**1. Introduction**

226 Carcinogenesis, Diagnosis, and Molecular Targeted Treatment for Nasopharyngeal Carcinoma

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#### **1.1 The Nasopharynx (an unusual perspective of a peculiar structure)**

Nasopharynx is a peculiar anatomic structure with an essential role in respiratory, digestive and auditory systems. It is located between the respiratory and digestive tracts and its anatomic and physiologic characteristics depend on complex and not totally explained phylogenic, ontogenic and embryologic factors. Some of these factors demonstrated to be relevant not only on the pathogenesis, characteristics and development of nasopharyngeal carcinoma (NPC), but also on therapeutic options and their implications (Breda, Catarino et al. 2008).

In what concerns to phylogenic aspects, it is important to emphasize that either in chordates as even in the first aquatic vertebrates (agnate), the upper aerodigestive and respiratory tracts were associated, providing both the oxygenation and feeding supplies needed in an aquatic environment. However, according to Everett C. Olson (Rainger 1997), the appraisal of the oxygen and nourish requirements requested by the tetrapods, demanded the taking apart of oronasal cavity and the formation of primitive choanae; this was performed by the expansion of the branchial arches and the development of the mandible. Interestingly, this original functional ambivalence persists in mammals, and is visible in their anatomic relationships: nasal cavity is in a dorsal position relatively to the oral cavity but the larynx diverges ventrally from the pharynx.

In humans, as in most mammalians, pharyngeal and most of nasal cavity mucosa are the end result of a unique embryologic origin, depending from the cephalic portion of the primitive gut, a blind ending tube, lined with endodermic tissue, separated from the ectodermic *stomatodeum* by the buccopharyngeal membrane (Beasly 2008). Before the rupture of buccopharyngeal membrane happens, two recesses will form in this region: the Rathke pouch, which will origin the adenohypophysis and the Seesel pouch, representing the primordium of the future nasopharynx (Figure 1).

Nasopharyngeal Cancer – An Expanding Puzzle – Claiming for Answers 229

The nasopharynx is thus the end result of the several factors affecting its development, resulting in a trapezoid tubular about 128º angled structure, with average 4cm to 5,5cm transversal diameter, 2,5cm to 3,5cm anterior-posterior diameter e 4cm high(Snow Jr and Ballenger 2003). It has an anterior opening continuous with the posterior choanae and communicates with the oropharynx inferiorly, through a complex sphincteric mechanism. Superior wall is continuous with the posterior and in lateral walls an apparent Rosenmuller lateral pharyngeal recess posterior to the internal opening of pharyngotympanic tube are the

The nasopharyngeal wall is composed of mucous membrane (with pseudostratified columnar epithelium, *"lamina propria",* lymphoid tissue and stem cells), pharyngobasilar fascia, muscle layer and buccopharyngeal fascia(Standring 2008).Immediately posterior lie

When considering local anatomic implications on nasopharyngeal malignancies, the organization and function of lymphoid tissue, the potential oncologic relevance of stem cells and the significance of the cellular structure of the epithelium, muscles and neighboring

The mucosal immune system of upper aero-digestive tract provides the first line of defense against the ingress of microbial pathogens during the physiological processes of inhalation and ingestion. One of the major components of the mucosal immune system is secretory IgA (S-IgA),which is produced by the mucosal interaction of epithelial cells, IgA-committed B

Nasopharynx-associated lymphoid tissue (NALT) plays a pivotal role in the initiation of Agspecific immune responses at both systemic and mucosal sites, acting acts as an important inductive site for the generation of Ag-specific IgA-committed B cells(Fukuyama, Nagatake et al. 2006). The relationship between NALT system and Epstein-Barr virus (EBV) infection is being studied. There is now increasing evidence that defects in the immune control of EBV infection are linked to EBV associated malignancies, namely Hodgkin's disease and NPC, with the selective loss of EBV specific T cells and induction of local immunosuppressive microenvironment that favors tumor growth (Braz-Silva, Vitale et al.

In the identification of the malignant population that might be responsible for tumor initiation, the cancer stem cell (CSC) (Clarke, Dick et al. 2006), is other promising scientific advance. CSC is commonly assumed to have developed from a normal tissue stem cell. In normal organs, stem cells are defined as a subset of cells with the capacity of self-renewal to maintain the stem cell reservoir and of differentiation to generate various types of cells in the tissue. By self-renewal, stem cells divide symmetrically and perpetuate themselves by generating daughter cells with identical stem cell abilities of the parent. By differentiation, stem cells give rise to a hierarchy of limitedly proliferative but functional mature cells (Brady, Heilman et al. 2010). Zhang *et al* described for the first time stem cells in mouse nasopharyngeal epithelium; they represent less than 3% of cell population, and they lie mostly in basal and superbasal layers (Zhang, Wu et al. 1982). The polycomb group protein *Bmi*-1 is required for the maintenance and self-renewal of stem cells. The molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. However, according to Song *et al* (Song, Li et al. 2009)*,* previous upregulation of

salivary (minor and major) glands are examples of some topics waiting for answers.

the retropharyngeal lymph nodes, between this and the prevertebral fascia.

main anatomic evidences.

2011).

cells, and Th cells(Shikina, Hiroi et al. 2004).

Fig. 1. Human embryo (about 3 weeks): 1 - notocordium; 2 - Tornwaldt pouch origin; 3- Seesel pouch; 4 - Rathke pouch; 5 - buccopharyngeal membrane [Adapted from (Breda, Catarino et al. 2008)].

The muscle and osseous components of nasopharynx will take origin from the viscerocranium (pharyngeal or branchial arches and clefts), and neurocranium (Saddlet 1995). Pharyngeal arches contribute with mesenchymal tissue covered on the outside by surface ectoderm and on the inside by epithelium of endodermal origin. In addition, each arch receives substantial numbers of neural crest cells, which migrate into the arches to contribute to skeletal components of the face (Saddlet 2009). The development of Meckel cartilage is associated with a predominance of sagital craniofacial growth and increase of vertical and anterior-posterior length compared with transversal diameter.

Nasopharyngeal osseous superior and posterior limits (body of the shpenoid bone and the basilar part of occipital bone united in the sphenobasilarsynchondrosis) are derived from the prechordal chondrocranium (cartilages lying in front of the rostral limit of the notochord, which ends at the level of the pituitary gland in the center of the sella turcica, derived from neural crest cells) and the chordal chondrocranium, composed by those cartilages that lie posterior to this limit and arise from occipital sclerotomes formed by paraxial mesoderm (Saddlet 2009).

Intra-uterine increase of supratentorial and craniobasal angles and simultaneous shortness of infratentorial one (Jeffery 2005) contribute also to nasopharyngeal final shape, and these changes could be connected, according to Bull, to the development of occipital lobe, associated with bipedalism (Bull 1969) (Figure 2).

Fig. 2. Ontogenic aspects of cranial morphogenesis: TSA – supratentorial angle; ITA – infratentorial angle;CBA – craniobasal angle; *tc* – cranial attachment of tentorium cerebelli; *s* – sella; *fc* – foramen caecum [Adapted from (Breda, Catarino et al. 2008)].

Fig. 1. Human embryo (about 3 weeks): 1 - notocordium; 2 - Tornwaldt pouch origin; 3- Seesel pouch; 4 - Rathke pouch; 5 - buccopharyngeal membrane [Adapted from (Breda,

vertical and anterior-posterior length compared with transversal diameter.

The muscle and osseous components of nasopharynx will take origin from the viscerocranium (pharyngeal or branchial arches and clefts), and neurocranium (Saddlet 1995). Pharyngeal arches contribute with mesenchymal tissue covered on the outside by surface ectoderm and on the inside by epithelium of endodermal origin. In addition, each arch receives substantial numbers of neural crest cells, which migrate into the arches to contribute to skeletal components of the face (Saddlet 2009). The development of Meckel cartilage is associated with a predominance of sagital craniofacial growth and increase of

Nasopharyngeal osseous superior and posterior limits (body of the shpenoid bone and the basilar part of occipital bone united in the sphenobasilarsynchondrosis) are derived from the prechordal chondrocranium (cartilages lying in front of the rostral limit of the notochord, which ends at the level of the pituitary gland in the center of the sella turcica, derived from neural crest cells) and the chordal chondrocranium, composed by those cartilages that lie posterior to this limit and arise from occipital sclerotomes formed by

Intra-uterine increase of supratentorial and craniobasal angles and simultaneous shortness of infratentorial one (Jeffery 2005) contribute also to nasopharyngeal final shape, and these changes could be connected, according to Bull, to the development of occipital lobe,

Fig. 2. Ontogenic aspects of cranial morphogenesis: TSA – supratentorial angle; ITA – infratentorial angle;CBA – craniobasal angle; *tc* – cranial attachment of tentorium cerebelli;

*s* – sella; *fc* – foramen caecum [Adapted from (Breda, Catarino et al. 2008)].

Catarino et al. 2008)].

paraxial mesoderm (Saddlet 2009).

associated with bipedalism (Bull 1969) (Figure 2).

The nasopharynx is thus the end result of the several factors affecting its development, resulting in a trapezoid tubular about 128º angled structure, with average 4cm to 5,5cm transversal diameter, 2,5cm to 3,5cm anterior-posterior diameter e 4cm high(Snow Jr and Ballenger 2003). It has an anterior opening continuous with the posterior choanae and communicates with the oropharynx inferiorly, through a complex sphincteric mechanism. Superior wall is continuous with the posterior and in lateral walls an apparent Rosenmuller lateral pharyngeal recess posterior to the internal opening of pharyngotympanic tube are the main anatomic evidences.

The nasopharyngeal wall is composed of mucous membrane (with pseudostratified columnar epithelium, *"lamina propria",* lymphoid tissue and stem cells), pharyngobasilar fascia, muscle layer and buccopharyngeal fascia(Standring 2008).Immediately posterior lie the retropharyngeal lymph nodes, between this and the prevertebral fascia.

When considering local anatomic implications on nasopharyngeal malignancies, the organization and function of lymphoid tissue, the potential oncologic relevance of stem cells and the significance of the cellular structure of the epithelium, muscles and neighboring salivary (minor and major) glands are examples of some topics waiting for answers.

The mucosal immune system of upper aero-digestive tract provides the first line of defense against the ingress of microbial pathogens during the physiological processes of inhalation and ingestion. One of the major components of the mucosal immune system is secretory IgA (S-IgA),which is produced by the mucosal interaction of epithelial cells, IgA-committed B cells, and Th cells(Shikina, Hiroi et al. 2004).

Nasopharynx-associated lymphoid tissue (NALT) plays a pivotal role in the initiation of Agspecific immune responses at both systemic and mucosal sites, acting acts as an important inductive site for the generation of Ag-specific IgA-committed B cells(Fukuyama, Nagatake et al. 2006). The relationship between NALT system and Epstein-Barr virus (EBV) infection is being studied. There is now increasing evidence that defects in the immune control of EBV infection are linked to EBV associated malignancies, namely Hodgkin's disease and NPC, with the selective loss of EBV specific T cells and induction of local immunosuppressive microenvironment that favors tumor growth (Braz-Silva, Vitale et al. 2011).

In the identification of the malignant population that might be responsible for tumor initiation, the cancer stem cell (CSC) (Clarke, Dick et al. 2006), is other promising scientific advance. CSC is commonly assumed to have developed from a normal tissue stem cell. In normal organs, stem cells are defined as a subset of cells with the capacity of self-renewal to maintain the stem cell reservoir and of differentiation to generate various types of cells in the tissue. By self-renewal, stem cells divide symmetrically and perpetuate themselves by generating daughter cells with identical stem cell abilities of the parent. By differentiation, stem cells give rise to a hierarchy of limitedly proliferative but functional mature cells (Brady, Heilman et al. 2010). Zhang *et al* described for the first time stem cells in mouse nasopharyngeal epithelium; they represent less than 3% of cell population, and they lie mostly in basal and superbasal layers (Zhang, Wu et al. 1982). The polycomb group protein *Bmi*-1 is required for the maintenance and self-renewal of stem cells. The molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. However, according to Song *et al* (Song, Li et al. 2009)*,* previous upregulation of

Nasopharyngeal Cancer – An Expanding Puzzle – Claiming for Answers 231

EBV is a member of the herpesvirus family. As other herpesviruses, EBV is an enveloped virus that contains a DNA core surrounded by an icosahedral nucleocapsid and a tegument. Family members include herpes simplex 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) - alphavirus subfamily; cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) and 7 (HHV-7) - betaherpesvirus subfamily; and human herpesvirus 8 (HHV-8) and EBV gammaherpesvirus subfamily. The oncogenic potential of EBV was recognized through the association with numerous human malignancies. In addition to endemic Burkitt's lymphoma and NPC, EBV was later found in Hodgkin's lymphoma cases, post-transplant lymphoproliferative diseases, some T-cell lymphomas and a proportion of gastric carcinomas (Young and Rickinson 2004). Research is currently ongoing to determine the role of EBV-encoded gene products in these different cellular environments in an attempt to

It is now known that EBV infects 90% of the world's adult population. Although herpesviruses are ubiquitous in nature, humans are the only natural host for EBV. Upon infection, the individual remains a life-long carrier of the virus (Henle and Henle 1976). During acute infection, EBV seems to primarily infect and replicate in the stratified squamous epithelium of the oropharynx, followed by a latent infection of the B lymphocytes. EBV infection of B lymphocytes is thought to occur in the upper aerodigestive tract lymphoid organs and the virus persists in circulating memory B cells (Sixbey, Nedrud et al. 1984; Farrell 1995). Taking in account that EBV infection is a common event in the entire World, the reasons why only some individuals will develop EBV-related

Much of the known biology of EBV relates to its interaction with B-lymphocytes. This is mainly a result of the ability of EBV to readily infect and transform normal resting Blymphocytes in vitro, which also conrms the B-lymphotropic nature of this virus. EBV latent gene expression in various EBV-associated malignancies and EBV-derived cell lines has led to the identication of three different and distinct latency patterns. These latency patterns characterize a heterogeneous group of diseases and are based on EBV genome expression arrangements as the result of differential promoter activity inuenced by host cell factors (Thompson and Kurzrock 2004; Young and Rickinson 2004). Latency type I is generally associated with Burkitt's lymphoma and is characterized by expression of the EBV-encoded RNAs (EBERs) and the BamHI-A rightward transcripts (BARTs), in addition to EBV nuclear antigen-1 (EBNA1) expression. Latency type II is associated with NPC, gastric cancer and EBV-positive Hodgkin lymphoma and is characterized by expression of EBERs, BARTs, EBNA1 and the latent membrane proteins (LMP1, LMP2A and LMP2B). Latency type III is associated with lymphoblastoid cell lines and post-transplant lymphoproliferative diseases: the full range of latent gene products are expressed, including EBNAs 1, 2, 3A, 3B, 3C and -LP, the expression of all three latent membrane proteins (LMP1, LMP2A and LMP2B) and EBERs and BART RNAs. Although these classications of latency are useful in characterizing the distinct gene expression patterns, they are by no means completely denitive (Thompson and Kurzrock 2004). In recent years, there has been increasing interest in the presence of different viral and cellular micro-RNAs in EBVinfected B cells and epithelial cells. Future works should investigate the role of EBV-encoded micro-RNAs and its correlation with transcriptional regulation of both the viral and cellular

understand the role that EBV plays in the pathogenesis of these malignancies.

malignancies, are interesting aspects yet to clarify.

genome.

*Bmi*-1 correlates with invasion of NPC and otherwise a possible mechanism of tumorigenesis involves repression of the *Ink4a-Arf* locus, which encodes the tumor suppressors INK4A and ARF, by direct binding of *Bmi*-1.

Furthermore, there is an ongoing debate over whether CSCs represent a mature tissue stem cell which has undergone malignant change or whether more differentiated cells re-initiate a 'stemness' programme as part of, or following, malignant transformation (Bomken, Fiser et al. 2010).

These and other questions are yet to be answered, namely how to explain the relationship between CSC, NPC and EBV. The recent accomplishment that LMP2A EBV encoded latent protein, induces epithelial–mesenchymal transition and stem-like cell self-renewal in NPC, is a strong evidence of this association (Kong, Hu et al. 2010).
