Carcinoma (SCLC) accounts for approximately for 13-15%% of all newly diagnosed cases of lung cancer worldwide (in United States, more than 220.000 new cases lung carcinoma were diagnosed in 2010, with about 160.000 cancer-related deaths [American Cancer Society, 2010].

More than 90% of patients with SCLC are elderly, current or past heavy smokers, and risk rises with increasing duration and intensity of smoking [Devesa et al., 2005]; rare cases have been reported in people who never smoked [Antony et al., 2010].

Incidence of SCLC is decreasing compared to that of adenocarcinoma [Govindan, 2006]; this reflects the decreased prevalence of smoking in industrialised countries. However, the burden of disease is shifting to developing countries (Asia and Eastern Europe), where, on the contrary, an increase of incidence is expected in next years. Further investment in research against SCLC is therefore warranted.

A revision in the WHO classification of lung cancers might also have contributed to incidence falling of SCLC, as some borderline cases previously described as mixed subtypes are now classified as NSCLC [Travis et al., 2004].

Median survival of untreated patients ranges from 2 to 4 months from diagnosis: historical data of untreated patients come from an old study of VALSG (Veterans Association of Lung Study Group) comparing cyclophosphamide to placebo. In IASLC (International Association for Study of Lung Cancer) database for staging project (the largest series of SCLCs reported), 5-year survival rates were 38/21% for clinical stages IA/IB and 38/18% for clinical stages IIA/IIB, respectively. Considering only resected, fully staged patients, 5-years survival rates were 53/44% for p-stages IA/IB and 43/35% in p-stages IIA/IIB, respectively.

## **2.2 Clinical presentation**

Two thirds of SCLCs present as peribronchial lesions with infiltration of bronchial submucosa. Extensive mediastinal lymph node metastases are a common finding at diagnosis; sometimes mediastinal involvement presents as "bulky" disease, causing superior vena cava syndrome. Only in 4-12% of cases SCLC presents as a peripheral "coin lesion" [Quoix et al., 1990].

Clinical presentation of SCLC is strictly related to stage and presence of paraneoplastic syndrome. Common symptoms are cough, wheeze, dyspnoea, haemoptysis for hilar localization. Symptoms may reflect direct involvement of chest wall, superior vena cava, oesophagus, recurrent nerve (pain, mediastinal syndrome, dysphagia, dysphonia) or the site of metastasis (brain, liver, adrenal glands, bone and bone marrow). SCLC is more frequently associated to paraneoplastic syndromes than NSCLC [Gandhi et al., 2006]. The most common are syndrome of inappropriate antidiuresis (15-40% of SCLC patients) and Cushing's syndrome (2-5% of SCLC patients), that can be responsible of infective complications during chemotherapy. Sometimes SCLC presents with dermatological abnormalities as acquired tylosis, trip palms, erythema gyratum repens [Master et al., 2010]. Occasionally SCLC is associated to dermatomyositis, hyperglycemia or hypoglicemia, hypercalcemia and gynecomastia. Neurological syndromes, that may precede diagnosis of SCLC by several months, are caused by cross-reaction of auto-antibodies and T-lymphocytes specific for common tumour epitopes and nervous components [Darnell et al., 2003]. Antibodies directed against the P/Q- type voltage-gated calcium channel in the presynaptic nerve terminal (expressed by 3% of SCLC) are responsible of Lambert-Eaton syndrome [Payne et al., 2010], that should be differentiated from miastenia gravis, rarely associated to SCLC. Similarly, paraneoplastic encephalomyelitis and paraneoplastic sensory neuropathy have been associated with antibodies directed against Hu proteins, a family of DNAbinding proteins (<1% of SCLC patients) [Gultekin et al., 2000]; neurological symptoms are not always reversible with therapy.

#### **2.3 Diagnosis**

276 Lung Diseases – Selected State of the Art Reviews

Small-cell Lung Carcinoma (SCLC) accounts for approximately for 13-15%% of all newly diagnosed cases of lung cancer worldwide (in United States, more than 220.000 new cases lung carcinoma were diagnosed in 2010, with about 160.000 cancer-related deaths

More than 90% of patients with SCLC are elderly, current or past heavy smokers, and risk rises with increasing duration and intensity of smoking [Devesa et al., 2005]; rare cases have

Incidence of SCLC is decreasing compared to that of adenocarcinoma [Govindan, 2006]; this reflects the decreased prevalence of smoking in industrialised countries. However, the burden of disease is shifting to developing countries (Asia and Eastern Europe), where, on the contrary, an increase of incidence is expected in next years. Further investment in

A revision in the WHO classification of lung cancers might also have contributed to incidence falling of SCLC, as some borderline cases previously described as mixed subtypes

Median survival of untreated patients ranges from 2 to 4 months from diagnosis: historical data of untreated patients come from an old study of VALSG (Veterans Association of Lung Study Group) comparing cyclophosphamide to placebo. In IASLC (International Association for Study of Lung Cancer) database for staging project (the largest series of SCLCs reported), 5-year survival rates were 38/21% for clinical stages IA/IB and 38/18% for clinical stages IIA/IIB, respectively. Considering only resected, fully staged patients, 5-years survival rates

Two thirds of SCLCs present as peribronchial lesions with infiltration of bronchial submucosa. Extensive mediastinal lymph node metastases are a common finding at diagnosis; sometimes mediastinal involvement presents as "bulky" disease, causing superior vena cava syndrome. Only in 4-12% of cases SCLC presents as a peripheral "coin

Clinical presentation of SCLC is strictly related to stage and presence of paraneoplastic syndrome. Common symptoms are cough, wheeze, dyspnoea, haemoptysis for hilar localization. Symptoms may reflect direct involvement of chest wall, superior vena cava, oesophagus, recurrent nerve (pain, mediastinal syndrome, dysphagia, dysphonia) or the site of metastasis (brain, liver, adrenal glands, bone and bone marrow). SCLC is more frequently associated to paraneoplastic syndromes than NSCLC [Gandhi et al., 2006]. The most common are syndrome of inappropriate antidiuresis (15-40% of SCLC patients) and Cushing's syndrome (2-5% of SCLC patients), that can be responsible of infective complications during chemotherapy. Sometimes SCLC presents with dermatological abnormalities as acquired tylosis, trip palms, erythema gyratum repens [Master et al., 2010]. Occasionally SCLC is associated to dermatomyositis, hyperglycemia or hypoglicemia, hypercalcemia and gynecomastia. Neurological syndromes, that may precede diagnosis of SCLC by several months, are caused by cross-reaction of auto-antibodies and T-lymphocytes specific for common tumour epitopes and nervous components [Darnell et al., 2003]. Antibodies directed against the P/Q- type voltage-gated calcium channel in the presynaptic

were 53/44% for p-stages IA/IB and 43/35% in p-stages IIA/IIB, respectively.

been reported in people who never smoked [Antony et al., 2010].

**2. Clinical background** 

[American Cancer Society, 2010].

**2.2 Clinical presentation** 

lesion" [Quoix et al., 1990].

research against SCLC is therefore warranted.

are now classified as NSCLC [Travis et al., 2004].

**2.1 Epidemiology** 

Although diagnosis of SCLC could be suspected on the basis of clinical and radiological findings, histo-patological diagnosis is required prior to establish the proper treatment.

SCLC represents the extreme of spectrum of neuro-endocrine lung carcinomas and is defined as pure SCLC or combined SCLC if the non small-cell component accounts for at least 10%of burden disease.

While pre-invasive and in situ lesions are frequently found in NSCLC, they are uncommon in SCLC [Kumar et al., 2005].

Samples can be obtained by bronchoscopy biopsy or fine-needle aspiration from primary tumour, lymph nodes or other metastatic sites. Since tumour shows propensity to spread through tunica submucosa, superficial bronchoscopic biopsy or brush may be falsely negatives. Colliquative necrosis can sometimes hamper diagnosis, especially for cytological samples; however there is a good interobserver agreement among pathologists for differential diagnosis with NSCLC. Immunohistochemistry is used in difficult cases: less than 10% of SCLC tumours are negative for all neuroendocrine markers (chromogranin, synaptophysin, and CD56. Positivity for TTF-1 and cytokeratins helps to distinguish them from lymphomas and other small-cell tumours [van Meerbeeck et al., 2011]

#### **2.4 Staging**

Historically, SCLC had been classified according to the Veterans Administration Lung Study Group (VALSG). In 1957 the VALSG created a dichotomous staging system that took into account the aggressive behaviour of SCLC and the standard of care at that time. This classification underlined the highest importance of radiation therapy and allowed a better selection of patients for this kind of treatment.

The Limited Disease (LD) was characterized by a tumour volume encompassed in one radiation portal (30% of cases): all that was not comprised in one radiation portal was classified as extensive stage (ED), including malignant pleural effusion and haematogenous metastases. [Zelen et al., 1973]. In 1989 the International Association for the Study of Lung Cancer modified the VALSG staging including all non-metastatic patients in the limited stage [Stahel et al., 1991] (Tab.1).

More recently, in 2007, the IASLC based on a retrospective analysis of survival of 8088 patients with SCLC recommended the TNM classification system also for SCLC [Shepherd et al., 2007]. This suggestion comes from the evidence of significantly worse survival of patients with limited-stage disease and N2-N3 lymph node involvement, than for those with N0-N1 lymph node involvement. Patients with pleural effusion without extrathoracic metastasis showed a survival intermediate between stage III and stage IV. In the TNM classification patients with cytology-negative pleural effusion are classified as having stage III disease. This classification is used less frequently in clinical practice because it relies on surgical confirmation for its accuracy and patients with SCLC seldom present at a stage for which surgery is appropriate (in IASLC database only 349 patients out of 12.620 affected by SCLC were resected, representing only 2,8%).


Table 1. Comparison between VALSG and TNM staging

Considering the therapeutic options (chemotherapy and radiotherapy) representing, to date, the standard of care, it could be argued that the more precise staging of SCLC using the TNM system does not provide extra benefits to select the treatment modality. However TNM has been shown to be prognostic of outcome [Micke et al., 2002], and a more precise definition of nodal involvement may be relevant for radiation treatment. Moreover, surgery for limited disease (T1-2, N0, M0) is nowadays considered a valid therapeutic option [Varlotto et al., 2011]. For these reasons clinicians and cancer registrars recommend to classify SCLC with the TNM staging system [Shepherd et al., 2007]. This recommendation is particularly strong for those trials in LD addressing thoracic and prophylactic cranial irradiation questions and those that include a surgical treatment arm [Vallieres et al., 2009]. In the future a better definition of N stage is needed and prognostic difference in patients with or without cytology-positive pleural and/or pericardial effusions must be addressed.

To assess the extent of disease, which remains the main prognostic factor, several staging tests are available; their execution sequence should be guided by the patient's signs and symptoms at presentation and the availability of the diagnostic tests. Staging should be accurate and fast, considering the rapid growth of SCLC. Staging work-up should include full medical history, physical examination, chest X-ray, hematology and chemistry panels including differential blood count, liver and renal function tests, lactate dehydrogenase (LDH) and sodium levels (hyponatremia, due to ectopic production of antidiuretic hormone or atrial natriuretic peptide, is observed in up to 15% of patients), pulmonary function tests, and contrast-enhanced CT of the chest and upper abdomen. SCLC displays the propensity for early distant metastases to liver, bones, adrenal glands, and above all brain. Therefore, in patients with suspect for distant metastases, additional tests may include bone scintigraphy, CT scan with intravenous contrast or MRI of the brain, and bone marrow aspiration and biopsy. Bone marrow is involved in 15 to 30 percent of patients at presentation, but it represents rarely a solitary site of metastatic disease (2 to 6%). Bone-marrow infiltration should be suspected in presence of an isolated rise in lactate dehydrogenase (LDH) concentration or blood counts indicating otherwise unexplained anemia or a leucoerythroblastic response or if bone scan is positive [Campling et al., 1986]. CT or MRI of the brain are recommended if chemo-radiation with curative intent is under consideration. In one report, the prevalence of brain metastases was 10% with CT and 24% with MRI. In this series all CT-detected brain metastases were symptomatic, whereas 11% of those detected by MRI were asymptomatic [Seute et al., 2008]. Chest CT scan, abdominal CT scan, brain MRI and bone scintigraphy are mandatory in the evaluation of patients amenable to surgery [Koletsis et al., 2009]. Fluorodeoxyglucose (FDG) uptake is usually high in SCLC, leading to a sensitivity of nearly 100% but its routine use in SCLC remains controversial [Thomson et al., 2011]. PET is, anyway, useful to plan radiotherapy in some countries [Van Loon et al., 2010]. Pathological confirmation is still required for PET-detected lesions that could result in upstaging, in particular if radical resection could be offered. The role of combined fluorodeoxyglucose PET (FDG-PET) and CT scanning is yet to be completely defined but, if available, it may be useful to improve the accuracy of staging by the detection of mediastinal nodal and occult distant metastatic spread. In patients who present with pleural effusion, in the absence of extrathoracic disease, cytopathologic confirmation of tumour involvement is needed.

With improvements in staging through the use of PET/CT and magnetic resonance imaging (MRI), more patients are found to have ES-SCLC: the ratio of LS-SCLC to ES-SCLC was formerly 1:1 and is now 1:3 as more subtle lesions, such as silent adrenal and brain metastases, are identified.

If extensive disease is detected by one test, further staging can be omitted, although bone scintigraphy may be used to identify symptomatic lesions amenable of palliation radiotherapy and brain CT/MRI could be performed or repeated in patients who respond to treatment in order to plan a brain irradiation of symptomatic lesions or to plan PCI in absence of metastasis (since PCI has been extended to ED-SCLC responsive to primary treatments)

#### **2.5 Prognosis**

278 Lung Diseases – Selected State of the Art Reviews

which surgery is appropriate (in IASLC database only 349 patients out of 12.620 affected by

VALSG staging TNM staging

Considering the therapeutic options (chemotherapy and radiotherapy) representing, to date, the standard of care, it could be argued that the more precise staging of SCLC using the TNM system does not provide extra benefits to select the treatment modality. However TNM has been shown to be prognostic of outcome [Micke et al., 2002], and a more precise definition of nodal involvement may be relevant for radiation treatment. Moreover, surgery for limited disease (T1-2, N0, M0) is nowadays considered a valid therapeutic option [Varlotto et al., 2011]. For these reasons clinicians and cancer registrars recommend to classify SCLC with the TNM staging system [Shepherd et al., 2007]. This recommendation is particularly strong for those trials in LD addressing thoracic and prophylactic cranial irradiation questions and those that include a surgical treatment arm [Vallieres et al., 2009]. In the future a better definition of N stage is needed and prognostic difference in patients with or without cytology-positive pleural and/or pericardial effusions must be addressed. To assess the extent of disease, which remains the main prognostic factor, several staging tests are available; their execution sequence should be guided by the patient's signs and symptoms at presentation and the availability of the diagnostic tests. Staging should be accurate and fast, considering the rapid growth of SCLC. Staging work-up should include full medical history, physical examination, chest X-ray, hematology and chemistry panels including differential blood count, liver and renal function tests, lactate dehydrogenase (LDH) and sodium levels (hyponatremia, due to ectopic production of antidiuretic hormone or atrial natriuretic peptide, is observed in up to 15% of patients), pulmonary function tests, and contrast-enhanced CT of the chest and upper abdomen. SCLC displays the propensity for early distant metastases to liver, bones, adrenal glands, and above all brain. Therefore, in patients with suspect for distant metastases, additional tests may include bone scintigraphy, CT scan with intravenous contrast or MRI of the brain, and bone marrow aspiration and biopsy. Bone marrow is involved in 15 to 30 percent of patients at presentation, but it represents rarely a solitary site of metastatic disease (2 to 6%). Bone-marrow infiltration should be suspected in presence of an isolated rise in lactate dehydrogenase (LDH) concentration or blood counts indicating otherwise unexplained anemia or a leucoerythroblastic response or if bone scan is positive [Campling et al., 1986]. CT or MRI of the brain are recommended if chemo-radiation with curative intent is under consideration. In one report, the prevalence of brain metastases was 10% with CT and 24% with MRI. In

From IA to IIIB

SCLC were resected, representing only 2,8%).

**Limited Disease**: disease encompassed within a tolerable radiation therapy portal Tumour confined to one hemithorax Involvement of ipsilateral and contralateral mediastinal nodes

**Extensive Disease**: any other IV Table 1. Comparison between VALSG and TNM staging

 Involvement of ipsilateral supraclavicular nodes Ipsilateral pleural effusion

> Stage is the major prognostic factor of survival but some other prognostic factors have been identified: performance status, weight loss, sex and some laboratory tests (CEA, LDH, NSE, hypoalbuminemia, elevated alkaline phosphatase). No histological or molecular features have been validated yet. Several algorithms have been elaborated for predicting survival but the reliability for individual patients remains poor. Paraneoplastic syndromes are more frequently found in patients with limited-stage SCLC and they are considered positive prognostic factors.

### **3. Historical background of SCLC**

SCLC was firstly recognized by Barnard in 1926, who noted that "oat-cell sarcomas of the mediastinum" were metastatic carcinomas of the lung instead.

In the 85 years of SCLC life, some milestones should be reminded:

**1959**: Pathogists recognized SCLC as a separate entity among carcinomas of the lung and Azzopardi defined six features of cells at light microscopic examination [Azzopardi, 1959] **1963**: VALSG introduced the 2-stage system for SCLC

**1969**: British Medical Research Council reported better survival of radiotherapy arm versus surgical arm in the first clinical randomized trial on SCLC [Miller et al., 1969]. Cyclophosphamide showed to be effective against lung cancer [Green et al., 1969]

**1979**: Medical Research Council showed advantage in survival with combination of chemotherapy (cyclophosphamide) and radiation therapy compared with radiation alone in LD-SCLC [Medical Research Council Lung Cancer Working Party, 1979]

**1982**: Shields re-evaluated surgery as initial treatment in SCLC after introduction of TNM staging system [Shields, 1982]

**1987**: Adding RT to chemotherapy improved survival in LD-SCLC [Perry et al., 1987]

**1989**: The International Association for Study of Lung Cancer (IASLC) was revised for the first time by its introduction the Veterans Administration Lung Study Group staging system [Stahel, 1989]

**1994**: Lung Cancer Study Group published results of the first (and unique, so far) perspective randomized trial in LD-SCLC comparing surgery to RT after induction chemotherapy: Authors concluded that surgery offered no advantage in terms of either survival or local control of disease.

**1995-1999**: Prophylactic Cranial Irradiation (PCI) showed to improve overall and diseasefree survival in selected patients (responders to first-line treatments) [Aupérin et al., 1999]

**1999**: studies of dose fractionation of RT demonstrated that twice-daily administration was superior compared to once-daily in terms of local control and overall survival [Turrisi et al., 1999]

Combining this dates we can schematically recognize three periods.

#### **3.1 The "Surgical Era" (early 1900-1960s)**

The dramatic increase of tobacco consumption in early 900's produced a sharp increase of incidence of SCLC; on the other hand, prevalence of pulmonary tuberculosis and chest wounds during World Wars promoted advances in thoracic surgery and lung resections. Surgery was considered the standard of care for Small-Cell as well as Non small-cell lung cancer. However high rates of recurrence (both local and distant), even after apparently complete resection, were observed. Results published by British Medical Council Group in first clinical prospective trial randomizing patients affected by SCLC to either surgery or thoracic radiation therapy reported the slight advantage in survival of RT arm. This was enough to abandon surgery at least for primary therapy for SCLC. Dichotomous staging by Veterans Administration Lung Cancer Study Group (1968) reflected and anticipated the centrality of RT as primary treatment for local control of disease

#### **3.2 The "advent of Chemotherapy" (1960s-1980s)**

Few years later report of British Medical Council, the pathology study conducted by Matthews in autopsies of patients deceased within 30 days of attempted curative resection showed that SCLC had, in almost all patients, microscopic but widely metastatic disease [Matthews et al., 1973]. This enforced the importance of accurate staging of disease and promoted the need of systemic control of disease. The chemosensitivity of SCLC was first recognized in 1940s when nitrogen mustard (methyl-bis-B-Chloro-ethyl amine hydrochloride) demonstrated capability of inducing tumour regression in more than 50% of patients. Despite a large amount of active drugs towards SCLC, it was perceived quite early that single-agent regimens were associated to frail remissions and high rates of precocious recurrences. In 1970s combination chemotherapy, mainly based on cyclophosphamide showed dramatic responses, even in very incapacitated patients, although long term disease-free survival remained disappointing and natural history of disease did not seem to change. Chemotherapy became the standard of care also in limited disease when Medical Research Council demonstrated the advantage of a combination of cyclophosphamide and radiation compared with radiation alone. During the 1970s thoracic radiation was relegated to an adjuvant, "consolidative" role in LD-SCLC.

**1982**: Shields re-evaluated surgery as initial treatment in SCLC after introduction of TNM

**1989**: The International Association for Study of Lung Cancer (IASLC) was revised for the first time by its introduction the Veterans Administration Lung Study Group staging system

**1994**: Lung Cancer Study Group published results of the first (and unique, so far) perspective randomized trial in LD-SCLC comparing surgery to RT after induction chemotherapy: Authors concluded that surgery offered no advantage in terms of either

**1995-1999**: Prophylactic Cranial Irradiation (PCI) showed to improve overall and diseasefree survival in selected patients (responders to first-line treatments) [Aupérin et al., 1999] **1999**: studies of dose fractionation of RT demonstrated that twice-daily administration was superior compared to once-daily in terms of local control and overall survival [Turrisi et al.,

The dramatic increase of tobacco consumption in early 900's produced a sharp increase of incidence of SCLC; on the other hand, prevalence of pulmonary tuberculosis and chest wounds during World Wars promoted advances in thoracic surgery and lung resections. Surgery was considered the standard of care for Small-Cell as well as Non small-cell lung cancer. However high rates of recurrence (both local and distant), even after apparently complete resection, were observed. Results published by British Medical Council Group in first clinical prospective trial randomizing patients affected by SCLC to either surgery or thoracic radiation therapy reported the slight advantage in survival of RT arm. This was enough to abandon surgery at least for primary therapy for SCLC. Dichotomous staging by Veterans Administration Lung Cancer Study Group (1968) reflected and anticipated the

Few years later report of British Medical Council, the pathology study conducted by Matthews in autopsies of patients deceased within 30 days of attempted curative resection showed that SCLC had, in almost all patients, microscopic but widely metastatic disease [Matthews et al., 1973]. This enforced the importance of accurate staging of disease and promoted the need of systemic control of disease. The chemosensitivity of SCLC was first recognized in 1940s when nitrogen mustard (methyl-bis-B-Chloro-ethyl amine hydrochloride) demonstrated capability of inducing tumour regression in more than 50% of patients. Despite a large amount of active drugs towards SCLC, it was perceived quite early that single-agent regimens were associated to frail remissions and high rates of precocious recurrences. In 1970s combination chemotherapy, mainly based on cyclophosphamide showed dramatic responses, even in very incapacitated patients, although long term disease-free survival remained disappointing and natural history of disease did not seem to change. Chemotherapy became the standard of care also in limited disease when Medical Research Council demonstrated the advantage of a combination of cyclophosphamide and radiation compared with radiation alone. During the 1970s thoracic radiation was relegated

Combining this dates we can schematically recognize three periods.

centrality of RT as primary treatment for local control of disease

**3.2 The "advent of Chemotherapy" (1960s-1980s)** 

to an adjuvant, "consolidative" role in LD-SCLC.

**1987**: Adding RT to chemotherapy improved survival in LD-SCLC [Perry et al., 1987]

staging system [Shields, 1982]

survival or local control of disease.

**3.1 The "Surgical Era" (early 1900-1960s)** 

[Stahel, 1989]

1999]

### **3.3 Evolution of combined modality treatment (1980s-today)**

The last period could be considered as "quiescent": no tools against disease proved to be revolutionary and efforts were made towards integration of multimodal approach. Paradigms and flow charts of treatment did not reflect the chronological sequence of most important studies but rather controlled studies and consensus expressed by expertise panels of Authors. Etoposide and platinum became from 1980s the first-line treatment. An important study from National Cancer Institute (NCI) in 1976 firstly tested a very aggressive protocol involving simultaneous irradiation of brain, primary tumour and mediastinum and concomitant CAV chemotherapy (cyclophosphamide/doxorubicine/vincristine). Despite unacceptable toxicity (radiation pneumonitis in 38%, mielodepression with fatal sepsis in 24%), this regimen showed the best results, never reached before (nearly 100% complete remissions, 80% of long term survival) [Greco et al., 1979]. This work firstly recognized the importance of tumour repopulation by clones of cells whose resistance was allowed by sequentiality of treatments. Efforts were made in order to minimize tissue interaction without compromising delivery of both radiation and chemo-therapy: timing of radiation therapy [Perry et al., 1987] and dose fractioning [Turrisi et al. 1999] were largely studied by various Authors.

In this period surgery revived with first report from Shields and coll. who concluded that primary surgery and adjuvant chemotherapy could be offered to patients with SCLC in early stages (i.e. T1N0). A large number of subsequent studies (discussed in the chapter) reported favourable long-term survival after surgery with improved local control of disease: one of the most remarkable experience was that of Toronto Group [Shepherd, 1983].

Undoubtedly, advances in both imaging and invasive staging tools (spiral CT, PET/CT, FNA-EBUS) lead to a more precise stratification of disease, allowing selection of patients in really "limited" disease amenable for radical resection. Application of TNM staging as used for NSCLC eliminated one of the barriers that divided SCLC and NSCLC and contributed to better define prognosis of patients previously classified as having Limited Disease (an heterogeneous stage ranging from isolated coin lesion to extensive hilar mass with supraclavicular node metastasis).

Times were mature for another clinical perspective randomized trial involving surgery: it was started by Lung Cancer Study Group in 1983 and ended in 1994. Results again excluded a benefit of surgery even on a multimodal approach, but did not prevent further Authors to report their retrospective experiences on surgically resected SCLC, criticizing at the same time this study.

Main changes of treatment modalities in SCLC are summarized in Fig. 1

Fig. 1. Evolution of treatment modalities through decades

## **4. Current guidelines for treatment (NCCN/ACCP)**

It should be noted that guidelines for the treatment of SCLC are based on review of a literature that lacks of big randomized prospective trials and is heterogeneous regarding to kind of treatment, timing, endpoints and selection of patients.

The National Comprehensive Cancer Network (NCCN) has recently developed a review of its guidelines, declaring that every recommendation for SCLC has to be considered category 2A, because they are "based upon lower-level evidence" but "uniform NCCN consensus that the intervention is appropriate" (see Table 2). [NCCN Clinical Practice Guidelines in Oncology]


Table 2. NCCN category of recommendation.

The American College of Chest Physicians (ACCP) in 2007 produced a systematic literature review resulting in evidence–based guidelines, graded upon the "ACCP grading system for guideline recommendations"

Both documents debate on diagnostic and therapeutic options for SCLC and are quite similar in recommendations, except for the use of PET (not yet standardized in 2007 ACCP guidelines) and for the choice of staging system (NCCN recommending the use of the new TNM instead of Veterans Administration Lung Study Group classification).

Guidelines for treatment have a key point in the use of platinum-based chemotherapy plus radiotherapy for all fit patients (Performance Status 0-2) with limited stage disease, followed by Prophylactic Cranial Irradiation (PCI); topics of discussion are timing of radiotherapy (concurrent versus sequential, early versus late), volume, dose and fractionation of radiations, treatment of unfit-elderly patients, maintenance and second line chemotherapy and surgery.

Cisplatin has to be preferred to Carboplatin in combination with Etoposide in first line treatment; in phase III randomized trials, Irinotecan was substituted for Etoposide in combination with Carboplatin in advanced disease with mild improve in survival and thus being added to guidelines as an option for patients with extensive-stage disease. In these patients, where the treatment of choice is chemotherapy alone with initial response of about 60-70% but median survival of 9-11 months due to early relapse, maintenance or consolidation chemotherapy beyond 4 to 6 cycles is currently not recommended outside clinical trials (grade of recommendation IB); likewise, the introduction of a third agent (alkylating agent with or without anthracycline) showed minor advantage in duration of response without improving survival and carried greater cumulative toxicity. The use of cytochine or anti-angiogenetic agents (i.e. Bevacizumab) is not currently recommended in first line treatment, even though randomized phase III trials are currently running.

Second line treatment is generally a single agent therapy, administered with an interval of at least 3 month since initial therapy (otherwise disease has to be considered refractory or resistant) and should be given until 2 cycles beyond best response, progression of disease, development of major toxicity.

It should be noted that guidelines for the treatment of SCLC are based on review of a literature that lacks of big randomized prospective trials and is heterogeneous regarding to

The National Comprehensive Cancer Network (NCCN) has recently developed a review of its guidelines, declaring that every recommendation for SCLC has to be considered category 2A, because they are "based upon lower-level evidence" but "uniform NCCN consensus that the intervention is appropriate" (see Table 2). [NCCN Clinical Practice Guidelines in

The American College of Chest Physicians (ACCP) in 2007 produced a systematic literature review resulting in evidence–based guidelines, graded upon the "ACCP grading system for

Both documents debate on diagnostic and therapeutic options for SCLC and are quite similar in recommendations, except for the use of PET (not yet standardized in 2007 ACCP guidelines) and for the choice of staging system (NCCN recommending the use of the new

Guidelines for treatment have a key point in the use of platinum-based chemotherapy plus radiotherapy for all fit patients (Performance Status 0-2) with limited stage disease, followed by Prophylactic Cranial Irradiation (PCI); topics of discussion are timing of radiotherapy (concurrent versus sequential, early versus late), volume, dose and fractionation of radiations, treatment of unfit-elderly patients, maintenance and second line chemotherapy and surgery. Cisplatin has to be preferred to Carboplatin in combination with Etoposide in first line treatment; in phase III randomized trials, Irinotecan was substituted for Etoposide in combination with Carboplatin in advanced disease with mild improve in survival and thus being added to guidelines as an option for patients with extensive-stage disease. In these patients, where the treatment of choice is chemotherapy alone with initial response of about 60-70% but median survival of 9-11 months due to early relapse, maintenance or consolidation chemotherapy beyond 4 to 6 cycles is currently not recommended outside clinical trials (grade of recommendation IB); likewise, the introduction of a third agent (alkylating agent with or without anthracycline) showed minor advantage in duration of response without improving survival and carried greater cumulative toxicity. The use of cytochine or anti-angiogenetic agents (i.e. Bevacizumab) is not currently recommended in

TNM instead of Veterans Administration Lung Study Group classification).

first line treatment, even though randomized phase III trials are currently running.

Second line treatment is generally a single agent therapy, administered with an interval of at least 3 month since initial therapy (otherwise disease has to be considered refractory or resistant) and should be given until 2 cycles beyond best response, progression of disease,

**4. Current guidelines for treatment (NCCN/ACCP)** 

kind of treatment, timing, endpoints and selection of patients.

Table 2. NCCN category of recommendation.

guideline recommendations"

development of major toxicity.

Oncology]

The addition of thoracic radiotherapy has improved survival in patients with limited stage disease (grade of recommendation 1A); staging of SCLC actually involves relationship between extension of disease and radiation port field. As far as timing is concerned, early (within 30 days to 9 weeks since the beginning of chemotherapy) concurrent chemoradiotherapy is recommended for patients with limited-stage disease (grade of recommendation 1A). Hyper-fractionation of radiation dose has not yet being correctly compared to once-daily administration in available trials, according to NCCN review of literature.

PCI given after completion of chemotherapy in a low dose per fraction causes less neurological toxicity and prevent the emergence of brain metastasis, both in limited and extensive stage disease; it is recommended for fit patients who achieve a complete or partial response to initial treatment (Grade of recommendation 1B).

Radiotherapy is also recommended in relapse for palliation of symptoms.

The European Society of Medical Oncology (ESMO) in the 1st consensus conference in lung cancer stated guidelines for diagnosis, treatment and follow up of SCLC, underlying the absence of randomized trials comparing surgery with concurrent chemo-radiotherapy. Nevertheless, with grade of recommendation III D, surgical resection may be considered for very limited disease (T1-2, N0), only after histological confirmation of N parameters by mediastinoscopy, followed by PCI.

ACCP guidelines stress the importance of invasive mediastinal staging, with grade of recommendation 1A. NCCN guidelines specify that lobectomy has to be preferred if resection is performed and that adjuvant chemotherapy alone is recommended for patients without nodal metastasis, while mediastinal RT has to be added in case of nodal involvement (Fig. 2)

## **5. Studies regarding surgery in SCLC**

Except from those by British Medical Council and Lung Cancer Study Group, all studies focused on surgery in SCLC are retrospective or prospective non-randomized, so conclusions should be interpreted cautiously.

However, also those phase III trials have been criticised on several points.

The British Medical Council study, dated 1963, did not include patients with peripheral lung lesions, since preoperative evaluation and diagnosis were made using rigid bronchoscopy available at that time; secondly, rate of complete (R0) resection was low (approximately 50%) compared to resections for NSCLC; finally, staging was performed without modern tools, like contrast CT-scan or PET, nor histological confirmation by mediastinoscopy was obtained. So it is likely that a remarkable number of patients with occult intrathoracic and extrathoracic disease was included in the study and randomized to surgery, compromising the long term survival results.

Twenty years later, after advent of chemotherapy as standard primary treatment, times were mature for another randomized trials, set up by Lung Cancer Study Group in 1983. Eligibility criteria include LD stage, according to VALSG staging: this means that also patients with clinical evident mediastinal adenopaties were included in the study. Conversely, patients with peripheral nodules and normal bronchoscopy were specifically excluded from the study. Induction therapy was quite heterogeneous (anthracycline based regimen) and only 144 of 340 patients (42%) accrued in the trial were randomized (68 to surgery arm and 76 to radiotherapy arm). Six patients randomized to surgery refused

Fig. 2. NCCN clinical practice guidelines

thoracotomy and, on the contrary, eight patients requested surgical intervention and received off-randomization surgery, representing a significant cross-over (10%) between the two arms. Moreover, few other data should be pointed out: only 65% of patients responded to induction chemotherapy, in 17% of patients assigned to surgery only exploratory thoracotomy was performed and, above all, the high proportion of patients included with bulky N2 or N3 nodal metastasis (who unlikely achieved a complete mediastinal downstaging). Interestingly N status and post-treatment clinical stage did not influence resectability (a result correlated with clinical understaging).

The conclusion of the study was that surgery did not add any benefit neither in terms of survival nor of pattern of recurrence.

The same results were anticipated in a retrospective study of 1985 based on 33 operated patients compared to 46 patients who fulfilled criteria of operability and resectability but were treated with non-surgical management. However microscopic or macroscopic residual disease was left in half of patients, suggesting that clinical staging was not so accurate in predicting resectability of disease [Østerlind, 1985]

Considering these weak points, rejection of surgery did not discourage several Authors, which continued to report good results with surgery combined to either induction or adjuvant chemotherapy and radiotherapy in some cases.

**SCLC or mixed SCLC/NSCLC on biopsy OR cytology of primary metastatic site**

> **PS 0-2: chemotherapy and cuncurrent thoracic radiotherapy PS 3-4: chemotherapy ± RT**

**Without localized symptomatic sites or brain metastasis: COMBINATION CHEMOTHERAPY**

**N0: chemotherapy N+: chemotherapy ± mediastinal RT IF COMPLETE OR PARTIAL RESPONSE:** 

thoracotomy and, on the contrary, eight patients requested surgical intervention and received off-randomization surgery, representing a significant cross-over (10%) between the two arms. Moreover, few other data should be pointed out: only 65% of patients responded to induction chemotherapy, in 17% of patients assigned to surgery only exploratory thoracotomy was performed and, above all, the high proportion of patients included with bulky N2 or N3 nodal metastasis (who unlikely achieved a complete mediastinal downstaging). Interestingly N status and post-treatment clinical stage did not influence

The conclusion of the study was that surgery did not add any benefit neither in terms of

The same results were anticipated in a retrospective study of 1985 based on 33 operated patients compared to 46 patients who fulfilled criteria of operability and resectability but were treated with non-surgical management. However microscopic or macroscopic residual disease was left in half of patients, suggesting that clinical staging was not so accurate in

Considering these weak points, rejection of surgery did not discourage several Authors, which continued to report good results with surgery combined to either induction or

**PATHOLOGICAL MEDIASTINAL STAGING POSITIVE OR MEDICALLY INOPERABLE**

> **PROPHILACTIC CRANIAL IRRADIATION**

resectability (a result correlated with clinical understaging).

predicting resectability of disease [Østerlind, 1985]

adjuvant chemotherapy and radiotherapy in some cases.

**EXTENSIVE STAGE Any T Any N M1 a/b**

> **With localized symptomatic sites or brain metastasis: CHEMOTHERAPY ± RT on symptomatic sites**

**LIMITED DISEASE Any T (except T3-T4 due to multiple lung nodules that not fit in a tolerable radiation field) Any N M0**

**Lobectomy and mediastinal lymph node dissection**

Fig. 2. NCCN clinical practice guidelines

survival nor of pattern of recurrence.

**PATHOLOGICAL MEDIASTINAL STAGING NEGATIVE**

Rostad et al. analyzed 2442 patients with SCLC in a national survey in Norway, 38 of which were surgically resected (25 received adjuvant therapies). For stage I addition of surgery to conventional treatments (chemo-radiation) improved 5-year survival rate from 11.3% to 44.9%, so Authors concluded that patients with resectable disease in stage I should be referred to surgery [Rostad, 2004].

The same conclusion is reported by Leo and Pastorino in their review [Leo & Pastorino, 2003] for T1-2N0M0 patients which can be treated by surgery and adjuvant chemotherapy, while patients in stage II-III should undergo surgery only in the context of clinical trials.

Rea et al. reported a 32% overall survival in 104 patients with SCLC surgically resected, despite a remarkable percentage of stage III (43.3%), which resulted as a major negative prognostic factors [Rea, 1998]

Brock et al reviewed their institutional experience of 1415 SCLC among whom 82 (6%) underwent surgery with curative intent from 1976 to 2002 [Brock, 2005]. Surgery was accompanied by induction or adjuvant chemotherapy in 77% of patients. Authors found a 5 year survival of 85.7% for stage I SCLC (similar to that historically expected for completely resected stage I NSCLC without adjuvant chemotherapy). Favourable prognostic factors were early stage, lobectomy as surgical procedure, female gender and date of intervention after 1987 (a surrogate marker for availability of platinum based chemotherapy). Pattern of recurrence was not reported in this study, however Authors concluded that lobectomy plus platinum based chemotherapy is a feasible option yielding excellent results in T1-T2N0-M0 SCLC.

High 5 years survival rate was reported also by Tsuchiya in a retrospective series of patients treated with a similar protocol [Tsuchiya, 2005]. Interestingly, Authors reported only 10% of local failure after surgery, which is lower than commonly reported after chemo-radiation therapies alone.

Badzio and coll. in a retrospective series of patients treated with surgery or non-surgical management reported a significative improvement in survival in surgical resected patients (22 months Vs 11, P<0.001) [Badzio, 2004]. The control group of patients treated with chemoradiotherapy was built using pair-matched case-control according to main prognostic factors, stage and resectability; moreover diagnosis of surgical treated patients was established only postoperatively, thus minimizing some bias related to selection of patients.

In 2006 the Bronchogenic Carcinoma Cooperative Group of the Spanish Society of Pneumology and Thoracic Surgery (GCCB-S) presented a multicenter study on 47 patients with SCLC out a total of 2994 lung cancers resected [Gomez de Antonio, 2006]. Thirty-three % of patients had incomplete resection suggesting that the criteria for surgery were not predictable enough for resectability. The routine use of mediastinoscopy (performed only in 19% of cases) might have identified some of the patients whose clinical staging was underestimated and which added to low rate of respectability. Moreover a low proportion of patients received adjuvant treatments. These factors accounts for an overall survival which does not exceed that reported in literature for patients with possibly more advanced stage managed with chemo-radiotherapy.

Recently, Schreiber and coll. analyzed patients included in the Surveillance, Epidemiology and End Results registry (SEER), which is the cancer registry representative of United States [Schreiber, 2010]. Among 14.179 patients affected by SCLC coded as localized (T1-2Nx-0) or regional disease (T3-4Nx-0), 863 (6%) had undergone surgical resection, making them one of the larger population based cohort ever reported in literature. Overall 5-year survival rates were 26.3 Vs 9.3% (P=0.01) in favour of surgery with median survival of 22 Vs 12 months, respectively. Advantage of surgery to survival was much more substantial in N0 patients (median survival 40 Vs 15 months), which on the other hand did show benefit from addition of PORT (Post-Operative Radiation Therapy). These data seem to suggest that radical surgery alone could be adequate for local control of disease and, conversely PORT may be detrimental for survival. Another retrospective analysis of SEER database conducted in 2010 and focused only on stage I confirmed reasonable outcome in patients who underwent lobectomy without PORT [Yu et al., 2010]. Unfortunately both studies, although based on large population, lack of data concerning chemotherapy, margins of resection, pathologic confirmation of diagnosis and performance status (not recorded in SEER database).


S: surgery, CT: chemotherapy, CTRx: chemoradiation therapy, PE: Platinum-Etoposide, HfRTx: Hyperfractioned Radiation therapy

Table 3. Some of the recent trials supporting the role of surgery

A current year report on a small series of patients (28) underlines the low rate of local failure (3/28) after surgery compared to distant (especially brain) metastasis, which confirms the role of PCI [Ogawa et al. 2011]

## **5.1 Rationale of surgery in SCLC**

286 Lung Diseases – Selected State of the Art Reviews

respectively. Advantage of surgery to survival was much more substantial in N0 patients (median survival 40 Vs 15 months), which on the other hand did show benefit from addition of PORT (Post-Operative Radiation Therapy). These data seem to suggest that radical surgery alone could be adequate for local control of disease and, conversely PORT may be detrimental for survival. Another retrospective analysis of SEER database conducted in 2010 and focused only on stage I confirmed reasonable outcome in patients who underwent lobectomy without PORT [Yu et al., 2010]. Unfortunately both studies, although based on large population, lack of data concerning chemotherapy, margins of resection, pathologic

confirmation of diagnosis and performance status (not recorded in SEER database).

S CT (RT) (92) CT S CT (15)

Stage IIB-IIIA: PE x 3 CTRx (HfRTx) S

46 Stage I-IIA: PE x 4 S

CTRx (67)

S adjuvant

95 S CTRx (Stage I)

47 CT S (3)

863 S

Hyperfractioned Radiation therapy

Brock, 2005 82 (CT) S (CT) Stage I: 58%

CT S CTRx (Stage III)

S CT (30) S (14)

S: surgery, CT: chemotherapy, CTRx: chemoradiation therapy, PE: Platinum-Etoposide, HfRTx:

S RT CT data n/a

Table 3. Some of the recent trials supporting the role of surgery

Tsuchiya, 2005 62 S PE x 4 10% Stage IA/IB: 73/67%

**Treatment Modality Local** 

Fujimori, 1997 22 PE x 2/4 S 5% Cumulative: 66.7% (3-year)

**recurrence** 

4.1% (10.6% local+dista nt)

5% 15% **5-year survival** 

Stage I-II: 73.3% (3-year) Stage IIIA: 42.9%

Cumulative: 22.6% 47% in stage I 0% in N2 patients 0% with surgery alone

Cumulative: 27% (4%

0% Cumulative: 39%

CTRx) Stage IA: 59%

Stage II: 18% Stage III: 23% Stage IV: 0%

Stage II: 38% Stage III: 39%

n/a Median survival (months) 31.3 (Stage I) 31.7 (Stage III – downstaged) 12.4 (Stage III – not downstaged)

> R0 resection: 31% Stage I: 36%

Cumulative: 34.6% Stage I: 44.8% Stage II-III: 26.3%

n/a Cumulative: 26%

n/a Stage I: 44.9%

**Study Number of** 

Eberhardt, 2003

Granetzny, 2006

Gomez De Antonio, 2006

Schreiber, 2009

**patients** 

Badzio, 2004 134 S CT (67)

Rostad, 2004 38 S

Lucchi, 1997 127 S (15)

As pointed out by Anraku and Waddell [Anraku, 2006] in their review, rationale of surgery can be can summarized in the following clinical presentations.


#### **5.2 Patient selection criteria and choice of surgical procedure**

If a diagnosis is obtained preoperatively, pathologists should exclude any possible coexisting NSCLC component. Even more than in NSCLC, accurate staging is crucial before planning surgery in fit patients, as discussed before. Toronto Group demonstrated better 5 year survival (18% vs 6%) in patients without mediastinal nodes involvement preoperatively [Shepherd, 1991]; in a recent phase II trial on surgery after induction chemotherapy only patients who achieved complete nodal downstaging had a fair survival. Mediastinoscopy and eventually re-mediastinoscopy after primary treatment is therefore recommended to detect nodal involvement which is microscopic and subclinical in a considerable percentage of patients. This matter accounts for discordance between clinical and pathological staging in SCLC.

If a SCLC is diagnosed in operative theatre at frozen section of specimens, radical resection (preferably lobectomy) is recommended if intraoperative histological examination of mediastinal lymph nodes does not reveal metastasis; however, surgery can be proposed to patients with mediastinal nodes micrometastasis if they can easily tolerate the procedure. Sublobar resections are recommended in less fit patients or in presence of nodal involvement.

## **6. Targeting the complex biology of SCLC**

First cytogenetic studies, dated 1982, found that a deletion on chromosome 3p was present in 95% of SCLC [Whang, 1982]. This chromosomal region contains tumour-suppressor genes relevant to the pathogenesis of the tumour e.g. RARβ [Naylor 1987, Kok 1987] and FITH [Franklin, 2010]. The 3p loss alone is not specific of SCLC, since it is frequent encountered also in other tumours. Thanks to the high-throughput technology as comparative genomic hybridization (CGH) and gene expression arrays, researchers discovered that in 90% of SCLC samples, the most frequent sites of chromosome loss are 3p, 5q and 13q, the last determining the loss of retinoblastoma gene (RB1) [Ried, 1994]. Mutation studies on biology of SCLC showed that this type of tumour has frequent mutation in the gene encoding for p53 protein (TP53) [Hanahan, 200] but rarely presents mutation in the tyrosine-kinase signalling gene including KRAS and EGFR [Franklin, 2010], which are actually the most known pathways with larger number of active drugs (mainly tested in lung adenocarcinomas).

Targeted cancer therapies are drugs or antibodies that block the growth and the spread of cancer by interfering with specific molecules involved in tumour growth and progression. These therapies are being studied for use alone, in combination with other targeted therapies, and in combination with other cancer treatments, such as chemotherapy. By blocking signals that make cancer cell grow and replicate, targeted cancer therapies can help to stop cancer progression and may induce cancer cell death through apoptosis.

To give an example the PI3K/AKT/mTOR intracellular pathway is chronically activated in SCLC through inactivating mutations in PTEN gene and this activation pathway correlates with sensitivity to Everolimus in vitro [Marinov, 2009]. Furthermore, the amplification of BCL-2 genes seems to be correlate with the sensitivity to the highly potent small-molecule called ABT-737 suggesting that patients with bcl-2 protein over expression, may have some benefits from bcl-2 inhibitors [Olejniczak, 2007]. In SCLC cell lines, microRNA and geneexpression signatures of chemo-resistance have been described [Guo, 2010] and several inhibitors of growth factor pathways implicated in SCLC are in clinical development (NCT00896752); the compound PD173074 induces apoptosis in vitro and in vivo in SCLC by inhibition of FGF2 signalling [Pardo, 2009].

The targeting of specific molecules has been already studied in several trials, even though with limited success. One of the emerging need in clinical research dealing with SCLC is inadequacy of preclinical models of disease used to date. Cancer-cell-line-based xenograft models employed as standard testing ground especially for drugs have revealed as a surrogate of disease. In vitro activity level is then weakly predictable of clinical efficacy thus explaining the high rates of failure of new drugs tested in clinical studies. Human cancers cells, compared to cancer-cell cultures, grow up in hypoxic and nutrient-poor complex environments which promotes continue selection of more aggressive cell-clones. This explains the reason why cell-line-based SCLC xenografts tend to grow as relatively indolent tumours, while corresponding human disease is characterized by aggressive clinical and biological behaviour.

Moreover, as for standard chemotherapy, the use of a single molecular target drug in such complex malignancy as SCLC, is wrong. The inhibition of multiple targets or the combination with standard chemotherapic agents are likely to have greater potential. The customizing of therapy with novel agent to individual patient's characteristics is becoming the most beneficial approach to treatment of SCLC. In addition to new agents, biomarkers of chemosensitivity need be identified to efficaciously assess single agents for relapse after first-line therapy or as a maintenance therapy in placebo-controlled, randomised designs. Several studies have identified genes or proteins in lung cancer whose expression levels are associated with response to antitumor drugs. The breast cancer resistant protein (BCRP), is one of the ABC transporters reported to be associated with resistance to anticancer drugs like doxorubicin, irinotecan, mitoxantrone, and its expression was found to be associated with a poor clinical outcome in SCLC patients undergoing chemotherapy [Kim, 2009]. Chiappori et al. reported that RRM1 and Topo2 alpha proteins expression are biomarkers of chemotherapeutic efficacy in SCLC [Chiappori, 2010] and, recently, Usuda et al. demonstrated that the expression levels of Klotho protein was correlated with the prognosis following resection in SCLC patients [Usuda, 2011]. These results are encouraging; however, this findings need to be incorporated into common signatures for individual therapies and further tested in prospective clinical trials. Of course, biostatistical concerns still exist for predicting response to drugs as for predicting patient prognosis.

## **7. Conclusions**

288 Lung Diseases – Selected State of the Art Reviews

recommended to detect nodal involvement which is microscopic and subclinical in a considerable percentage of patients. This matter accounts for discordance between clinical

If a SCLC is diagnosed in operative theatre at frozen section of specimens, radical resection (preferably lobectomy) is recommended if intraoperative histological examination of mediastinal lymph nodes does not reveal metastasis; however, surgery can be proposed to patients with mediastinal nodes micrometastasis if they can easily tolerate the procedure. Sublobar resections are recommended in less fit patients or in presence of nodal

First cytogenetic studies, dated 1982, found that a deletion on chromosome 3p was present in 95% of SCLC [Whang, 1982]. This chromosomal region contains tumour-suppressor genes relevant to the pathogenesis of the tumour e.g. RARβ [Naylor 1987, Kok 1987] and FITH [Franklin, 2010]. The 3p loss alone is not specific of SCLC, since it is frequent encountered also in other tumours. Thanks to the high-throughput technology as comparative genomic hybridization (CGH) and gene expression arrays, researchers discovered that in 90% of SCLC samples, the most frequent sites of chromosome loss are 3p, 5q and 13q, the last determining the loss of retinoblastoma gene (RB1) [Ried, 1994]. Mutation studies on biology of SCLC showed that this type of tumour has frequent mutation in the gene encoding for p53 protein (TP53) [Hanahan, 200] but rarely presents mutation in the tyrosine-kinase signalling gene including KRAS and EGFR [Franklin, 2010], which are actually the most known pathways with larger number of active drugs (mainly tested in lung

Targeted cancer therapies are drugs or antibodies that block the growth and the spread of cancer by interfering with specific molecules involved in tumour growth and progression. These therapies are being studied for use alone, in combination with other targeted therapies, and in combination with other cancer treatments, such as chemotherapy. By blocking signals that make cancer cell grow and replicate, targeted cancer therapies can help

To give an example the PI3K/AKT/mTOR intracellular pathway is chronically activated in SCLC through inactivating mutations in PTEN gene and this activation pathway correlates with sensitivity to Everolimus in vitro [Marinov, 2009]. Furthermore, the amplification of BCL-2 genes seems to be correlate with the sensitivity to the highly potent small-molecule called ABT-737 suggesting that patients with bcl-2 protein over expression, may have some benefits from bcl-2 inhibitors [Olejniczak, 2007]. In SCLC cell lines, microRNA and geneexpression signatures of chemo-resistance have been described [Guo, 2010] and several inhibitors of growth factor pathways implicated in SCLC are in clinical development (NCT00896752); the compound PD173074 induces apoptosis in vitro and in vivo in SCLC by

The targeting of specific molecules has been already studied in several trials, even though with limited success. One of the emerging need in clinical research dealing with SCLC is inadequacy of preclinical models of disease used to date. Cancer-cell-line-based xenograft models employed as standard testing ground especially for drugs have revealed as a surrogate of disease. In vitro activity level is then weakly predictable of clinical efficacy thus explaining the high rates of failure of new drugs tested in clinical studies. Human cancers

to stop cancer progression and may induce cancer cell death through apoptosis.

and pathological staging in SCLC.

**6. Targeting the complex biology of SCLC** 

inhibition of FGF2 signalling [Pardo, 2009].

involvement.

adenocarcinomas).

Although the incidence of SCLC has been steadily decreasing over time, it continues to represent a relevant problem of public health due to its aggressive clinical behaviour and the lack of effective therapies; it is considered one of the most elusive cancers. Twenty-five years ago it was considered to be the next malignancy added to the list of curable cancers, because of the effectiveness of several chemotherapeutic agents and radiation therapy, and the discover of central nervous system sanctuary, which would require distinct treatment.

Conversely, despite active and ongoing research involving novel approaches to treat SCLC, few discovers had a successful translation in clinical practice over the past 25 years, with cumulative improvement of only 15% in survival which remains dismal and moreover seems to have now reached a plateau.

## **7.1 Current evidences**

Over the last 25 years, few landmarks in therapy have been provided by clinical trials focused on LS-SCLC. Minimal progress was noted in ES-SCLC.

Main strategies that demonstrated able to add small but significant improvements in survival can be summarized in the followings:

 **Advantage of addition of radiotherapy to chemotherapy**: The defining report, Cancer and Leukemia Group B (CALGB) 8083 [14], published in the New England Journal of Medicine, showed, for patients with LS-SCLC, a local control, failure-free survival, and overall survival benefit with the addition of thoracic radiation to chemotherapy using a cyclophosphamide and doxorubicin–based regimen. At 2 years, only 13% of patients who received chemotherapy alone maintained local control, compared with 54% of patients receiving chest radiotherapy. Although the likelihood of relapse in the chest may be reduced by up to 50% when thoracic irradiation is administered after chemotherapy, 20–36% of patients will have local recurrence even after combined modality treatment [12,13]. An analysis of the site of first relapse demonstrated that, even for patients who have achieved clinical complete response, the primary tumour bed and hilar or mediastinal lymph node areas are the most frequent single sites of failure [Elliott, 1987]. Failure to achieve control at the primary site remains the most important obstacle to cure in patients with limited SCLC [Shepherd, 1991].


## **7.2 Take home messages**

There are other recommendations without strong evidence, but for which there is general agreement:


#### **7.3 Challenge to systemic disease**

Since the overwhelming majority of patients with LD-SCLC have subclinical metastatic foci at the time of diagnosis, chemotherapy is an essential part of multimodal treatment to control systemic disease.

important obstacle to cure in patients with limited SCLC [Shepherd, 1991].

managed patients

**7.2 Take home messages** 

clinical trials.

agreement:

patients (also with ES-SCLC)

 **superiority of twice-daily radiation therapy over daily fractionation**: hyperfractioning radiation therapy was defined and experimented in order to respond to the need of improve local control of disease, which remains a serious issue in non-surgical

**advantage of prophylactic central nervous system radiation** (PCI) in all responding

There are other recommendations without strong evidence, but for which there is general

 **Resection is a reasonable option as initial treatments for early stages T1-2N0-M0 patients**: if preoperative diagnosis of SCLC has been obtained, nodal or distant metastasis must be excluded using by either non invasive or invasive staging, avoiding futile thoracotomies. Induction chemo-radiotherapy did not demonstrated superior to adjuvant setting. In any other stage of presentation, surgery is contemplated only in

 **Clinical understaging is frequent in SCLC** (concordance with pathological stage is only 58% in IASLC database) and common criteria of resectability often fails to predict complete resections in SCLC, due to propensity to spread through peribronchial lymphatic vessels to regional lymph-nodes. Introduction and more liberal use of EUS in flexible bronchoscopy or mediastinoscopy may help to rule out patients with

 **Surgery improves local control of disease** if radical resection is achieved. Local failure rates vary in literature from 0 to 15%, which is considerably lesser than 35-50% reported for chemo-therapy with twice-daily radiation therapy (the standard of care, to date) [Pijls-Johannesma et al., 2007; Turrisi et al., 1999]. In resected specimens after concurrent CT with HfRTx, Eberhardt found persistence of vital disease at the primary site [Eberhardt & Korfee, 2003]. However, there is no evidence, to date, that this translates in an improvement of overall and disease-free survival, which could eventually derive

Since the overwhelming majority of patients with LD-SCLC have subclinical metastatic foci at the time of diagnosis, chemotherapy is an essential part of multimodal treatment to

micrometastasis not amenable of surgical resection

from progresses in systemic treatments.

**7.3 Challenge to systemic disease** 

control systemic disease.

overall survival benefit with the addition of thoracic radiation to chemotherapy using a cyclophosphamide and doxorubicin–based regimen. At 2 years, only 13% of patients who received chemotherapy alone maintained local control, compared with 54% of patients receiving chest radiotherapy. Although the likelihood of relapse in the chest may be reduced by up to 50% when thoracic irradiation is administered after chemotherapy, 20–36% of patients will have local recurrence even after combined modality treatment [12,13]. An analysis of the site of first relapse demonstrated that, even for patients who have achieved clinical complete response, the primary tumour bed and hilar or mediastinal lymph node areas are the most frequent single sites of failure [Elliott, 1987]. Failure to achieve control at the primary site remains the most Cytotoxic drugs active in SCLC discovered in the early 1980s remains a standard of care at present. Advances in supportive care and technical advances in radiation therapy have allowed the application of therapies with less toxicity and well tolerated by most patients. Fall in treatment-related deaths implies an improvement on survival (compared to history of 20-yrs ago) which is actually difficult to be discriminated by the true prolongation of survival due to change in natural history of disease.

At now, most of ongoing clinical trials deal with various combination of active cytotoxic agents, but it has become evident that only small improvements in survival should be expected by such protocols. The issue is not spectrum of active drugs, as is the case in melanoma, for example but rather the rapid development of drug resistance and the failure of second-line therapy, especially in case of no response to primary treatment or early relapse of disease (< 3 months). Therefore clinical research on SCLC is shifting towards target-therapy, although most efforts have been and are still spent on lung adenocarcinoma, because its arise in incidence makes it more attractive in terms of cost/effectiveness of research.

Another issue in SCLC-related researches is paradoxically the introduction of fine-needle aspiration techniques rather than biopsy for histologic diagnosis: this has dramatically diminished the material available for studying compared with other lung cancers and other epithelial tumors.

## **7.4 Current perspectives: "Time to fish or cut bait"**

"Immortalization" of old randomized trials which refused surgery in multimodal treatment of SCLC in others era is no more acceptable today. A different staging system, lack or routine diagnostic tools and some evident weak points make these milestones anachronistic at now.

However, due to small number of cases and bias in selection of patients, retrospective series starts to carry a small usefulness in adding greater evidence in any type of treatment not contemplated in current guidelines. Meta-analyses too seem not to be reliable on studies that encompass sometimes two or three decades: lack of homogeneity between cohorts characteristics, staging system, diagnostic tools and type of treatments administered are a major issue to significance of results reported.

As brilliantly stated from Shepherd in comment to another retrospective trial on SEER database [Yu, 2010] "it is time to fish or cut bait": thoracic oncologists have to seriously face the question of role of surgery in multimodal approach to SCLC,: if they decide to fish, a large multi-center, international prospective randomized trial seem the only feasible option to achieve powerful statistical results [Shepherd, 2010]. Trial should accrue a large number of patients in a limited period of time with strict eligibility criteria in order to ensure as more homogeneity as possible among centres regarding patients selection, staging criteria and treatment modalities. In the forthcoming seventh edition of the TNM staging system, the IASLC has recommended to apply TNM stratification to SCLC in future clinical trials on LD-SCLC. At least one of the arm should be treated with chemo-radiation therapy followed by PCI, which represents actually the standard of care.

The small number of patients eligible for surgical trials makes accrual extremely slow, so that 3 trials started before 2003 have not been published yet (Essen Thoracic Oncology Group, West Japan Thoracic Oncology group, German Multicenter Randomised Trial).

Meanwhile advances in comprehension of molecular biology of SCLC will perhaps improve systemic control of disease, supporting and not excluding the role of surgery as the most powerful tool of cytoreduction local strategy even in more locally advanced disease in order to eliminate potential residual clones of resistant cells. It seems in fact still REMOTE the possibility discover of definitive systemic therapy capable of achieve complete local and distant, permanent remission of disease

## **8. Acknowledgment**

Many thanks to Dr. Giorgio Sgarbi, Chief of Thoracic Surgery Unit, Dr. Salvatore De Franco, Clinical Director of IRCCS and "Associazione Vittorio Lodini per la ricerca in chirurgia" for supporting our research activity.

Many thanks to Ms Giulia Mazzi for her English revision.

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