**Radioresistance in Head and Neck Squamous Cell Carcinoma — Possible Molecular Markers for Local Recurrence and New Putative Therapeutic Strategies**

Federica Ganci, Andrea Sacconi, Valentina Manciocco, Giuseppe Spriano, Giulia Fontemaggi, Paolo Carlini and Giovanni Blandino

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60081

## **1. Introduction**

Head and neck squamous cell carcinoma (HNSCC) comprises 5.5% of all incidence cancers and is the sixth leading cancer worldwide with approximately 600,000 cases reported annually [1, 2]. The vast majority of them are squamous cell carcinomas that originate in the epithelium of the oral cavity, pharynx and larynx. There is a higher incidence rate in males compared to females and the median age of patients with HNSCC is about 60 years [3]. The main risk factors for HNSCC are tobacco smoking and heavy use of alcohol. In particular, alcohol consumption and tobacco smoking have a synergic effect [4]. The contribution of tobacco exposure to HNSCC carcinogenesis is strongly correlated with the time and rate of the person who smokes and has showed to have site-specific differences according to the anatomical regions, with an increase in sensitivity from the oral cavity down to the larynx [5]. In addition, high-risk infection types of human papillomavirus (especially HPV-16 and 18) is emerging as a major cause of a subgroup of HNSCC, particularly those of the oropharynx and oral cavity [2, 6]. The traditional risk factors, tobacco and alcohol use, do not appear to play a contributing role in HPV-positive cancers [7]. However, it is known that HPV-positive and negative tumors have different clinical, pathological and molecular characteristics and that HPV-positive tumors are associated with a more favorable outcome [2, 6] and better response to standard therapy.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Many molecular studies show that these HNSCC may not be as homogeneous as previously supposed. This indicates the need to obtain a more detailed molecular characterization in order to stratify patients better. This ultimately is likely to provide a more rational therapeutic approach, potentially relevant to diagnosis and prognosis of this poorly defined subset of HNSCC cancer.

## **2. Therapy strategies and molecular mechanisms of radioresistance**

HNSCC is typically characterized by locoregional diffusion and low propensity to develop distant metastasis. Due to the lack of symptoms in the early stage of the disease, about two thirds of patients are diagnosed in advanced stage with lymph node metastases. Local recurrence affects about 50-60% of patients and metastases develop in 15-20% of cases [8], with the five-year overall survival rate less than 50% [8, 9]. Locoregional failure is the most common cause of death in patients affected by HNSCC [10]. Recurrence may arise from residual neoplastic cells that survive to the treatment or from underlying field cancerization. Indeed, one key feature of HNSCC is the insurgence of recurrences after seemingly complete surgical resection, probably due to the existence of preneoplastic processes at multiple sites in the mucosa ("field cancerization" hypothesis). These preneoplastic tissues are apparently tumorfree when analyzed at histological level but present several genetic alterations when analyzed at a molecular level [11, 12].

Typically, HNSCC treatment consists of surgical resection followed by ionizing radiation or chemoradiation, or chemoradiation alone. Therapeutic strategy choice depends on disease stage: tumors at early stage are treated with surgery or radiotherapy. Surgery can be performed if complete tumor excision is possible and radiation can be used postoperatively when surgical margins are positive for the presence of tumor cells and/or if lymphovascular invasion by tumor is found. Platinum-based agents, in particular cisplatin (CDDP), are the conventional chemotherapeutic drugs for HNSCC treatment. More advanced cancers require multimodality therapy combining surgery, radiation and chemotherapy. Concurrent chemoradiation is the preferred treatment for advanced inoperable HNSCC [13-15]. These standard therapies have some limitations; they have several side effects and generally more than 50% of HNSCC patients relapse. The toxicities are mainly due to non-selective nature of treatment. However, resistance to chemoradiotherapy frequently occurs and is associated with poor outcome. This is the major clinical problem in HNSCC patients and relies on the fact that recurrence is often related to an intrinsic tumor radioresistance [14].

Molecular mechanisms underlying the resistance to radiotherapy or combined treatments mainly involve intracellular pathways related to cell proliferation, apoptosis, DNA repair and angiogenesis [13, 14, 16, 17]. To date, the main molecular mechanisms for radioresistance are:


#### **2.1. Hypoxia**

Many molecular studies show that these HNSCC may not be as homogeneous as previously supposed. This indicates the need to obtain a more detailed molecular characterization in order to stratify patients better. This ultimately is likely to provide a more rational therapeutic approach, potentially relevant to diagnosis and prognosis of this poorly defined subset of

**2. Therapy strategies and molecular mechanisms of radioresistance**

HNSCC is typically characterized by locoregional diffusion and low propensity to develop distant metastasis. Due to the lack of symptoms in the early stage of the disease, about two thirds of patients are diagnosed in advanced stage with lymph node metastases. Local recurrence affects about 50-60% of patients and metastases develop in 15-20% of cases [8], with the five-year overall survival rate less than 50% [8, 9]. Locoregional failure is the most common cause of death in patients affected by HNSCC [10]. Recurrence may arise from residual neoplastic cells that survive to the treatment or from underlying field cancerization. Indeed, one key feature of HNSCC is the insurgence of recurrences after seemingly complete surgical resection, probably due to the existence of preneoplastic processes at multiple sites in the mucosa ("field cancerization" hypothesis). These preneoplastic tissues are apparently tumorfree when analyzed at histological level but present several genetic alterations when analyzed

Typically, HNSCC treatment consists of surgical resection followed by ionizing radiation or chemoradiation, or chemoradiation alone. Therapeutic strategy choice depends on disease stage: tumors at early stage are treated with surgery or radiotherapy. Surgery can be performed if complete tumor excision is possible and radiation can be used postoperatively when surgical margins are positive for the presence of tumor cells and/or if lymphovascular invasion by tumor is found. Platinum-based agents, in particular cisplatin (CDDP), are the conventional chemotherapeutic drugs for HNSCC treatment. More advanced cancers require multimodality therapy combining surgery, radiation and chemotherapy. Concurrent chemoradiation is the preferred treatment for advanced inoperable HNSCC [13-15]. These standard therapies have some limitations; they have several side effects and generally more than 50% of HNSCC patients relapse. The toxicities are mainly due to non-selective nature of treatment. However, resistance to chemoradiotherapy frequently occurs and is associated with poor outcome. This is the major clinical problem in HNSCC patients and relies on the fact that recurrence is often

Molecular mechanisms underlying the resistance to radiotherapy or combined treatments mainly involve intracellular pathways related to cell proliferation, apoptosis, DNA repair and angiogenesis [13, 14, 16, 17]. To date, the main molecular mechanisms for radioresistance are:

**•** Alterations in the Epidermal Growth Factor Receptor (EGFR)- PI3K/Akt pathway

HNSCC cancer.

4 Contemporary Issues in Head and Neck Cancer Management

at a molecular level [11, 12].

**•** The hypoxia phenomenon

related to an intrinsic tumor radioresistance [14].

**•** Epithelial Mesenchymal Transition (EMT) process

Hypoxia is a common phenomenon present in many tumors and is associated with poor prognosis, malignant transformation and therapy resistance [18, 19]. In solid tumors, including HNSCC, oxygen is frequently reduced as the result of intermittent blood flow arising from the abnormal tumor microvasculature. Under oxygen deficiency, hypoxic tumor cells can activate the expression of hypoxia-inducible genes, functionally related to pro-survival, anti-apoptosis, angiogenesis, DNA-repair and metabolism signaling pathways [18, 20]. In particular, tumor cells switch their glucose metabolism from the oxygen-dependent tricarboxylic acid (TCA) cycle to oxygen-independent glycolysis metabolic pathway; as a consequence, hypoxic cells use glycolysis as main mechanism to produce ATP.

A key transcription factor having a central role in hypoxia-related gene expression changes is hypoxia-inducible transcription factor 1 (HIF-1). In normoxia, HIF-1α undergoes rapid hydroxylation and degradation. In hypoxia, hydroxylation is prevented, stabilized HIF-1α binds to HIF-1β and the heterodimer binds to hypoxia response elements in target genes, such as glycolytic enzymes, angiogenic molecules (among which VEGFA), survival and growth factors (among which EGF, PDGF and TGF-β), chaperons and other apoptosis resistancerelated proteins [13, 18, 21].

DNA double-stranded breaks (DSB) are the main DNA lesions leading to cell killing after radiotherapy. Oxygen is known to be a potent radiosensitizer and, through interaction with the radicals formed following radiation, it is essential for the promotion of radiation-induced DNA damage. Oxygen deficiency causes a reduction in reactive oxygen species (ROS) production and a deficit in radiation-induced DNA damage [20]. In agreement with these evidences, cells irradiated in the presence of air are about three times more sensitive than cells irradiated under conditions of severe hypoxia [22].

One of the evidences linking hypoxia to radiation response is a correlation between tumor control and hemoglobin levels [23], which is also related to oxygenation of solid tumors. Indeed, high hemoglobin (Hb) level, prior to and during treatment, has been associated with good prognosis in HNSCC patients treated with radiotherapy [23].

Hypoxia problem is particularly relevant in smoker patients. Indeed, in these HNSCC patients, the low oxygen level is also influenced by the formation of carboxyhemoglobin (COHb) and nicotine vase constrictive effect. As a consequence, the response to treatment and survival of smoker patients is significantly reduced compared to nonsmokers [23].

Given the influence of hemoglobin on tumor oxygenation and radiotherapy response, many researches tried to find methods able to increase Hb level in HNSCC patients having low Hb level, prior to and during radiation treatment; transfusion, or erythropoietin stimulating agents, are some of them, but unfortunately did not result in improved outcome or response to therapy [23]. To date, the main radiosensitizing and cytotoxic agents used in the clinical practice for hypoxic cells targeting are nitroimidazoles, which have also been shown to improve locoregional control, when applied in conjunction with radiation [20].

There is also interest in the use of nitroimidazoles as noninvasive hypoxia markers [24, 25]. Indeed, it remains difficult to identify hypoxic tumors and those patients most likely to benefit from hypoxia modification therapy. Under hypoxic conditions, nitroimidazoles are converted into reactive intermediates, which then become covalently bound to macromolecules within the cell. Nitroimidazoles labeling with an appropriate isotope or immunologically recogniza‐ ble marker allows the bioreduced compound to be detected, indicating the presence of hypoxia.

Additional indirect non-invasive techniques being explored to identify hypoxic tumors include measuring the immunohistochemical expression of hypoxia-regulated proteins, such as carbonic anhydrase 9 (CA9) and HIF-1α [26, 27]. This represents an attractive approach for routine clinical use, but is limited by the variability of expression of these markers within a tumor and by the lack of hypoxia specificity of individual proteins. An attempt to overcome these problems has been carried out by searching for tumor hypoxia gene signatures by metaanalysis of transcriptome datasets [28-30]. Winter and colleagues defined an *in vivo* hypoxia metagene by clustering around the RNA expression of a set of known *in vitro* hypoxiaregulated genes; this signature was also validated as a prognostic factor for recurrence-freesurvival in an independent data set [30].

#### **2.2. Alterations in the Epidermal Growth Factor Receptor (EGFR)-PI3K/Akt pathway**

Epidermal growth factor receptor (EGFR) is a transmembrane protein with tyrosine kinase activity that is overexpressed in about 90% of HNSCC, even if its expression is highly variable according to different subgroups of head and neck tumors as well as within the same tumor type [2, 14]. Stimulation by extracellular soluble ligands as epidermal growth factor (EGF) and transforming growth factors (TGFs) induces a conformational change leading to receptor heterodimerization with one of its family members (ErbB2, ErbB3, ErbB4); this causes auto‐ phosphorylation of the receptor intracellular domain and subsequent internalization followed by the activation of multiple signaling pathways, such as Ras-MAPKs (mitogen-activated protein kinases), extracellular signal-regulated kinases (ERKs), phosphatidylinositol-3-kinase-AKT (PI3-K/AKT), signal transducers and activators of transcription (STAT) and phospholi‐ pase C gamma (PLC-g) pathways [20].

High EGFR expression correlates with poor prognosis and resistance to conventional radio‐ therapy. EGFR expression can also be activated by the ionizing radiation itself, leading to increased radioresistance [20]. EGFR activation is also involved in increased proliferation rate and consequent repopulation, rendering radiotherapy ineffective [14].

Key proteins activated by EGFR are AKT and Ras; the first one is a kinase which phosphory‐ lates multiple downstream effectors, stimulating cell survival and inhibiting apoptosis; Ras is a cell membrane protein able to stimulate a tyrosine kinase cascade, including B-RAF, MEK, MAPK proteins, by which Myc, FOS and Jun translocate in the nucleus finally promoting cell proliferation. This cascade is also able to stimulate the production of EGFR monomers, TGFs and amphiregulin (AREG), contributing to paracrine EGFR activation [14]. Other proteins activated by EGFR are cyclin D1 and Pim-1, involved in cell cycle progression and inhibition of apoptosis; for the activation of both, the signal is mediated by STATs proteins [31, 32]. In addition, the interaction between EGFR-PI3-K/AKT and HIF pathways was also observed under hypoxic conditions, providing evidences on the correlation between EGFR signaling and the induction of angiogenic proteins, such as VEGFA, which is a downstream target of HIF-1 [20]

A subgroup of HNSCC (40%) expresses a truncated splicing variant of the EGFR, called EGFRvIII, in which the ligand-binding domain is altered, due to the deletion of amino acids 6-273. This alteration causes a permanent phosphorylation and activation of the receptor, also in the absence of EGF and TGFs ligands binding. As wild-type EGFR, EGFRvIII is implicated in increased cell proliferation, cell survival, motility and invasion. This variant is absent in normal tissues [17].

Besides EGFR overexpression, other mechanisms are involved in PI3K/Akt signaling hyperactivation, such as Ras activation, PI3-K gene mutation, Akt gene amplification and loss of tumor suppressor protein PTEN [14].

### **2.3. Epithelial Mesenchymal Transition (EMT) process**

Another important mechanism by which radiotherapy can fail in HNSCC is epithelial to mesenchymal transition (EMT) process. When EMT occurs, epithelial cells change in mesen‐ chymal phenotype which is characterized by reduction of the matrix contact, cell–cell adhesion followed by an increase in cell migration and motility. A crucial step of EMT is the loss of Ecadherin, a strong epithelial marker involved in adherens junction that anchors epithelial cells to each other [33]. A reduction of E-cadherin level was observed in HNSCC, especially in poorly differentiated tumors. In addition, many studies have demonstrated that aberrant E-cadherin expression is associated with poor outcome and local recurrence in HNSCC [34]. Loss or decrease of E-cadherin expression causes the translocation of β-catenin protein from the cell membrane to the nucleus to induce transcription of EMT-related genes, such as TWIST and SNAIL1 [33]. Another important protein involved in EMT is vimentin, which is an intermediate filament protein used as a marker for mesenchymal cells and is associated with the migratory phenotype, local recurrence and survival in HNSCC [34, 35]. Also fibronectin, a glycoprotein which mediates cellular interaction with extracellular matrix, plays an important role in migration, growth and adhesion of cells; its expression can be promoted by SNAIL and TWIST transcription factors [33]. Fibronectin is expressed at high level in tumors and blood plasma of HNSCC patients and has been proposed as biomarker of poor response to radiotherapy [36].

#### **2.4.** *TP53* **gene deregulation**

to therapy [23]. To date, the main radiosensitizing and cytotoxic agents used in the clinical practice for hypoxic cells targeting are nitroimidazoles, which have also been shown to

There is also interest in the use of nitroimidazoles as noninvasive hypoxia markers [24, 25]. Indeed, it remains difficult to identify hypoxic tumors and those patients most likely to benefit from hypoxia modification therapy. Under hypoxic conditions, nitroimidazoles are converted into reactive intermediates, which then become covalently bound to macromolecules within the cell. Nitroimidazoles labeling with an appropriate isotope or immunologically recogniza‐ ble marker allows the bioreduced compound to be detected, indicating the presence of

Additional indirect non-invasive techniques being explored to identify hypoxic tumors include measuring the immunohistochemical expression of hypoxia-regulated proteins, such as carbonic anhydrase 9 (CA9) and HIF-1α [26, 27]. This represents an attractive approach for routine clinical use, but is limited by the variability of expression of these markers within a tumor and by the lack of hypoxia specificity of individual proteins. An attempt to overcome these problems has been carried out by searching for tumor hypoxia gene signatures by metaanalysis of transcriptome datasets [28-30]. Winter and colleagues defined an *in vivo* hypoxia metagene by clustering around the RNA expression of a set of known *in vitro* hypoxiaregulated genes; this signature was also validated as a prognostic factor for recurrence-free-

**2.2. Alterations in the Epidermal Growth Factor Receptor (EGFR)-PI3K/Akt pathway**

Epidermal growth factor receptor (EGFR) is a transmembrane protein with tyrosine kinase activity that is overexpressed in about 90% of HNSCC, even if its expression is highly variable according to different subgroups of head and neck tumors as well as within the same tumor type [2, 14]. Stimulation by extracellular soluble ligands as epidermal growth factor (EGF) and transforming growth factors (TGFs) induces a conformational change leading to receptor heterodimerization with one of its family members (ErbB2, ErbB3, ErbB4); this causes auto‐ phosphorylation of the receptor intracellular domain and subsequent internalization followed by the activation of multiple signaling pathways, such as Ras-MAPKs (mitogen-activated protein kinases), extracellular signal-regulated kinases (ERKs), phosphatidylinositol-3-kinase-AKT (PI3-K/AKT), signal transducers and activators of transcription (STAT) and phospholi‐

High EGFR expression correlates with poor prognosis and resistance to conventional radio‐ therapy. EGFR expression can also be activated by the ionizing radiation itself, leading to increased radioresistance [20]. EGFR activation is also involved in increased proliferation rate

Key proteins activated by EGFR are AKT and Ras; the first one is a kinase which phosphory‐ lates multiple downstream effectors, stimulating cell survival and inhibiting apoptosis; Ras is a cell membrane protein able to stimulate a tyrosine kinase cascade, including B-RAF, MEK, MAPK proteins, by which Myc, FOS and Jun translocate in the nucleus finally promoting cell

and consequent repopulation, rendering radiotherapy ineffective [14].

improve locoregional control, when applied in conjunction with radiation [20].

hypoxia.

survival in an independent data set [30].

6 Contemporary Issues in Head and Neck Cancer Management

pase C gamma (PLC-g) pathways [20].

*TP*53 is a tumor suppressor gene, which functions in carcinogenesis by initiating G1 arrest in response to certain DNA damages and apoptosis. About 40-70% of HNSCC has mutation in *TP53* gene, leading to inactivation of its product [37]. Indeed, mutant p53 proteins are unable to transcriptionally regulate wt-p53 target genes and to exert antitumor effects such as apoptosis, growth arrest, differentiation and senescence. On the other hand, countless evidence has demonstrated that at least certain mutant forms of the p53 protein may possess gain of function activity, thereby positively contributing to the development, maintenance and spreading of many types of tumor, including HNSCC [38, 39]. The prognostic role of p53 alteration in HNSCC is controversial. However, generally, deregulation of p53 protein predicts shorter overall survival, local recurrence and cancer treatment failure [40-44].

In particular, p53 alteration leads to an impaired capability to arrest cell cycle and to inhibit apoptosis. In addition, in this condition also DNA damage repair results compromised. As a consequence, tumor cells carrying *TP53* mutation are less sensitive to radiation-induced cell death and are unable to restore DNA integrity, thus accumulating several genetic mutations which lead to increased tumor heterogeneity and finally to resistance to conventional therapy [14].

In addition, several evidences show that its prognostic value depends on the *TP53* protein domain affected by mutation [43-46]. One of the main classifications of *TP53* mutation used in HNSCC is "disruptive" versus "not-disruptive"; any mutation in L2 or L3 loop of the DNAbinding domain resulting in a polarity change of the protein or any stop codon was classified as disruptive [44]. Disruptive *TP53* mutations were associated with poor outcome and increased radioresistance [44, 46]. Other studies have proposed an alternative classification by which mutations in DNA-binding regions, especially in L2 and L3+LSH motifs, were associ‐ ated with poorer prognosis and clinical response to radiotherapy [45].

Of note, emerging evidences show that senescence may play a role in the radiation response by wild-type p53 [47]. Senescence is a form of cell cycle arrest in which cells lack replicative potential while remaining metabolically active, and was found to correlate with radiosensi‐ tivity in HNSCC [46]. In the proposed model, in the presence of *TP53* wild type or nondis‐ ruptive mutation, radiation promotes the induction of ROS production and p21 protein expression, which are critical mediators of cellular senescence. *TP53* disruptive mutations cause cellular senescence inhibition by reduction of radiation-induced ROS, thus driving resistance to radiotherapy [46].

#### **2.5. Alterations in the expression of angiogenic factors**

Angiogenesis is a process by which new blood vessels grow up from preexisting capillaries. Because expanding tumors have a continuous need for oxygen and nutrients, tumor cells induce angiogenesis. In particular, by secreting a variety of growth factors they activate the endothelial cells, constituting the inner lining of blood vessels, which produce proteases that degrade the basal membrane and extracellular matrix components. As a consequence, the endothelial cells can proliferate and migrate forming new capillary beds. Because in tumors new blood vessels are irregular and disorganized, the oxygen supply inside the tissue is not homogenous, resulting in continuous angiogenesis stimulation [48].

The main actors of this process are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and matrix metalloprotease (MPP) family proteins.

VEGF family consists of seven ligands, which play a central role in the formation of new blood vessels; VEGFA is the best known agent that induces angiogenesis by binding two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. VEGFA is able to promote development of the vascular system, cell migration, survival and induction of MMPs [13]; it also activates PI3K/AKT and Ras/MAPK signaling pathways [49]. There are increasing evidences that angiogenic response of irradiated tumor cells is related with decreasing radiation sensitivity and head and neck cancer progression. In a meta-analysis of 12 studies including 1002 patients affected by cancer of oral cavity, pharynx and larynx, VEGF expression positivity was associated with a two folds higher risk of death at 2 years of follow-up [50].

Release of VEGF and bEGF by epithelial tumor cells after irradiation is a common response mechanism by which cancer cells may survive and become protected from radiation-induced cell death [51]. Therefore, the level of VEGF and bEGF prior to and during treatment may be relevant for successful therapy.

## **2.6. Cancer Stem Cells (CSCs)**

to transcriptionally regulate wt-p53 target genes and to exert antitumor effects such as apoptosis, growth arrest, differentiation and senescence. On the other hand, countless evidence has demonstrated that at least certain mutant forms of the p53 protein may possess gain of function activity, thereby positively contributing to the development, maintenance and spreading of many types of tumor, including HNSCC [38, 39]. The prognostic role of p53 alteration in HNSCC is controversial. However, generally, deregulation of p53 protein predicts

In particular, p53 alteration leads to an impaired capability to arrest cell cycle and to inhibit apoptosis. In addition, in this condition also DNA damage repair results compromised. As a consequence, tumor cells carrying *TP53* mutation are less sensitive to radiation-induced cell death and are unable to restore DNA integrity, thus accumulating several genetic mutations which lead to increased tumor heterogeneity and finally to resistance to

In addition, several evidences show that its prognostic value depends on the *TP53* protein domain affected by mutation [43-46]. One of the main classifications of *TP53* mutation used in HNSCC is "disruptive" versus "not-disruptive"; any mutation in L2 or L3 loop of the DNAbinding domain resulting in a polarity change of the protein or any stop codon was classified as disruptive [44]. Disruptive *TP53* mutations were associated with poor outcome and increased radioresistance [44, 46]. Other studies have proposed an alternative classification by which mutations in DNA-binding regions, especially in L2 and L3+LSH motifs, were associ‐

Of note, emerging evidences show that senescence may play a role in the radiation response by wild-type p53 [47]. Senescence is a form of cell cycle arrest in which cells lack replicative potential while remaining metabolically active, and was found to correlate with radiosensi‐ tivity in HNSCC [46]. In the proposed model, in the presence of *TP53* wild type or nondis‐ ruptive mutation, radiation promotes the induction of ROS production and p21 protein expression, which are critical mediators of cellular senescence. *TP53* disruptive mutations cause cellular senescence inhibition by reduction of radiation-induced ROS, thus driving

Angiogenesis is a process by which new blood vessels grow up from preexisting capillaries. Because expanding tumors have a continuous need for oxygen and nutrients, tumor cells induce angiogenesis. In particular, by secreting a variety of growth factors they activate the endothelial cells, constituting the inner lining of blood vessels, which produce proteases that degrade the basal membrane and extracellular matrix components. As a consequence, the endothelial cells can proliferate and migrate forming new capillary beds. Because in tumors new blood vessels are irregular and disorganized, the oxygen supply inside the tissue is not

The main actors of this process are vascular endothelial growth factor (VEGF), fibroblast

shorter overall survival, local recurrence and cancer treatment failure [40-44].

ated with poorer prognosis and clinical response to radiotherapy [45].

**2.5. Alterations in the expression of angiogenic factors**

homogenous, resulting in continuous angiogenesis stimulation [48].

growth factor (FGF) and matrix metalloprotease (MPP) family proteins.

conventional therapy [14].

8 Contemporary Issues in Head and Neck Cancer Management

resistance to radiotherapy [46].

Cancer stem cells (CSCs) have been defined by Clarke et al., as a small tumor subpopulation possessing the capability to self-renewal and causing the heterogeneous lineage of cancer cells inside the tumor [52]. They are functionally defined as a subset of tumor cells with ability of self-renewal and multipotency, serving as progenitors of cancer cells. The characteristics by which CSCs can be distinguished to other cancer cells are the following [53]:


The presence of this subpopulation has been identified in several tumors, including HNSCC where its ability to maintain tumor population, metastasize and to be resistant to radioche‐ motherapy has been shown [53-55].

The origin of CSCs has not been clearly defined; in HNSCC, it has been proposed that a chronic inflammation caused by permanent tobacco, alcohol use, mechanic irritation or viral infection, in association with genetic predisposition, lead to the accumulation of various genetic alterations and finally to the manifestation of a malignant phenotype [53].

In addition, during tumor progression, some CSCs, through an EMT process, can acquire the ability to infiltrate and metastasize. On the other hand, EMT is involved in the acquisition of cancer stem cells properties; at the molecular level, the transcription factor Twist induces downregulation of E-cadherin while promoting expression of Bim1, which has an essential role in self-renewal of CSCs. In agreement with these data, high expression of Bim1 and Twist are associated with a poor prognosis in HNSCC [53].

In HNSCC patients, high percentage of CD44 positive cells was associated with higher rate of treatment failure in general, while cells expressing CD44, CD24, Oct4 and integrin β1 were associated with poor outcome after radiotherapy [56, 57]. From a clinical point of view, these evidences suggest that the patients can be cured if CSCs are completely eliminated.

## **3. Potential molecular markers for local recurrence and radioresistance**

One of the current major research questions in the management of HNSCC disease addresses the prediction and treatment of local recurrence. As described before, mortality of patients with HNSCC is primarily driven by tumor cell radioresistance leading to local recurrence. Due to the heterogeneous nature of tumors, the identification of markers with prognostic or predictive value to be used as a complement to conventional diagnostic methods is a complex challenge. Indeed, although advance in expression technologies, current studies have provid‐ ed ambiguous results.

Among the prognostic markers proposed in HNSCC, as described in the previous paragraph, the presence of mutation in *TP53* gene predicts the development of locoregional recurrence by increasing the radioresistance in tumor cells (Table 1).

Additional molecular markers predicting high local recurrence development and response to radiotherapy are summarized in Table 1.


Radioresistance in Head and Neck Squamous Cell Carcinoma — Possible Molecular Markers for Local Recurrence… http://dx.doi.org/10.5772/60081 11

In HNSCC patients, high percentage of CD44 positive cells was associated with higher rate of treatment failure in general, while cells expressing CD44, CD24, Oct4 and integrin β1 were associated with poor outcome after radiotherapy [56, 57]. From a clinical point of view, these

evidences suggest that the patients can be cured if CSCs are completely eliminated.

ed ambiguous results.

EGFR

increasing the radioresistance in tumor cells (Table 1).

radiotherapy are summarized in Table 1.

10 Contemporary Issues in Head and Neck Cancer Management

**3. Potential molecular markers for local recurrence and radioresistance**

One of the current major research questions in the management of HNSCC disease addresses the prediction and treatment of local recurrence. As described before, mortality of patients with HNSCC is primarily driven by tumor cell radioresistance leading to local recurrence. Due to the heterogeneous nature of tumors, the identification of markers with prognostic or predictive value to be used as a complement to conventional diagnostic methods is a complex challenge. Indeed, although advance in expression technologies, current studies have provid‐

Among the prognostic markers proposed in HNSCC, as described in the previous paragraph, the presence of mutation in *TP53* gene predicts the development of locoregional recurrence by

Additional molecular markers predicting high local recurrence development and response to

**Gene Function References**

Transmembrane TK acting as a central transducer in multiple pathways that mediate cell cycle progression, angiogenesis, inhibition of apoptosis, tumor invasion and metastasis.

cell survival. [2, 42, 44, 46, 58]

promoting EMT, angiogenesis, cell migration and metastasis. [16]

cell proliferation and apoptosis. [59-62]

role in cell adhesion, growth, migration, and differentiation. [36]

migration and survival of endothelial cells during tumor growth. [13, 14, 20, 51]

and important for tumor progression. [63]

[14, 20]

*TP*<sup>53</sup> A tumor-suppressor regulating cell cycle progression, apoptosis and

PTEN A tumor suppressor gene regulating signaling pathways controlling

Fibronectin It is a glycoprotein of the extracellular matrix, which plays a major

VEGFs Ligands of transmembrane TK promoting cell proliferation,

Cox2 Catalytic enzyme decreasing apoptosis, increasing inflammation

HIF-1α A transcription factor induced under hypoxic condition and


**Table 1.** Biomarkers predicting local recurrence and radioresistance in head and neck cancers.

## **3.1. MicroRNAs as new potential biomarkers predicting radiotherapy response**

A class of small non-coding RNAs termed microRNAs (miRNAs) has recently been indicated as biomarker of some type of cancers [73]. miRNAs are endogenous, small, non-coding RNAs of 17-25 nucleotides that are thought to regulate approximately 30% of human genes at posttranscriptional level, primarily through their partial complementarity with the coding region or 3' untranslated region (UTR) of target mRNAs. This leads to translational repression and/or degradation of target mRNA, therefore regulating gene expression [74]. They are involved in essential biological activities such as cellular differentiation, proliferation, development, apoptosis and cell cycle regulation. The roles of miRNAs in cancer have been extensively investigated in the past few years. The relevance of miRNAs in cancer was suggested by the observed changes in expression patterns and recurrent amplification as well as deletion of miRNA genes in cancer [75]. It has been shown that there are two types of cancerrelated miRNAs: oncogenic or tumor suppressor miRNAs [74].

Several investigators have empathized the role of miRs as biomarkers for HNSCC [42] and the usefulness of miRs as prognostic factors has only begun to be explored. Moreover, miRNA expression may predict the efficacy of therapies, including radiotherapy [76]. Data from the study of miR-205 and let-7d expression showed their association with locoregional occurrence and shorter survival [77]. In addition, high expression of miR-205 can be used to detect positive lymph nodes, suggesting that this miR can be considered as a marker for metastatic HNSCC [78]. A similar study has shown that lower expression levels of miR-451 in HNSCC tumors are associated with recurrence [79]. Another recent work reported that downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signaling [80]. In addition, miR-17-5p expression has been shown to be induced in irradiated oral cancer cells and it downregulates p21 protein expression, contribu‐ ting to radioresistance [81].

Furthermore, we also identified microRNAs signatures (miR-17-3p, miR-18b-5p, miR-324-5p, miR-19a-3p, miR-200a-3p, miR-331-3p, miR-21-3p, miR-21-5p, miR-205-5p, miR-151a-3p, miR-96-5p and miR-429) that are able to predict the risk of local recurrence and poor outcome in HNSCC tumors, and that are more powerful as biomarkers when compared to traditional prognostic indicators [42]. Finally, some evidences support the possibility to use miRNA detected in plasma as radio-responsive biomarkers for different types of cancer, including HNSCC. Accordingly, in HNSCC patients, the authors have detected changes in the abun‐ dance of circulating miRNAs (miR-425-5p and miR-93-5p) during radiochemotherapy. In addition, the researchers have demonstrated that the altered plasma miRNA changes after the therapy are the results of miRNAs release from damaged tumor cells [82].

## **4. Molecular strategies and future application in the treatment of HNSCC**

Conventional HNSCC treatment consists of surgical resection followed by ionizing radiation or chemoradiation. In case of local advance/inoperable HNSCC, the typical treatment is concomitant platinum-based chemoradiotherapy. These standard therapies have some limitations; the surgery can result in disfigurement and functional impairment, while the radiochemotherapy, although it is an organ-preserving treatment, can cause several side effects including mucositis, oral candidiasis, loss of taste, xerostomia and osteoradionecrosis [83, 84]. In addition, overall five-year survival rate is lower than 50% in HNSCC patients. Therefore, resistance to chemoradiotherapy often occurs and is associated with recurrences and poor outcomes; this represents a major clinical problem for HNSCC patients [14].

The understanding of the molecular perturbations in the cells of carcinomas recurring after irradiation could help to identify more specific target proteins and design novel therapeutic agents that will help improving therapy outcome in patients with HNSCC recurrences.

Tumor cells repopulation is a common effect observed in radiotherapy failure. A method to decrease this phenomenon, called Accelerated Radiotherapy (AR), is the reduction of overall radiation treatment time maintaining the total dose constant [14]. This therapeutic approach has produced excellent results in patients with advanced HNSCC [85]. In addition, several studies have shown that patients overexpressing EGFR protein, result to be more sensitive and consequently to have a better response to AR [14].

Besides the modification of radiotherapy modalities, there are several therapeutic strategies, such as, for example, immunotherapy, that can be combined with radiation, and are subjected to clinical development [86] (Table 2). Currently, two of the main intriguing targets for new targeted therapy are EGFR and VEGFR [86]. Both targeted therapies can be subdivided in monoclonal antibodies and tyrosine kinase inhibitors.

## **4.1. EGFR targeted therapy**

**3.1. MicroRNAs as new potential biomarkers predicting radiotherapy response**

related miRNAs: oncogenic or tumor suppressor miRNAs [74].

12 Contemporary Issues in Head and Neck Cancer Management

ting to radioresistance [81].

A class of small non-coding RNAs termed microRNAs (miRNAs) has recently been indicated as biomarker of some type of cancers [73]. miRNAs are endogenous, small, non-coding RNAs of 17-25 nucleotides that are thought to regulate approximately 30% of human genes at posttranscriptional level, primarily through their partial complementarity with the coding region or 3' untranslated region (UTR) of target mRNAs. This leads to translational repression and/or degradation of target mRNA, therefore regulating gene expression [74]. They are involved in essential biological activities such as cellular differentiation, proliferation, development, apoptosis and cell cycle regulation. The roles of miRNAs in cancer have been extensively investigated in the past few years. The relevance of miRNAs in cancer was suggested by the observed changes in expression patterns and recurrent amplification as well as deletion of miRNA genes in cancer [75]. It has been shown that there are two types of cancer-

Several investigators have empathized the role of miRs as biomarkers for HNSCC [42] and the usefulness of miRs as prognostic factors has only begun to be explored. Moreover, miRNA expression may predict the efficacy of therapies, including radiotherapy [76]. Data from the study of miR-205 and let-7d expression showed their association with locoregional occurrence and shorter survival [77]. In addition, high expression of miR-205 can be used to detect positive lymph nodes, suggesting that this miR can be considered as a marker for metastatic HNSCC [78]. A similar study has shown that lower expression levels of miR-451 in HNSCC tumors are associated with recurrence [79]. Another recent work reported that downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signaling [80]. In addition, miR-17-5p expression has been shown to be induced in irradiated oral cancer cells and it downregulates p21 protein expression, contribu‐

Furthermore, we also identified microRNAs signatures (miR-17-3p, miR-18b-5p, miR-324-5p, miR-19a-3p, miR-200a-3p, miR-331-3p, miR-21-3p, miR-21-5p, miR-205-5p, miR-151a-3p, miR-96-5p and miR-429) that are able to predict the risk of local recurrence and poor outcome in HNSCC tumors, and that are more powerful as biomarkers when compared to traditional prognostic indicators [42]. Finally, some evidences support the possibility to use miRNA detected in plasma as radio-responsive biomarkers for different types of cancer, including HNSCC. Accordingly, in HNSCC patients, the authors have detected changes in the abun‐ dance of circulating miRNAs (miR-425-5p and miR-93-5p) during radiochemotherapy. In addition, the researchers have demonstrated that the altered plasma miRNA changes after the

**4. Molecular strategies and future application in the treatment of HNSCC**

Conventional HNSCC treatment consists of surgical resection followed by ionizing radiation or chemoradiation. In case of local advance/inoperable HNSCC, the typical treatment is concomitant platinum-based chemoradiotherapy. These standard therapies have some

therapy are the results of miRNAs release from damaged tumor cells [82].

The role of EGFR signaling in radioresistance was widely discussed in the paragraph 2.2. Many evidences suggest that the use of EGFR inhibitors in combination with radiotherapy improves the outcomes of HNSCC patients respect to those treated with radiotherapy alone [14].

#### *4.1.1. EGFR monoclonal antibodies*

One of the main antibodies targeting EGFR is called cetuximab. Other anti-EGFR antibodies under active investigations in combination with chemoradiotherapy in HNSCC are panitu‐ mumab, zalutumumab and nimotuzumab (Table 2).

*Cetuximab:* It is a chimeric IgG1 mAb, which by the recognition of determinants expressed on the extracellular domain of EGFR, antagonize normal receptor interaction, preventing the activation of the downstream signaling pathways [17]. Based on the results obtained from the clinical trials, since 2006 it has been approved by the Food and Drug Administration (FDA) in association with radiotherapy [14, 86]. However, a meta-analysis studying 15 trials and focusing on the comparison of the two currently combined modality therapies show that chemoradiotherapy respect to radiotherapy plus cetuximab is associated with a better overall survival and locoregional recurrence in advanced HNSCC [87]. In addition, some HNSCC patients develop a resistance to anti-EGFR therapy mainly due to k-Ras deregulation in absence of its mutation [14, 17, 88] and the presence of the variant EGFRvIII in tumor cells [17]. In this last case, the deletion presents in this variant cause a reduction in the binding affinity of monoclonal antibodies raised with wild type EGFR [17].

*Panitumumab*: Preclinical evidences show that it increases radiosensitivity by the radiationinduced DNA damage and preventing the translocation of EGFR in the nucleus. Currently, therapy combining radiation in combination with panitumumab is undergoing phase III clinical trials [86]. In addition, a phase III trial performed in advanced HNSCC patients to compare 5-FU and cisplatin treatment in presence and not of panitumumab have not shown an important improvement of the clinical outcome [89].

*Zalutumumab:* Several studies on phase I/II trial were performed using this drug at different doses in combination to radiation and/or chemotherapy; the results are encouraging and a phase III is ongoing [86].

*Nimotuzumab:* Preclinical studies show that it has antiproliferative, antiangiogenic and proapoptotic effects and it is well tolerated in HNSCC patients treated with radiation [86]. However, it has been demonstrated that cetuximab is more effective in comparison to nimo‐ tuzumab in enhancing radiosensitivity in high-EGFR expressing cells [90].

In conclusion, antibody anti-EGFR in combination with radiation therapy was well tolerated in HNSCC patients; currently, the best-studied mAb are cetuximab and panitumumab. Both improve radiosensitivity and overall survival in advanced HNSCC treated with radiation. However, the addition of cetuximab to conventional chemoradiotherapy has not shown a significant improvement in clinical outcome and the results obtained from the treatment of a large number of patients in multi-centered trials has shown that the treatment is effective in about 20% of cases [17].

To date, the use of cetuximab in combination with radiation represents a standard clinical approach, particularly in HNSCC patients who cannot tolerate chemotherapy [86].

## *4.1.2. EGFR tyrosine kinase inhibitors*

Another group of agents targeting EGFR are small molecule tyrosine kinase inhibitors (TKIs). They act preventing EGFR autophosphorylation and consequently its activation by the occupation of the EGFR intracellular ATP-binding domain [17]. The two best studied TKIs are gefitinib (Iressa) and erlotinib (Tarceva). Others are called lapatinib and afanitib (Table 2).

*Gefitinib:* Preclinical studies show that gefitinib treatment on HNSCC cells can inhibit cell proliferation, decrease cell survival and enhance tumor cell radiosensitivity [91]. In addition, encouraging results were obtained in the clinical studies when gefitinib was combined with VEGFR inhibitors or other targets, suggesting the possibility to use it as possible neoadjuvant agent. Besides that, clinical trials combining gefitinib with chemoradiotherapy have not yet demonstrated a significant improvement respect to conventional therapy [86].

*Erlotinib:* Encouraging results were obtained from preclinical studies showing that the combination of erlotinib with radiation and/or VEGFR inhibitors improve treatment efficacy by the inhibition of tumor growth, proliferation and vessel density [92, 93]. However, to date, there is no convincing clinical evidence that the addition of erlotinib to conventional therapy is universally beneficial [86]

*Lapatinib and Afatinib:* They are orally active EGFR and HER2 inhibitors, which seem to be well tolerated from HNSCC patients. Interestingly, in p16-negative HNSCC patients, a large difference in clinical outcome was observed in patients treated with lapatinib versus placebo. Phase III trials are ongoing in HNSCC for both molecules [86].

## **4.2. VEGF targeted therapy**

In this last case, the deletion presents in this variant cause a reduction in the binding affinity

*Panitumumab*: Preclinical evidences show that it increases radiosensitivity by the radiationinduced DNA damage and preventing the translocation of EGFR in the nucleus. Currently, therapy combining radiation in combination with panitumumab is undergoing phase III clinical trials [86]. In addition, a phase III trial performed in advanced HNSCC patients to compare 5-FU and cisplatin treatment in presence and not of panitumumab have not shown

*Zalutumumab:* Several studies on phase I/II trial were performed using this drug at different doses in combination to radiation and/or chemotherapy; the results are encouraging and a

*Nimotuzumab:* Preclinical studies show that it has antiproliferative, antiangiogenic and proapoptotic effects and it is well tolerated in HNSCC patients treated with radiation [86]. However, it has been demonstrated that cetuximab is more effective in comparison to nimo‐

In conclusion, antibody anti-EGFR in combination with radiation therapy was well tolerated in HNSCC patients; currently, the best-studied mAb are cetuximab and panitumumab. Both improve radiosensitivity and overall survival in advanced HNSCC treated with radiation. However, the addition of cetuximab to conventional chemoradiotherapy has not shown a significant improvement in clinical outcome and the results obtained from the treatment of a large number of patients in multi-centered trials has shown that the treatment is effective in

To date, the use of cetuximab in combination with radiation represents a standard clinical

Another group of agents targeting EGFR are small molecule tyrosine kinase inhibitors (TKIs). They act preventing EGFR autophosphorylation and consequently its activation by the occupation of the EGFR intracellular ATP-binding domain [17]. The two best studied TKIs are gefitinib (Iressa) and erlotinib (Tarceva). Others are called lapatinib and afanitib (Table 2).

*Gefitinib:* Preclinical studies show that gefitinib treatment on HNSCC cells can inhibit cell proliferation, decrease cell survival and enhance tumor cell radiosensitivity [91]. In addition, encouraging results were obtained in the clinical studies when gefitinib was combined with VEGFR inhibitors or other targets, suggesting the possibility to use it as possible neoadjuvant agent. Besides that, clinical trials combining gefitinib with chemoradiotherapy have not yet

*Erlotinib:* Encouraging results were obtained from preclinical studies showing that the combination of erlotinib with radiation and/or VEGFR inhibitors improve treatment efficacy by the inhibition of tumor growth, proliferation and vessel density [92, 93]. However, to date,

demonstrated a significant improvement respect to conventional therapy [86].

approach, particularly in HNSCC patients who cannot tolerate chemotherapy [86].

tuzumab in enhancing radiosensitivity in high-EGFR expressing cells [90].

of monoclonal antibodies raised with wild type EGFR [17].

14 Contemporary Issues in Head and Neck Cancer Management

an important improvement of the clinical outcome [89].

phase III is ongoing [86].

about 20% of cases [17].

*4.1.2. EGFR tyrosine kinase inhibitors*

As explained in the paragraph 2.5, VEGF is one of the most important regulators of angiogen‐ esis; its upregulation is a common event in HNSCC and it is associated with radioresistance and poor prognosis.

## *4.2.1. VEGF monoclonal antibodies*

Bevacizumab (Avastin) (Table 2) is the main recombinant anti-VEGFA monoclonal antibody under active investigation for HNSCC therapy. Preclinical evidences show that bevacizumab is able to act as radiation sensitizer in HNSCC cells, to reduce angiogenesis and tumor growth [86]. Phase I/II clinical trials performed using bevacizumab in combination with conventional chemoradiotherapy in HNSCC have shown that although this combined modality therapy is possible, to date there is no strong evidence that the addition of bevacizumab to chemoradio‐ therapy causes an improvement of the overall survival in HNSCC patients [13]. Future investigations are necessary to define the effectiveness of this molecule in the treatment of HNSCC.

### *4.2.2. VEGFR tyrosine kinase inhibitors*

To date, the known VEGF tyrosine kinase inhibitors are: vandetanib (ZD6474), sunitinib, sorafenib and linifanib (ABT-869) (Table 2).

*Vandetanib:* It is an orally multi-kinase inhibitor targeting EGFR, VEGFR-2 and RET tyrosine kinases. Preclinical evidences show that the administration of vandetanib enhances the antitumor effects of radiation therapy by inhibition of both EGFR and VEGFR signaling in HNSCC human tumor xenografts; in particular, the authors demonstrate that radiation plus vandetanib treatment is effective in both overexpressing EGFR tumor cells and EGFR- null cells [94]. In addition, vandetanib restores HNSCC cells sensitivity to cisplatin and radiation *in vivo* and *in vitro* by promoting an increase of apoptosis and a decrease of microvessel density [95]. A randomized phase II clinical trial using a combination of cisplatin and radiation with or without vandetanib in advanced HNSCC is under consideration [13].

*Sunitinib:* It is an orally multi-kinase inhibitor targeting VEGFR, PDGFR and c-Kit tyrosine kinases. Preclinical and clinical studies show that sunitinib has low activity as monotherapy, but in combination with cetuximab and radiation, it causes a strong tumor inhibition effect by a complete abolition of tumor growth. Specifically, the combination of cetuximab and sunitinib causes a decrease of cell proliferation and enhances cell differentiation, while a decrease in tumor vessels number was observed when the radiation treatment was added [96]. These results encourage future clinical investigations regarding the sunitinib and chemoradiother‐ apy treatment combination.

*Sorafenib:* It is an oral inhibitor of serine/threonine protein kinase b-Raf, C-Raf, VEGFR and PDGFR. Preclinical evidences show that sorafenib in combination with chemoradiation is able to enhance a more effective antitumor effect by the inhibition of cell growth, clone formation, cell migration and invasion compared to chemoradiation or radiation alone. This therapy combination is also able to inhibit tumor angiogenesis [97]. In addition, sunitinib can increase the antiproliferative effect of chemoradiotherapy by inhibiting the Raf/MEK/ERK signaling pathway and consequently downregulating the expression of the DNA repair proteins ERCC-1 and XRCC-1 [13]. Although these results suggest that sorafenib could enhance the effectiveness of chemoradiotherapy, ongoing phase I/II clinical trials will determine the real efficacy of sorafenib in HNSCC patients.

*Linifanib:* It is a novel ATP-competitive tyrosine kinase inhibitor of the VEGF and PDGF receptor family members. Preliminary data on HNSCC cells show that linifanib can act as radiation sensitizer since its combination with radiation is more effective compared to radiation or chemoradiation alone [13].

#### **4.3. Other targeted therapies**

As explained in the paragraph 2 relative to molecular mechanisms of radioresistance, there are many actors playing a key role in the failure of radiotherapy in HNSCC. As a consequence, targeted therapies against other molecules besides EGFR and VEGF family proteins were developed and their characterization is still ongoing. Among them, there are Src-family kinase inhibitors such as dosatinib; proteasome inhibitors as bortezomib, cyclooxygenase(Cox)-2 inhibitor (colecoxib); PI3K/Akt/mTOR inhibitors as wortmannin, perifostine and temsiroli‐ mus; and therapies targeting c-Met signaling pathway [14, 86] (Table 2).

Briefly, Src-kinase inhibitor dasatinib promotes radiosensitization by decreasing EGFR phosphorylation, its translocation in the nucleus and consequently, its association with DNA– protein kinases, blocking DNA repair pathways [98, 99]. Evidences on proteasome inhibitor bortezomib show its capability to act as radiosensitizer; specifically, it promotes the upregu‐ lation of PTEN activity and downregulation of p-Akt, leading to an increase of apoptosis of tumor cells [100-102]. Cox inhibitor colecoxib leads to a decrease of VEGFR expression and angiogenesis [103]. Next, mTOR inhibitors cause a reduction of angiogenesis and an induction of cell death by autophagy [86, 104]. Finally, given the important role discussed in the paragraph 2 on the significance of CSCs subpopulation in radioresistance, an emerging concept is the combined use of standard chemoradiotherapy with cancer stem cells targeted therapy. Preclinical study on CD44 expressing HNSCC cells combine radiation with anti-CD44 antibodies; the results show an increase in local tumor control in patients treated with radiation plus anti-CD44 antibodies compared to those treated with radiation alone in vivo [105].

Radioresistance in Head and Neck Squamous Cell Carcinoma — Possible Molecular Markers for Local Recurrence… http://dx.doi.org/10.5772/60081 17

tumor vessels number was observed when the radiation treatment was added [96]. These results encourage future clinical investigations regarding the sunitinib and chemoradiother‐

*Sorafenib:* It is an oral inhibitor of serine/threonine protein kinase b-Raf, C-Raf, VEGFR and PDGFR. Preclinical evidences show that sorafenib in combination with chemoradiation is able to enhance a more effective antitumor effect by the inhibition of cell growth, clone formation, cell migration and invasion compared to chemoradiation or radiation alone. This therapy combination is also able to inhibit tumor angiogenesis [97]. In addition, sunitinib can increase the antiproliferative effect of chemoradiotherapy by inhibiting the Raf/MEK/ERK signaling pathway and consequently downregulating the expression of the DNA repair proteins ERCC-1 and XRCC-1 [13]. Although these results suggest that sorafenib could enhance the effectiveness of chemoradiotherapy, ongoing phase I/II clinical trials will determine the real efficacy of

*Linifanib:* It is a novel ATP-competitive tyrosine kinase inhibitor of the VEGF and PDGF receptor family members. Preliminary data on HNSCC cells show that linifanib can act as radiation sensitizer since its combination with radiation is more effective compared to

As explained in the paragraph 2 relative to molecular mechanisms of radioresistance, there are many actors playing a key role in the failure of radiotherapy in HNSCC. As a consequence, targeted therapies against other molecules besides EGFR and VEGF family proteins were developed and their characterization is still ongoing. Among them, there are Src-family kinase inhibitors such as dosatinib; proteasome inhibitors as bortezomib, cyclooxygenase(Cox)-2 inhibitor (colecoxib); PI3K/Akt/mTOR inhibitors as wortmannin, perifostine and temsiroli‐

Briefly, Src-kinase inhibitor dasatinib promotes radiosensitization by decreasing EGFR phosphorylation, its translocation in the nucleus and consequently, its association with DNA– protein kinases, blocking DNA repair pathways [98, 99]. Evidences on proteasome inhibitor bortezomib show its capability to act as radiosensitizer; specifically, it promotes the upregu‐ lation of PTEN activity and downregulation of p-Akt, leading to an increase of apoptosis of tumor cells [100-102]. Cox inhibitor colecoxib leads to a decrease of VEGFR expression and angiogenesis [103]. Next, mTOR inhibitors cause a reduction of angiogenesis and an induction of cell death by autophagy [86, 104]. Finally, given the important role discussed in the paragraph 2 on the significance of CSCs subpopulation in radioresistance, an emerging concept is the combined use of standard chemoradiotherapy with cancer stem cells targeted therapy. Preclinical study on CD44 expressing HNSCC cells combine radiation with anti-CD44 antibodies; the results show an increase in local tumor control in patients treated with radiation plus anti-CD44 antibodies compared to those treated with radiation alone in vivo [105].

mus; and therapies targeting c-Met signaling pathway [14, 86] (Table 2).

apy treatment combination.

16 Contemporary Issues in Head and Neck Cancer Management

sorafenib in HNSCC patients.

**4.3. Other targeted therapies**

radiation or chemoradiation alone [13].


**Table 2.** List of molecular targeted therapies combined with radiotherapy under consideration for treatment of HNSCC patients (clinicaltrials.gov) [13, 86, 106].

#### **4.4. Therapy by reactivation or elimination of mutant p53 protein**

The vast majority of HNSCC show mutations in *TP53* gene; several evidences have shown that mutant p53 protein loses its function as tumor suppressor and acquires new oncogenic functions by which it promotes resistance to cisplatin and radiation treatment. The transfection of wild-type *TP53* into cell lines induces growth arrest and reduces tumorigenicity in nude mice. This suggested that restoring p53 function in HNSCC could inhibit cell growth [107]. HNSCC has been one of the first tumor localities to benefit from gene transfer therapy. Several strategies have been developed to restore p53 function in HNSCC [14, 108].

**Gene therapy**: The most used vector for p53 gene therapy in HNSCC is the adenovirus, for its high affinity with the cells of the upper aerodigestive tract. A series of modified p53 adenovi‐ ruses (Ad-p53) are able to induce apoptosis and sensitize HNSCC cells to radiotherapy [109, 110]. Therefore, a phase I/II clinical trial based on the injection of Ad-p53 in HNSCC patients was performed and has shown that Ad-p53 is a promising therapeutic strategy [111, 112]. A phase III study based on the comparison of Ad-p53 to methotrexate treatment in advanced HNSCC show that overall, there is no significant difference in clinical outcome between these two subgroups of treated HNSCC, but, interestingly, Ad-p53 treatment was associated with a significant increase of survival in specific subgroup of HNSCC patients, having *TP53* wild type but inactivated by the upregulation of p53 inhibitors Mdm-2 or Mdm-4 [113]. This evidence suggests the possibility to select HNSCC patients who are most likely to benefit from Ad-p53 therapy. Another phase III clinical trial based on the use of recombinant Ad-p53 (gendicine) injection in combination with radiation shows encouraging results [114].

**Virus targeting p53 deficient cells**: This therapeutic strategy is based on the elimination of mutant p53. The efficient replication of adenovirus requires the neutralization of p53 function through E1B viral protein. ONYX-015 is an engineered adenovirus that does not express E1B protein and consequently is able to induce viral replication and cell death only in tumor cells carrying *TP53* mutations. Phase I/II clinical trials performed in HNSCC patients have shown that intravenous administration of ONYX-15 is feasible and while the treatment with ONYX-15 alone gave only marginal effects, its combination with cisplatin and 5-fluorouracil had a more profound impact on the response of patients [115]. Other clinical trials will be necessary to evaluate its real effectiveness in HNSCC treatment.

**Molecules reactivating mutant p53:** They are small molecules able to alter the conformation of mutant p53 to wild type, leading to the restoration of its tumor suppression function. Among them, glycerol treatment is able to reactivate p53 wild-type functions in HNSCC cell lines carrying mutant p53 by its ability to refold proteins [116]. Due to its toxicity, glycerol use is not so feasible in HNSCC patients. As a consequence, a series of other similar molecules was developed. Among them, PRIMA and CP-31389 were tested in HNSCC cell lines carrying mutant p53 and have demonstrated to inhibit proliferation and promote apoptosis by the induction of p53-related genes expression, including p21, Bax, Puma and Noxa [117]. Cur‐ rently, there are no clinical data testing real effectiveness of these molecules in the treatment of HNSCC patients.

**Molecules disrupting p53 inhibitors**: In tumor cells, the function of p53 protein can be compromised not only by the presence of mutation on its gene, but also by upregulation of its inhibitors. The main p53 natural inhibitor is MDM2, which functions binding p53 protein and promoting its degradation. Nutlins and their derivate RITA are a class of small molecules able to prevent the binding MDM2-p53, restoring p53 tumor suppressor function. Therefore, Nutlins and RITA treatment leads to an increase of nuclear p53 levels, inhibition of prolifera‐ tion, increase of cell death and antitumor efficacy of cisplatin [108]. Therapy treatment based on these molecules is more effective in tumor cells carrying p53 wild-type compared with mutant p53-carrying cells.

In addition, in a subset of HPV-related HNSCC, the activity of p53 can be also inhibited by the exogenous viral oncoprotein E6. Specifically, it acts by interacting with E6AP protein to degrade p53 via proteasome pathway and with p300 to prevent p53 acetylation. Treatment of HNSCC cell lines with the small molecule CH1iB, disrupting the binding of E6 HPV16 protein and p300, promotes an increase of the p53 acetylation levels and therefore an increase of p53 transcriptional activity. Additionally, Ch1iB shows an anticancer effect also due to its capa‐ bility to reduce cancer stem cells population and by sensitizing tumor cells to cisplatin treatment in HPV positive cells [14, 108].

## **4.5. microRNAs as therapeutic agents**

of wild-type *TP53* into cell lines induces growth arrest and reduces tumorigenicity in nude mice. This suggested that restoring p53 function in HNSCC could inhibit cell growth [107]. HNSCC has been one of the first tumor localities to benefit from gene transfer therapy. Several

**Gene therapy**: The most used vector for p53 gene therapy in HNSCC is the adenovirus, for its high affinity with the cells of the upper aerodigestive tract. A series of modified p53 adenovi‐ ruses (Ad-p53) are able to induce apoptosis and sensitize HNSCC cells to radiotherapy [109, 110]. Therefore, a phase I/II clinical trial based on the injection of Ad-p53 in HNSCC patients was performed and has shown that Ad-p53 is a promising therapeutic strategy [111, 112]. A phase III study based on the comparison of Ad-p53 to methotrexate treatment in advanced HNSCC show that overall, there is no significant difference in clinical outcome between these two subgroups of treated HNSCC, but, interestingly, Ad-p53 treatment was associated with a significant increase of survival in specific subgroup of HNSCC patients, having *TP53* wild type but inactivated by the upregulation of p53 inhibitors Mdm-2 or Mdm-4 [113]. This evidence suggests the possibility to select HNSCC patients who are most likely to benefit from Ad-p53 therapy. Another phase III clinical trial based on the use of recombinant Ad-p53 (gendicine)

**Virus targeting p53 deficient cells**: This therapeutic strategy is based on the elimination of mutant p53. The efficient replication of adenovirus requires the neutralization of p53 function through E1B viral protein. ONYX-015 is an engineered adenovirus that does not express E1B protein and consequently is able to induce viral replication and cell death only in tumor cells carrying *TP53* mutations. Phase I/II clinical trials performed in HNSCC patients have shown that intravenous administration of ONYX-15 is feasible and while the treatment with ONYX-15 alone gave only marginal effects, its combination with cisplatin and 5-fluorouracil had a more profound impact on the response of patients [115]. Other clinical trials will be necessary to

**Molecules reactivating mutant p53:** They are small molecules able to alter the conformation of mutant p53 to wild type, leading to the restoration of its tumor suppression function. Among them, glycerol treatment is able to reactivate p53 wild-type functions in HNSCC cell lines carrying mutant p53 by its ability to refold proteins [116]. Due to its toxicity, glycerol use is not so feasible in HNSCC patients. As a consequence, a series of other similar molecules was developed. Among them, PRIMA and CP-31389 were tested in HNSCC cell lines carrying mutant p53 and have demonstrated to inhibit proliferation and promote apoptosis by the induction of p53-related genes expression, including p21, Bax, Puma and Noxa [117]. Cur‐ rently, there are no clinical data testing real effectiveness of these molecules in the treatment

**Molecules disrupting p53 inhibitors**: In tumor cells, the function of p53 protein can be compromised not only by the presence of mutation on its gene, but also by upregulation of its inhibitors. The main p53 natural inhibitor is MDM2, which functions binding p53 protein and promoting its degradation. Nutlins and their derivate RITA are a class of small molecules able to prevent the binding MDM2-p53, restoring p53 tumor suppressor function. Therefore, Nutlins and RITA treatment leads to an increase of nuclear p53 levels, inhibition of prolifera‐

strategies have been developed to restore p53 function in HNSCC [14, 108].

18 Contemporary Issues in Head and Neck Cancer Management

injection in combination with radiation shows encouraging results [114].

evaluate its real effectiveness in HNSCC treatment.

of HNSCC patients.

The role of microRNAs as predictors and modifiers of chemoradiotherapy in several kinds of human cancers, including HNSCC, has been shown [118]. For instance, miR-125b transfection on oral cancer cell lines enhances radiosensitivity to X-ray irradiation [80]. In addition, changes in the abundance of circulating miRNAs during radiochemotherapy has been detected and has been shown to reflect the therapy response of primary HNSCC cells after an in vitro treatment [82]. Finally, in our laboratory, we have demonstrated that the expression of signatures of *TP53* mutation-associated miRNAs, composed of 12 and 4 miRNAs, predicts, respectively, the risk of local recurrence insurgence and poor outcome, independently from other relevant prognostic indicators [42]. These evidences suggest the possibility of monitoring changes in miRNAs expression before to and during treatment in order to estimate the effectiveness of certain therapies. At the same time, another possibility for future application of miRNAs in therapy is the modulation of deregulated miRNAs concentration by molecules that replace downregulated miRNAs or using antagonists that binds overexpressed miRNAs [119]. Evidence supporting this possibility has been shown for the treatment of HCV infection; this phase II clinical study is based on the treatment of HCV infected patients with Miravirsen by which miR-122 is sequestered [120]. Miravirsen is the first miR-targeted drug to receive Investigation New Drug (IND) acceptance from FDA [121]. To date, there is only one clinical trial available in cancer patients; in particular, the treatment of liver cancer with MRX34, which is a molecule mimicking miR-34, is ongoing, in order to evaluate its maximum tolerated dose and its pharmacokinetics in patients [119].

#### **4.6. TRAIL and Smac mimetics molecules**

Recently, two classes of novel therapeutic agents targeting specific molecules involved in apoptosis pathway have emerged. The first one is the tumor necrosis factor-related apoptosisinducing ligand (TRAIL). It is able to induce cell death by binding to its corresponding cell surface receptor TRAIL-R1/R2 and activating the apoptotic pathways [122-124]. A second class of targeted anticancer agents is composed by Smac mimetics (SMs). They mimic the function of endogenous proapoptotic mitochondrial protein Smac/Diablo [125]. In response to a death stimulus, it is released in the cytoplasm and inhibits the antiapoptotic activity of IAP proteins [126]. Both TRAIL and SMs have been tested in several cancer models [123, 125, 127]. A study testing the sensitivity to TRAIL and SMs treatment on HNSCC cell lines show that both molecules are highly effective in killing tumor cells. In addition, caspase 8 and TNF-α expres‐ sion was identified as biomarker for predicting, respectively, TRAIL and SMs sensitivity [128]. These preliminary results encourage future investigations on the possibility to use them as targeted HNSCC treatment.

#### **4.7. Therapeutic activity of molecules derived from plants**

Antineoplastic effects of molecules derived from plant extracts have recently gained increasing attention as an additive to traditional therapies of cancer, including HNSCC.

One of the most studied molecules derived from plants for HNSCC treatment is curcu‐ min (diferuloylmethane). It is a polyphenol derived from the *Curcuma longa* plant, common‐ ly known as turmeric. Curcumin, which has been used extensively in Ayurvedic medicine for centuries, is a pleiotropic molecule able to interact with multiple molecular targets and signal transduction pathways, and has a variety of therapeutic properties, including antioxidant, analgesic, anti-inflammatory and antiseptic activity [15]. More recently, curcumin has been found to possess anti-cancer activities, acting on several biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigen‐ esis and metastasis [15]. For instance, it is able to inhibit the transcription factor NF-kB and downstream gene products (including c-myc, bcl-2, COX-2, NOS, cyclin D1, TNF-alpha, interleukins and MMP-9). Additionally, curcumin affects a variety of growth factor receptors and cell adhesion molecules involved in tumor growth, angiogenesis and metastasis [15]. As a natural product, curcumin is no toxic. It is a potent antitumor agent also in HNSCC and can be used to overcome chemoradiotherapy resistance. Indeed, the treatment of HNSCC cell lines with a molecule derived from curcumin (H-4073) inhibits cell prolifera‐ tion, angiogenesis and significantly sensitizes the cells to cisplatin treatment. H-4073 mediated its antitumor effects by inhibiting JAK/STAT3, FAK, Akt and VEGF signaling pathways that play important role in cell proliferation, migration, survival and angiogene‐ sis [129]. Another study shows that curcumin sensitizes to radiation HPV-negative HNSCC cells with high levels of Thioredoxin reductase (TrxRs). Indeed, in this work it has been demonstrated that the efficacy of curcumin in sensitizing tumor cells to radiation de‐ pends on its ability to inhibit TrxRd1. TrxRs are a family of NADPH-dependent flavopro‐ teins, which are involved in several redox-regulated cellular functions as transcription, DNA repair, proliferation, angiogenesis and apoptosis. Specifically, high levels of TrxRd1 isoform were found in HNSCC and were associated with poor outcome [130]. Finally, data from a very recent study shows that curcumin is more effective, in terms of inhibition of cancer growth, when combined with another non-flavonoid polyphenol called Resveratrol [131].

Another intriguing natural anticancer Chinese medicine is Gamboge. It acts as antiinflammatory agent, detoxifying and apoptotic inducer in different type of cancer cells. Interestingly, the Gamboge derivate Compound 2 (C2) is able to inhibit growth also in HNSCC stem cells. Indeed, it can inhibit formation of tumor spheres and repress the

expression of multiples genes related to cancer stem cell phenotype by blocking the activation of EGFR pathways [132]. Since one of the main causes of failure in HNSCC treatment is the enrichment of CSCs population, which are resistant to current therapy, the future use of this molecule in combination with chemoradiotherapy could prevent the selective enrichment of CSCs after HNSCC conventional treatment.

## **5. Conclusions**

[126]. Both TRAIL and SMs have been tested in several cancer models [123, 125, 127]. A study testing the sensitivity to TRAIL and SMs treatment on HNSCC cell lines show that both molecules are highly effective in killing tumor cells. In addition, caspase 8 and TNF-α expres‐ sion was identified as biomarker for predicting, respectively, TRAIL and SMs sensitivity [128]. These preliminary results encourage future investigations on the possibility to use them as

Antineoplastic effects of molecules derived from plant extracts have recently gained increasing

One of the most studied molecules derived from plants for HNSCC treatment is curcu‐ min (diferuloylmethane). It is a polyphenol derived from the *Curcuma longa* plant, common‐ ly known as turmeric. Curcumin, which has been used extensively in Ayurvedic medicine for centuries, is a pleiotropic molecule able to interact with multiple molecular targets and signal transduction pathways, and has a variety of therapeutic properties, including antioxidant, analgesic, anti-inflammatory and antiseptic activity [15]. More recently, curcumin has been found to possess anti-cancer activities, acting on several biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigen‐ esis and metastasis [15]. For instance, it is able to inhibit the transcription factor NF-kB and downstream gene products (including c-myc, bcl-2, COX-2, NOS, cyclin D1, TNF-alpha, interleukins and MMP-9). Additionally, curcumin affects a variety of growth factor receptors and cell adhesion molecules involved in tumor growth, angiogenesis and metastasis [15]. As a natural product, curcumin is no toxic. It is a potent antitumor agent also in HNSCC and can be used to overcome chemoradiotherapy resistance. Indeed, the treatment of HNSCC cell lines with a molecule derived from curcumin (H-4073) inhibits cell prolifera‐ tion, angiogenesis and significantly sensitizes the cells to cisplatin treatment. H-4073 mediated its antitumor effects by inhibiting JAK/STAT3, FAK, Akt and VEGF signaling pathways that play important role in cell proliferation, migration, survival and angiogene‐ sis [129]. Another study shows that curcumin sensitizes to radiation HPV-negative HNSCC cells with high levels of Thioredoxin reductase (TrxRs). Indeed, in this work it has been demonstrated that the efficacy of curcumin in sensitizing tumor cells to radiation de‐ pends on its ability to inhibit TrxRd1. TrxRs are a family of NADPH-dependent flavopro‐ teins, which are involved in several redox-regulated cellular functions as transcription, DNA repair, proliferation, angiogenesis and apoptosis. Specifically, high levels of TrxRd1 isoform were found in HNSCC and were associated with poor outcome [130]. Finally, data from a very recent study shows that curcumin is more effective, in terms of inhibition of cancer growth, when combined with another non-flavonoid polyphenol called Resveratrol [131]. Another intriguing natural anticancer Chinese medicine is Gamboge. It acts as antiinflammatory agent, detoxifying and apoptotic inducer in different type of cancer cells. Interestingly, the Gamboge derivate Compound 2 (C2) is able to inhibit growth also in HNSCC stem cells. Indeed, it can inhibit formation of tumor spheres and repress the

attention as an additive to traditional therapies of cancer, including HNSCC.

targeted HNSCC treatment.

20 Contemporary Issues in Head and Neck Cancer Management

**4.7. Therapeutic activity of molecules derived from plants**

Radioresistance strongly affects the clinical outcome of HNSCC patients. The key mechanisms by which radioresistance occur have been associated with deregulation of several molecular signaling pathways such as EGFR, VEGFR and p53. Recently, it has been shown that the enrichment of a small population of tumor cells, named cancer stem cells, also plays an important role in the failure of conventional HNSCC treatment. In addition, current treatments are associated with high toxicity and side effects. The basis of treatment decisions are mainly based on TNM staging, but patients with the same staging have different response to therapy. Several molecular targeted therapies are actively under investigation in order to improve the effectiveness of current therapy. Only a few of these strategies have been tested in clinical trials and to date cetuximab is the unique targeted therapy approved from FDA. However, this treatment showed efficacy in about 20% of HNSCC patients. In addition, due to the heteroge‐ neous nature of these tumors, the study of molecular prognostic and predictive factors has been motivated by the necessity to predict radiosensitivity of patients and to define more homogenous groups of patients for treatment selection. Indeed, personalized treatment plans based on biomarkers could improve overall survival and reduce morbidity. Although several evidences have shown that many molecules, as proteins and microRNAs, can potentially predict response to therapy and clinical outcome, to date, the HNSCC treatment decision is uniquely based on TNM staging and HPV infection. One of the reasons of the difficulties to find efficacious biomarkers is the disagreement between these studies; this mainly relies on the variety of tumor sites, sensitivity of the techniques used, quality of the specimens studied and the arbitrary cut-off values set.

## **Author details**

Federica Ganci1 , Andrea Sacconi1 , Valentina Manciocco1 , Giuseppe Spriano1 , Giulia Fontemaggi1 , Paolo Carlini2 and Giovanni Blandino1\*

\*Address all correspondence to: blandino@ifo.it

1 Translational Oncogenomics Unit, National Cancer Institute "Regina Elena", Rome, Italy

2 Medical Oncology A, National Cancer Institute "Regina Elena", Rome, Italy

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## **The Six Rs of Head and Neck Cancer Radiotherapy**

Loredana G. Marcu, Iuliana Toma Dasu and Alexandru Dasu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60015

## **1. Introduction**

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34 Contemporary Issues in Head and Neck Cancer Management

Locally advanced head and neck cancers are usually aggressive tumours, due to the presence of hypoxia and the ability of the tumour to repopulate during treatment. The aggressive behaviour generally requires aggressive treatment, which for head and neck carcinomas consist of altered radiotherapy fractionation schedules combined with chemotherapy. Treatment fractionation, based on the 4 Rs of radiotherapy [1] is a well-accepted concept that has been re-adjusted for head and neck cancer decades ago to accommodate new radiobio‐ logical findings. The 4 Rs in terms of *repair, repopulation, reoxygenation, and redistribution* along the cell cycle have been promoted to 5 Rs with the aid of *radiosensitivity* and more recently to 6 Rs with the experimental evidence of *remote* (bystander) *cellular effects*.

The paragaphs below aim to describe the major aspects concerning the six Rs of radiotherapy applied to head and neck cancer.

## **2. The 5 Rs revisited**

The 5 Rs of radiobiology represent a group of processes determining the response of cells and tissues to radiation, with great impact in fractionated irradiation. Balancing them one against the other has become one of the pillars of modern radiation therapy to maximise the therapeutic gain by either increasing radiation damage in the tumour cells or decreasing the damage to the normal tissues. The impact of the individual Rs varies across the different tissues, but for head and neck tumours all of them play important roles that have to be taken into account when designing successful radiotherapy schedules.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

#### **2.1. Repair**

Repair is a term covering a number of processes responsible for the identification and correc‐ tion of the damage to the DNA molecule induced by endogenous and exogenous factors. Sometimes the term "recovery" is used instead of "repair" because other processes are involved besides the actual repair of damage. The importance of recovery or repair in relation to irradiation has been determined in a series of experiments employing continuous irradiation at various dose-rates or split-dose experiments studying the variation of the cell survival with irradiation time or the time between individual fractions of radiation. Following these types of experiments radiation damage has been divided into two categories, non-repairable or lethal damage and repairable or sublethal damage. It should be remarked that these are operational terms that have not been correlated with any type of radiation-induced damage, with the possible exception of the irreparable clusters of damage. Sublethal damage could be removed as part of the cell recovery, unless fixated by interaction with other sublethal lesions or by being forced to be expressed by the cells. Consequently, cell survival following irradiation depends not only on the creation of irreparable lesions, but also on the competition between repair and fixation of sublethal damage that is influenced by many factors including the rate of generating new lesions and the cellular environment.

When describing the effect of radiation with the help of the well-known cell survival curves, the repairable component of damage is considered responsible for the shoulder of the curve as the accumulation of damage in the absence of the repair increases cell kill. Consequently the recovery capacity of cells could be quantified by relating the "bendiness" of the cell survival curve to its initial slope that is a measure of the irreparable or lethal damage induced by radiation. For the linear-quadratic (LQ) model [2,3] this is given by the beta/alpha ratio, which is low for acutely reacting normal tissues and most tumours including head and neck tumours and high for late reacting tissues [4]. This indicates the high potential for recovery of late reacting tissue that should be exploited in head and neck radiotherapy to limit the late complication rates.

Several models have been proposed to account for the process of recovery, including the incomplete repair model [5], the lethal-potentially-lethal model [6] or the repairable-condi‐ tionally repairable damage model [7], with the incomplete repair model, based itself on the LQ formalism, being mostly known and employed in clinical practice. According to the incomplete repair model [5], the effect of continuous irradiation delivering radiation dose *D* in time *t* is given by equation 1.

$$E = \alpha \dot{d}t + \chi(t)\beta (\dot{d}t)^2 \tag{1}$$

where *α* and *β* are parameters of the LQ model and *g* is a function describing the repair taking place in time *t*. If the repair is described as an exponential process, with repair constant *μ*, the *g* term is described by the expression in equation 2.

$$\log\left(\mu t\right) = 2\frac{\left[\mu t \cdot 1 + \exp\left(-\mu t\right)\right]}{(\mu t)^2} \tag{2}$$

Similar expressions could be derived for the case of fractionated irradiation, when the focus is on the repair taking place between the individual fractions. Thus, the effect in fractionated radiation with time *t* between fractions is given by the expression in equation 3.

**2.1. Repair**

36 Contemporary Issues in Head and Neck Cancer Management

complication rates.

in time *t* is given by equation 1.

*g* term is described by the expression in equation 2.

Repair is a term covering a number of processes responsible for the identification and correc‐ tion of the damage to the DNA molecule induced by endogenous and exogenous factors. Sometimes the term "recovery" is used instead of "repair" because other processes are involved besides the actual repair of damage. The importance of recovery or repair in relation to irradiation has been determined in a series of experiments employing continuous irradiation at various dose-rates or split-dose experiments studying the variation of the cell survival with irradiation time or the time between individual fractions of radiation. Following these types of experiments radiation damage has been divided into two categories, non-repairable or lethal damage and repairable or sublethal damage. It should be remarked that these are operational terms that have not been correlated with any type of radiation-induced damage, with the possible exception of the irreparable clusters of damage. Sublethal damage could be removed as part of the cell recovery, unless fixated by interaction with other sublethal lesions or by being forced to be expressed by the cells. Consequently, cell survival following irradiation depends not only on the creation of irreparable lesions, but also on the competition between repair and fixation of sublethal damage that is influenced by many factors including the rate

When describing the effect of radiation with the help of the well-known cell survival curves, the repairable component of damage is considered responsible for the shoulder of the curve as the accumulation of damage in the absence of the repair increases cell kill. Consequently the recovery capacity of cells could be quantified by relating the "bendiness" of the cell survival curve to its initial slope that is a measure of the irreparable or lethal damage induced by radiation. For the linear-quadratic (LQ) model [2,3] this is given by the beta/alpha ratio, which is low for acutely reacting normal tissues and most tumours including head and neck tumours and high for late reacting tissues [4]. This indicates the high potential for recovery of late reacting tissue that should be exploited in head and neck radiotherapy to limit the late

Several models have been proposed to account for the process of recovery, including the incomplete repair model [5], the lethal-potentially-lethal model [6] or the repairable-condi‐ tionally repairable damage model [7], with the incomplete repair model, based itself on the LQ formalism, being mostly known and employed in clinical practice. According to the incomplete repair model [5], the effect of continuous irradiation delivering radiation dose *D*

where *α* and *β* are parameters of the LQ model and *g* is a function describing the repair taking place in time *t*. If the repair is described as an exponential process, with repair constant *μ*, the

*<sup>g</sup>*(*μt*)=2 *μt* - <sup>1</sup> <sup>+</sup> *exp*(-*μt*)

*E* =*αd*˙*t* + *g*(*t*)*β*(*d*˙*t*)<sup>2</sup> (1)

(*μt*)2 (2)

of generating new lesions and the cellular environment.

$$E = n \Box \alpha d + h \begin{pmatrix} t \end{pmatrix} \beta d^2 \tag{3}$$

where *n* is the number of fractions, *d* is the dose per fraction and the repair term *h(t)* is given by the expression in equation 4.

$$\ln\left(t\right) = \frac{2}{n} \frac{\exp\left(-\mu t\right)}{1 - \exp\left(-\mu t\right)} \left[\ln - \frac{1 - \exp\left(-n\mu t\right)}{1 - \exp\left(-\mu t\right)}\right] \tag{4}$$

These expressions could be used together with the Biological Effective Dose (BED) formalism [8,9] to calculate the effectiveness of various treatment approaches. Thus, the expressions in equations 1 and 2 are mostly suited for brachytherapy, while expressions in equations 3 and 4 are suited for fractionated therapy when intra-fraction repair is considered negligible. Expressions have also been derived to account simultaneously for intra and inter-fraction repair [10].

A number of studies have determined relevant repair constants from clinical and experimental data, yielding values between 0.5 and 2 h for the repair half-life, depending on the tissue and the type of experiment used to derive them [11]. More recent studies indicated that the rate of repair might depend on the dose or that second order or bi- or even multi-exponential processes might exist [12-15]. The multi-exponential processes appear to be equally divided between the fast and the slow components with median repair half-lives of 0.3 h for the fast component and about 4 h for the slow component (for a summary see [16]). In this context it has been pointed out that identification of the relevant rates might depend on the design of the experiment as it has been suggested that split-dose experiments could easily miss a fast component of repair [16].

Repair or recovery of radiation damage has been extensively exploited to spare late reacting normal tissues in head and neck radiation therapy. Thus, the low alpha/beta ratio (or high beta/ alpha ratio) of these tissues in comparison to the tumours indicates their high capacity for repair in fractionated regimes if enough time is allowed between individual fractions for the recovery of sublethal damage. Consequently, decreasing the dose per fraction can protect late reacting normal tissues more than the tumour cells, this differential effect allowing an escalation of the dose to the tumours in comparison to conventional fractionation or a decrease in the expected complication rates to maximise the therapeutic gain. Indeed, several clinical studies initiated in the 1980s and 1990s have shown that increased fractionation in head and neck radiotherapy could increase the control rates for the same levels of complications or could even reduce the complication rates. Thus, Bourhis *et al* [17] performed a meta-analysis of hyperfractionated trials in head and neck cancer, and reported a significant benefit from hyperfractionation than with conventional fractionation on survival (8% at 5 years) and on locoregional control (6.4% at 5 years).

Hyperfractionated regimes employing very small fractions required that they are given twice or three times per day to keep the overall treatment times to manageable lengths that avoid problems from tumour repopulation (see section II.2). Some of these treatment schedules highlighted the clinical implications of not allowing enough time between fractions for complete recovery of sublethal damage. One of the most known examples is the Continuous Hyperfractionated Accelerated Radiotherapy (CHART) trial delivering 54 Gy in 36 fractions in 12 consecutive days [18]. While the trial demonstrated a similar level of local control with 66 Gy in 33 daily fractions for a significant reduction in late treatment-related morbidity, the reduction was much less than expected from BED calculations. Analyses of the complication rates in the conventional and hyperfractionated arms showed that the repair half-life might indeed be 4-5 h, which is quite long for the 6 h interfraction intervals in the hyperfractionated arm of CHART [19]. The high amount of residual damage after the slow component of recovery might also explain why twice daily fractionation schemes with 2 Gy per fraction sometimes led to too high rates of late effects [20]. Nevertheless, twice daily fractionation regimes employing 1.2 or even 1.6 Gy per fraction have been safely used for treatment [21-24] and illustrate how the differential recovery potential of late and acutely reacting tissue could be exploited to increase the therapeutic potential in the radiation therapy of head and neck cancers.

In fact the potential for improvement of any fractionation scheme employing *n* fractions of size *d* could be evaluated using the BED formalism. Thus, the biologically equivalent of total dose in 2 Gy fractions (EQD) could be derived for the effects in late reacting tissues using an alpha/ beta ratio of 3 Gy (equation 5) and in tumours and acutely reacting tissues using an alpha/beta of 10 Gy (equation 6).

$$EQD\_3 = nd \frac{1 + \frac{d}{3}}{1 + \frac{2}{3}} \tag{5}$$

$$EQD\_{10} = \nu d \frac{1 + \frac{d}{10}}{1 + \frac{2}{10}} \tag{6}$$

where1 <sup>+</sup> <sup>2</sup> <sup>3</sup> is the relative effectiveness of conventional fractionation regimes in late reacting tissues and 1 <sup>+</sup> <sup>2</sup> <sup>10</sup> is the relative effectiveness of conventional fractionation regimes in acutely reacting tissues and tumours. The expressions could be adapted to include the effect of incomplete recovery between fractions (equations 3 and 4) or for protracted irradiation (equations 1 and 2). In fact, the loss of effects due to protracted irradiation has been a cause of concern in some applications like intensity modulated radiotherapy (IMRT), although this loss might not be significant as long as the delivery time is shorter than the half-life of the quick component of recovery [16].

#### **2.2. Repopulation**

Hyperfractionated regimes employing very small fractions required that they are given twice or three times per day to keep the overall treatment times to manageable lengths that avoid problems from tumour repopulation (see section II.2). Some of these treatment schedules highlighted the clinical implications of not allowing enough time between fractions for complete recovery of sublethal damage. One of the most known examples is the Continuous Hyperfractionated Accelerated Radiotherapy (CHART) trial delivering 54 Gy in 36 fractions in 12 consecutive days [18]. While the trial demonstrated a similar level of local control with 66 Gy in 33 daily fractions for a significant reduction in late treatment-related morbidity, the reduction was much less than expected from BED calculations. Analyses of the complication rates in the conventional and hyperfractionated arms showed that the repair half-life might indeed be 4-5 h, which is quite long for the 6 h interfraction intervals in the hyperfractionated arm of CHART [19]. The high amount of residual damage after the slow component of recovery might also explain why twice daily fractionation schemes with 2 Gy per fraction sometimes led to too high rates of late effects [20]. Nevertheless, twice daily fractionation regimes employing 1.2 or even 1.6 Gy per fraction have been safely used for treatment [21-24] and illustrate how the differential recovery potential of late and acutely reacting tissue could be exploited to increase the therapeutic potential in the radiation therapy of head and neck

38 Contemporary Issues in Head and Neck Cancer Management

In fact the potential for improvement of any fractionation scheme employing *n* fractions of size *d* could be evaluated using the BED formalism. Thus, the biologically equivalent of total dose in 2 Gy fractions (EQD) could be derived for the effects in late reacting tissues using an alpha/ beta ratio of 3 Gy (equation 5) and in tumours and acutely reacting tissues using an alpha/beta

reacting tissues and tumours. The expressions could be adapted to include the effect of incomplete recovery between fractions (equations 3 and 4) or for protracted irradiation (equations 1 and 2). In fact, the loss of effects due to protracted irradiation has been a cause of concern in some applications like intensity modulated radiotherapy (IMRT), although this loss might not be significant as long as the delivery time is shorter than the half-life of the quick

<sup>3</sup> is the relative effectiveness of conventional fractionation regimes in late reacting

<sup>10</sup> is the relative effectiveness of conventional fractionation regimes in acutely

(5)

(6)

*EQD*<sup>3</sup> =*nd*

*EQD*<sup>10</sup> =*nd*

cancers.

where1 <sup>+</sup> <sup>2</sup>

tissues and 1 <sup>+</sup> <sup>2</sup>

component of recovery [16].

of 10 Gy (equation 6).

Besides repair, repopulation during treatment is another important factor that could modulate the response to fractionated regimes. Indeed, as the effects in radiotherapy are related to the inactivation of cells from tumours and normal tissues, any proliferative process taking place during treatment will increase the cell population and consequently diminish the effect of radiation therapy. The effect would therefore depend on the time available for proliferation, i.e., the overall treatment time, and will particularly be a problem for acutely reacting tissues and tumours that have significant proliferation. In late reacting tissues in contrast, proliferative activity is minimal during the few days or weeks required by most treatment schedules and the treatment duration will not influence the complication rates.

The existence of a time factor in clinical radiation therapy was recognised quite early and gave rise to the Strandquist plots [25] and the nominal standard dose (NSD) concept [26] attempting to relate the time, dose and fractionation of equivalent fractionation regimes. However, an important breakthrough linking the time factor to the proliferation of tumour cells was made following the publication by Withers and colleagues of a study analysing the total dose needed to achieve 50% control for squamous tumours of the head and neck as a function of the overall treatment time [27]. The results showed that there was an increase of the required dose at a rate of 0.5-0.6 Gy per day for treatments lasting more than about 21-28 days, which was attributed to accelerated proliferation taking place in tumours after the lag time. This report was accompanied by the development of the BED concept with proliferation [9]. Thus, the effect of proliferation with doubling time *Tp* for a treatment with *n* fractions of size *d* being delivered in time *T* could be accounted by equation 7

$$BED = nd \left[ 1 + \frac{d}{\alpha/\beta} \right] \cdot \frac{\ln \left( 2 \right)}{\alpha T\_p} \left( T - T\_k \right) \tag{7}$$

where *Tk* is the delay in the onset of proliferation. The equation could be further adapted to include the effects of repair as described in equations 1-4.

A series of clinical studies have investigated the impact of overall treatment time and prolif‐ eration on the outcome of radiation therapy for head and neck tumours. Studies investigating only the impact of acceleration, i.e., the delivery of more than 10 Gy per week that is the norm in conventional fractionation delivered in 5 daily fractions per week, have reported better control, but also an increase in acute reactions and sometimes an increase in late reactions when the interfraction interval was too short to allow full repair of sublethal damage [20,28,29]. More successful were schedules combining hyperfractionation and acceleration by delivering two or more fractions per day [18,22,30,31]. Nevertheless, an analysis performed by Bourhis and colleagues found a small but statistically significant benefit of 2% on survival at 5 years from acceleration itself in comparison to conventional fractionation [17].

The overall treatment time analysis of head and neck tumours indicates proliferation doubling time of the order of 3 to 5 days. This corresponds to the values determined from measurements of the potential doubling time of tumours, *Tpot* [32]. Whether *Tp* in equation 7 is *Tpot* is still a matter of debate. Experimental studies have shown that the effective doubling time for proliferation during radiotherapy could be either smaller or larger than *Tpot* [33]. A large multicentre analysis also failed to correlate experimental determinations of *Tpot* with treatment outcome in head and neck cancers [34]. However, a more recent analysis has shown that pretreatment proliferation parameters are better predictors of outcome when other factors like tumour size, individual radiosensitivity and overall treatment time are taken into account [35].

The increase in acute reactions when shortening the overall treatment time indicates that compensatory proliferation is also part of the mechanisms available for the rapidly prolifer‐ ating normal tissues to recover from radiation damage. However, it has been found that the kinetic parameters for acute reactions are significantly different from those from tumours [36]. Thus, acute mucosal reactions that may become a limiting factor in the radiotherapy of head and neck tumours have a *Tk* of 7 days and a *Tp* of 2.5 days. This difference has given the opportunity to search for an optimum overall treatment time that maximises tumour effect, without jeopardising the function of late and acute normal tissues [37,38].

Three mechanisms have been proposed to be behind accelerated proliferation, namely asymmetry loss and acceleration of divisions of the stem cell compartment, as well as abortive divisions of sterilised cells [39]. Recruitment of quiescent cells into the cell cycle has also been proposed. The molecular triggers for these mechanisms remain to be elucidated [40], although there are some indications that epidermal growth factor receptor (EGFR) and protein tyrosine phosphatase (PTEN) activation might be involved [41-44].

Ample modelling of the aforementioned proliferation mechanisms has been undertaken in order to quantify the extent of repopulation during treatment, to study the individual contri‐ bution of each mechanism as well as their interplay towards overall tumour repopulation [45,46]. It was shown that while cell recruitment does contribute towards repopulation to a small extent, the major mechanism responsible for accelerated proliferation of tumour cells during radiotherapy is the asymmetry loss of stem cell division.

#### **2.3. Reoxygenation**

Tumour oxygenation is known to be one of the main factors that determine the response to radiotherapy. For advanced head and neck cancer in particular, clinical trials have shown that pre-treatment polarographic measurements of tumour oxygenation indicating the presence of tumour hypoxia correlate with poor prognosis [47]. This clinical evidence of the role of tumour hypoxia in determining the outcome of the treatment has been further confirmed by several studies in which pre-treatment uptake of nitroimidazole compounds such as 18F-Fluoromiso‐ nidazole (18F-MISO) or Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) used as Positron Emission Tomography (PET) hypoxia imaging agents was shown to predict the outcome in head and neck cancer radiotherapy [48]. Furthermore, pre-treatment tumour hypoxia does not only correlate with poor local control due to the presence of resistant cells to radiotherapy, but also to chemotherapy and poor long time prognosis because of the locoregional spread and formation of distant metastases [49].

The mechanism of resistance of tumour hypoxic cells to radiation can be explained by the socalled oxygen effect related to the oxygen actions at the level of the free radicals formed after the interaction of charged particles with biological material. The free radicals, which are highly reactive molecules because of their unpaired valence electron, are responsible for the break of the DNA chemical bonds which might be further made permanent by molecular oxygen. The resulting biological damage depends thus on the presence or absence of oxygen, welloxygenated cells being more sensitive to radiation induced damage than hypoxic cells deprived of oxygen.

proliferation during radiotherapy could be either smaller or larger than *Tpot* [33]. A large multicentre analysis also failed to correlate experimental determinations of *Tpot* with treatment outcome in head and neck cancers [34]. However, a more recent analysis has shown that pretreatment proliferation parameters are better predictors of outcome when other factors like tumour size, individual radiosensitivity and overall treatment time are taken into account [35].

The increase in acute reactions when shortening the overall treatment time indicates that compensatory proliferation is also part of the mechanisms available for the rapidly prolifer‐ ating normal tissues to recover from radiation damage. However, it has been found that the kinetic parameters for acute reactions are significantly different from those from tumours [36]. Thus, acute mucosal reactions that may become a limiting factor in the radiotherapy of head and neck tumours have a *Tk* of 7 days and a *Tp* of 2.5 days. This difference has given the opportunity to search for an optimum overall treatment time that maximises tumour effect,

Three mechanisms have been proposed to be behind accelerated proliferation, namely asymmetry loss and acceleration of divisions of the stem cell compartment, as well as abortive divisions of sterilised cells [39]. Recruitment of quiescent cells into the cell cycle has also been proposed. The molecular triggers for these mechanisms remain to be elucidated [40], although there are some indications that epidermal growth factor receptor (EGFR) and protein tyrosine

Ample modelling of the aforementioned proliferation mechanisms has been undertaken in order to quantify the extent of repopulation during treatment, to study the individual contri‐ bution of each mechanism as well as their interplay towards overall tumour repopulation [45,46]. It was shown that while cell recruitment does contribute towards repopulation to a small extent, the major mechanism responsible for accelerated proliferation of tumour cells

Tumour oxygenation is known to be one of the main factors that determine the response to radiotherapy. For advanced head and neck cancer in particular, clinical trials have shown that pre-treatment polarographic measurements of tumour oxygenation indicating the presence of tumour hypoxia correlate with poor prognosis [47]. This clinical evidence of the role of tumour hypoxia in determining the outcome of the treatment has been further confirmed by several studies in which pre-treatment uptake of nitroimidazole compounds such as 18F-Fluoromiso‐ nidazole (18F-MISO) or Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) used as Positron Emission Tomography (PET) hypoxia imaging agents was shown to predict the outcome in head and neck cancer radiotherapy [48]. Furthermore, pre-treatment tumour hypoxia does not only correlate with poor local control due to the presence of resistant cells to radiotherapy, but also to chemotherapy and poor long time prognosis because of the loco-

The mechanism of resistance of tumour hypoxic cells to radiation can be explained by the socalled oxygen effect related to the oxygen actions at the level of the free radicals formed after

without jeopardising the function of late and acute normal tissues [37,38].

phosphatase (PTEN) activation might be involved [41-44].

40 Contemporary Issues in Head and Neck Cancer Management

during radiotherapy is the asymmetry loss of stem cell division.

regional spread and formation of distant metastases [49].

**2.3. Reoxygenation**

Given the clinically proven impact of the presence of hypoxia on the treatment outcome, it is important to investigate the mechanisms of the occurrence of tumour hypoxia and its dynam‐ ics. The impaired oxygen supply to tumour cells leading to the formation of tumour hypoxia originates in the particularities of the tumour vasculature formed mainly through parasitation of the normal tissue vasculature and angiogenesis. Consequently, the major mechanisms involved in the formation of hypoxia are related to either the actual architecture of the blood vessels and the diffusion-limited delivery of oxygen, or to the functional abnormalities of tumour capillaries leading to perfusion limitations [49]. The two main forms of hypoxia associated with them are known as chronic (diffusion-limited) or acute (perfusion-limited) hypoxia. Thus, chronic hypoxia will occur when the distance from the cells to the nearer capillaries is close to exceeding the maximum oxygen diffusion distance, which under normal rates of oxygen consumption by the cells is expected to be in the order of 100-150 μm as shown by the early studies of Thomlinson and Gray [50] and confirmed later by experimental and modelling studies [51-53]. Acute hypoxia arises near the blood vessels temporary occluded, and, by its nature, has a transient character, unless the blood vessels remain blocked a long time period, depriving the cells of oxygen beyond the limit for survival.

For head and neck squamous cell carcinomas (HNSCC) in particular, which appear to be formed from nonvascularized epithelium, relatively hypoxic under normal conditions, hypoxia might pose particular reasons of concern. Thus, heterogeneous distributions of oxygen throughout the cellular microenvironment are expected in HNSCC. The impairment in the oxygen supply is not only spatially but also temporally heterogeneous.

Regardless the mechanism through which hypoxia occurs in tumours, there is a general consensus correlated to the clinical evidence that hypoxia is a negative predictive factor for the treatment and that patients might benefit from treatment strategies adapted according to the oxygenation of their individual tumours. However, it has also been suggested that chronically and acutely hypoxic cells might respond differently to radiation on the grounds of their energy supply and viability. It is well known that the combined high rate of glycolytic metabolism and poor availability of glucose result in low energy reserves for tumour cells reflected by the relative levels of ATP, ADP, AMP, Pi and PCr [54, 55]. The energy supply of chronically hypoxic cells, however, appears to decrease after a couple of hours of glucose deprivation [56] while the energy of well oxygenated cells does not decrease significantly under glucose deprivation. Therefore, one could postulate that chronically hypoxic cells are less capable to activate their DNA repair mechanisms and therefore would be more radia‐ tion sensitive compared to the acutely hypoxic cell [57] and quote in support studies on nutrient deprived cells [58,59]. Consequently, particular attention has to be paid to the cells at intermediate oxygen levels which might possess a dangerous combination of viability and partial radioresistance that might in turn be reflected in the poor outcome to radiation treatment [60,61].

The oxygen status of the tumour cells is however not static with respect to both spatial and temporal patterns. Changes in the cellular oxygenation related to the dynamics of both chronic and acute hypoxia are generally known as tumour reoxygenation.

Reoxygenation manifests itself following two main patterns. Temporal heterogeneity in oxygenation arises in relation to acute or perfusion-limited hypoxia. Abnormal vasculature can lead to fluctuations in the blood flow due to the temporary occlusion or even backflow. These phenomena have a rather chaotic but transient character and are conventionally referred to as fast reoxygenation [62] although in some cases the change in oxygen supply might in fact be from poor to well and back to poor, thus not necessarily leading to an improvement of the oxygenation of the cells but rather to a re-hypoxiation. The temporal scale of changes in oxygenation related to acute, perfusion limited hypoxia, ranges from minutes to hours as demonstrated by several experiments using sequential injection of different fluorescent dyes for hypoxia and vascular perfusion [63,64]. Furthermore, in presence of irradiation, the dynamics of the oxygenation might be even more pronounced as shown in a study on human laryngeal squamous cell carcinoma tumour line grown as xenografts in nude mice by Bussink *et al* [51] indicating that irradiation could lead to rapid changes in oxygenation and perfusion. Chronically hypoxic cells might also change their oxygenation status during the course of fractionated therapy through the so-called slow reoxygenation. In a mixed tumour cell population with respect to oxygenation, ionizing radiation will primarily kill the sensitive well-oxygenated cells. This would result in lower oxygen consumption and hence to larger distances of oxygen diffusion which independently or in conjunction to overall tumour shrinkage might lead to the improvement of the tumour oxygenation by reoxygenation of the chronically hypoxic cells. Furthermore, during long, fractionated, radiotherapy treatments, extending over several weeks, revascularisation of the tumour through angiogenesis might also occur resulting in the reoxygenation of cells that were chronically hypoxic.

Taking advantage of the changes in tumour oxygenation and expecting that they will result in an improvement of radiosensitivity by fractionating the dose and thus increasing the treatment duration is one of the first approaches clinically used for overcoming tumour hypoxia. However, the search for the optimal dose per fraction and number of fractions in which the treatment has to be delivered is far from being over considering that one has to find the right balance between the fractional dose that might overcome hypoxia and the number of fractions that will ensure proper reoxygenation accounting at the same time for the normal tissue and organs at risk.

There are several clinical studies indicating that the oxygenation of head and neck tumours is indeed dynamic. The early studies of changes in tumour oxygenation in advanced head and neck carcinoma using polarographic electrodes were inconclusive in proving that reoxygena‐ tion positively correlates to increased local control most likely due to the inherent limitations of the technique [65-67]. More recent studies, however, using PET imaging, started to shed more light on the clinical evidence of oxygenation changes and reoxygenation in head and neck tumours [68-70]. The general consensus is that hypoxic subvolumes identified with the use of PET imaging in head and neck cancer are inversely correlated with the response to radiotherapy and generally with the treatment outcome. The improvement in the tumour oxygenation together with the observed geometrical stability of the persistent hypoxic regions during the course of radiotherapy suggest that head and neck tumours are strong candidates for treatment strategies accounting for tumour hypoxia at the time of treatment planning and/ or treatment adaptation based on hypoxia PET imaging.

Clinical implementation of hypoxia-driven radiotherapy is, however, still in its infancy. Several strategies for dose-painting approaches based on hypoxia in head and neck tumours have been proposed and they are under various stages of validation. Among them one could mention the planning study by Thorwarth *et al*[71] on dose escalation to head and neck hypoxic subvolumes based on PET imaging which was followed by a still ongoing clinical trial and the dose prescription and treatment planning method based on hypoxia PET imaging proposed by Toma-Dasu *et al* [72] which is currently under clinical validation.

In addition to dose escalation, treatment strategies focusing on overcoming hypoxia could include radiosensitizers or hypoxic cytotoxins. Lin and Hahn [48] presented a conceptual multimodal adaptive clinical trial approach focusing on radiation dose escalation to hypox‐ ic regions highlighting the importance of pretreatment hypoxia imaging in order to proper‐ ly select the patients that would be expected to benefit from hypoxia targeted treatments. They envisaged that serial imaging should be performed during therapy to evaluate treatment response and to select in a step-wise manner the highest-risk areas warranting treatment modifications, such as radiation dose escalation and to select the candidates for radiosensitiz‐ ers.

A special class of strategy in the management of advanced head and neck is represented by the anti-angiogenic treatment which addresses the vascular endothelial growth factors and their respective receptors on endothelial cells as well as their role in role in promoting the growth and progression of carcinoma of the head and neck. Several anti-angiogenic treatments have shown promising results in the clinical setting such as those using tyrosine kinase inhibitors or bevacizumab [73]. Nevertheless, the current results suggest that multimodal therapies combining anti-angiogenic agents with chemo/radiotherapy have the potential to further increase the overall clinical benefit.

## **2.4. Redistribution**

partial radioresistance that might in turn be reflected in the poor outcome to radiation

The oxygen status of the tumour cells is however not static with respect to both spatial and temporal patterns. Changes in the cellular oxygenation related to the dynamics of both chronic

Reoxygenation manifests itself following two main patterns. Temporal heterogeneity in oxygenation arises in relation to acute or perfusion-limited hypoxia. Abnormal vasculature can lead to fluctuations in the blood flow due to the temporary occlusion or even backflow. These phenomena have a rather chaotic but transient character and are conventionally referred to as fast reoxygenation [62] although in some cases the change in oxygen supply might in fact be from poor to well and back to poor, thus not necessarily leading to an improvement of the oxygenation of the cells but rather to a re-hypoxiation. The temporal scale of changes in oxygenation related to acute, perfusion limited hypoxia, ranges from minutes to hours as demonstrated by several experiments using sequential injection of different fluorescent dyes for hypoxia and vascular perfusion [63,64]. Furthermore, in presence of irradiation, the dynamics of the oxygenation might be even more pronounced as shown in a study on human laryngeal squamous cell carcinoma tumour line grown as xenografts in nude mice by Bussink *et al* [51] indicating that irradiation could lead to rapid changes in oxygenation and perfusion. Chronically hypoxic cells might also change their oxygenation status during the course of fractionated therapy through the so-called slow reoxygenation. In a mixed tumour cell population with respect to oxygenation, ionizing radiation will primarily kill the sensitive well-oxygenated cells. This would result in lower oxygen consumption and hence to larger distances of oxygen diffusion which independently or in conjunction to overall tumour shrinkage might lead to the improvement of the tumour oxygenation by reoxygenation of the chronically hypoxic cells. Furthermore, during long, fractionated, radiotherapy treatments, extending over several weeks, revascularisation of the tumour through angiogenesis might

also occur resulting in the reoxygenation of cells that were chronically hypoxic.

Taking advantage of the changes in tumour oxygenation and expecting that they will result in an improvement of radiosensitivity by fractionating the dose and thus increasing the treatment duration is one of the first approaches clinically used for overcoming tumour hypoxia. However, the search for the optimal dose per fraction and number of fractions in which the treatment has to be delivered is far from being over considering that one has to find the right balance between the fractional dose that might overcome hypoxia and the number of fractions that will ensure proper reoxygenation accounting at the same time for the normal tissue and

There are several clinical studies indicating that the oxygenation of head and neck tumours is indeed dynamic. The early studies of changes in tumour oxygenation in advanced head and neck carcinoma using polarographic electrodes were inconclusive in proving that reoxygena‐ tion positively correlates to increased local control most likely due to the inherent limitations of the technique [65-67]. More recent studies, however, using PET imaging, started to shed more light on the clinical evidence of oxygenation changes and reoxygenation in head and neck tumours [68-70]. The general consensus is that hypoxic subvolumes identified with the

and acute hypoxia are generally known as tumour reoxygenation.

treatment [60,61].

42 Contemporary Issues in Head and Neck Cancer Management

organs at risk.

Similar to other tumour cell populations, squamous cell carcinomas of the head and neck proliferate in asynchronous growth. Therefore cells will be distributed unevenly through the cell cycle phases. Yet, the most probable distribution is the exponential one, with the largest population in G1 and smallest in mitosis. Partial synchronisation can occur as a result of cell arrest in one or more cycle phases due to the effect of radiotherapy or chemotherapy.

Cellular redistribution or reassortment along the cell cycle plays an important role in the success of fractionated radiotherapy, given that cells present various radiosensitivities along the four phases of the cell cycle. Given the relatively short average cell cycle time of squamous cell carcinomas (around 33 hours) [62], cells that survive a first dose of radiation will tend to be in a resistant phase however, within a few hours they may progress into a more sensitive phase where they can be hit by radiation and killed.

Cells situated in the S phase are known to be about three times more radioresistant than cells undergoing mitosis. Since the duration of the S phase is about one third of the cell cycle length, there are large numbers of cells escaping the effect of radiation during a single hit. Fractionated radiotherapy assists in overcoming this challenge due to cellular redistribution between two consecutive doses.

Head and neck tumour cells have a high cell turnover, thus a relatively short cell cycle time. Cellular redistribution along the cell cycle for rapidly proliferating tumours consents to a more uniform cell kill than in slowly growing tumours. This rationale justifies the implementation of hyperfractionated radiotherapy schedules in head and neck cancers, which also hinders tumour repopulation during treatment.

Cells in the quiescent phase also play an important role during treatment as they can be triggered back into the cycle by cell loss due to radio- or chemotherapy. Quiescent cells are usually more resistant to radiation than cycling cells, fact that makes cellular recruitment (i.e. the process whereby quiescent cells re-enter the cell cycle) a double-edged sword: once they reach mitosis, newly cycling cells can increase the pool of tumour cells via cell division and, at the same time, cell killing can be more effective among cycling cells (as compared to quiescent cells) due to an overall higher radiosensitivity.

One of the known risk factors in head and neck cancer is the infection with HPV (human papillomavirus). A large number of studies have proven that head and neck cancers that are positive for HPV have higher cellular radiosensitivity than their non-HPV counterparts [74,75]. One of the explanations for this behaviour is the impaired DNA repair ability found among HPV-positive tumours and a considerable G2 arrest. These experimental studies have shown that irradiated HPV-positive cells progress faster through the S phase and then accrue in G2/M [75]. This unusual behaviour alters the expected cellular distribution along the cell cycle, accumulating the HPV cells in the more radiosensitive phases. Therefore, patients that tested positive for HPV respond better to the effect of radiotherapy and have a more favourable prognosis than non-HPV patients [74].

#### **2.5. Radiosensitivity**

Radiosensitivity is the tumour feature which aims to account for the fact that tumours respond differently to radiation therapy in a manner correlated with the intrinsic radiosensitivity of the cells derived as derived from *in vitro* experiments.

The intrinsic radiosensitivity influences the overall response of tumours to (chemo-) radio‐ therapy. The origins of the intrinsic radiosensitivity are related to the genetic instability of individual tumours leading to variations in response even among tumours of the same histological type [76]. Therefore, identifying *a priori* the alterations in the intracellular path‐ ways involved in the DNA response, regulation of cell cycle and cell proliferation or respon‐ sible for activating the apoptotic signal, might offer the possibility of identifying the patients expected to respond poorly to radiation therapy due to intrinsic radioresistance and custom‐ ising the treatment based on individual radiobiological and genetic features of the tumours.

The most often mentioned pathways that were identified as clinically relevant in relation to the intrinsic radiosensitivity are the activation of Epidermal Growth Factor Receptor (EGFR), p53 and Ki-67 proteins signalling cascades.

For head and neck in particular, the activation of the phosphatidylinositol-3-kinase (PI3-K)/ protein kinase B (AKT) pathway has been shown to be associated not only with intrinsic radioresistance but also with other well-known tumour features responsible to poor outcome, cell proliferation and tumour hypoxia. This is because the PI3-K/AKT is a key element for the regulation of several cellular processes like apoptosis, invasion and proliferation. Consequent‐ ly, it has been proposed that the manipulation of this signal-transduction pathway could be used in the management of head and neck cancers. Given the activation of this pathway by the stimulation of receptor tyrosine kinases like the EGFR, it has been suggested that markers for PI3-K/AKT activation should be related to predictors of EGFR sensitivity. Furthermore, inhibiting the PI3-K/AKT pathway will antagonise radiation-induced cellular defence mech‐ anisms that in turn will result in enhancing the effectiveness of radiation therapy [77].

More recently, a biomarker that encodes the p53 protein, TP53, has been identified as the most commonly altered gene in squamous cell carcinomas of the head and neck leading to radio‐ resistance [78].

As already mentioned above, the presence of Human Papilloma Virus (HPV) influences the response of the response to radiotherapy for head and neck squamous cell carcinoma [79]. Thus, the HPV status of the tumour could be regarded as a strong and independent prognostic factor for the success of the treatment, both in terms of local regional control and overall survival. This is due the increased cellular radiosensitivity caused by compromised DNA repair capacity in HPV-positive cells [74]. This might indicate that radiosensitivity and repair in cells should be correlated. Two main mechanisms have been identified for the repair of the double strand breaks of the DNA, homologous recombination (HR) and nonhomologous endjoining (NHEJ). Nevertheless, it has been shown that mutations in genes that impair HR often cause only modest or no radiation hypersensitivity. In contrast, mutations in NHEJ genes appear to lead to greater radiation hypersensitivity [80]. These complex relationships may in fact be the reason for difficulties in finding a correlation between repair and radiosensitivity [81] and why these are considered as two independent Rs in radiation biology.

## **3. The 6th R: Remote bystander effects**

be in a resistant phase however, within a few hours they may progress into a more sensitive

Cells situated in the S phase are known to be about three times more radioresistant than cells undergoing mitosis. Since the duration of the S phase is about one third of the cell cycle length, there are large numbers of cells escaping the effect of radiation during a single hit. Fractionated radiotherapy assists in overcoming this challenge due to cellular redistribution between two

Head and neck tumour cells have a high cell turnover, thus a relatively short cell cycle time. Cellular redistribution along the cell cycle for rapidly proliferating tumours consents to a more uniform cell kill than in slowly growing tumours. This rationale justifies the implementation of hyperfractionated radiotherapy schedules in head and neck cancers, which also hinders

Cells in the quiescent phase also play an important role during treatment as they can be triggered back into the cycle by cell loss due to radio- or chemotherapy. Quiescent cells are usually more resistant to radiation than cycling cells, fact that makes cellular recruitment (i.e. the process whereby quiescent cells re-enter the cell cycle) a double-edged sword: once they reach mitosis, newly cycling cells can increase the pool of tumour cells via cell division and, at the same time, cell killing can be more effective among cycling cells (as compared to

One of the known risk factors in head and neck cancer is the infection with HPV (human papillomavirus). A large number of studies have proven that head and neck cancers that are positive for HPV have higher cellular radiosensitivity than their non-HPV counterparts [74,75]. One of the explanations for this behaviour is the impaired DNA repair ability found among HPV-positive tumours and a considerable G2 arrest. These experimental studies have shown that irradiated HPV-positive cells progress faster through the S phase and then accrue in G2/M [75]. This unusual behaviour alters the expected cellular distribution along the cell cycle, accumulating the HPV cells in the more radiosensitive phases. Therefore, patients that tested positive for HPV respond better to the effect of radiotherapy and have a more favourable

Radiosensitivity is the tumour feature which aims to account for the fact that tumours respond differently to radiation therapy in a manner correlated with the intrinsic radiosensitivity of the

The intrinsic radiosensitivity influences the overall response of tumours to (chemo-) radio‐ therapy. The origins of the intrinsic radiosensitivity are related to the genetic instability of individual tumours leading to variations in response even among tumours of the same histological type [76]. Therefore, identifying *a priori* the alterations in the intracellular path‐ ways involved in the DNA response, regulation of cell cycle and cell proliferation or respon‐ sible for activating the apoptotic signal, might offer the possibility of identifying the patients

phase where they can be hit by radiation and killed.

44 Contemporary Issues in Head and Neck Cancer Management

tumour repopulation during treatment.

prognosis than non-HPV patients [74].

cells derived as derived from *in vitro* experiments.

**2.5. Radiosensitivity**

quiescent cells) due to an overall higher radiosensitivity.

consecutive doses.

Remote cellular effects or bystander effects occur when non-irradiated cells that are located nearby irradiated cells undergo cellular damage similar to the irradiated cells. This experi‐ mental observation contradicts the formerly accepted theory of radiation-induced targeted cell kill [82]. While targeted cells can be killed by radiation, according to the bystander theory, nontargeted cells can also present signs of radiation damage that eventually kills the cell. This happens as a consequence of cellular communication when radiation-hit cells direct damage signals through gap junctions to the neighbouring non-targeted cells, which then act as being hit by radiation.

Bystander effects have been evidenced in both tumour and normal cells, which implies that such remote effects could have clinical implications. The finding that gamma-ray-induced bystander effects have influence on epithelial cells and not fibroblasts, suggested that tissue architecture and also cell communication play a significant role in this process [83]. Since squamous cell carcinomas originate from epithelial cells, the bystander effect becomes an important consideration in the treatment of head and neck tumours (table 1).

It is known that in normal tissues, gap junctions physiologically connect one cell to the adjacent one to enable the transmission of genetic signals between cells. Both metabolic cooperation between cells and the regulation of normal tissue homeostasis requires the involvement of gap junctions. This normal phenotype is usually lost during head and neck carcinogenesis. Although the complete function of gap junctions in head and neck neoplasms is not fully clarified, experimental studies demonstrate that gap-junctional intercellular communication (GJIC) could mediate apoptotic cell death in non-targeted squamous cell carcinoma adjacent to individually targeted squamous cell carcinomas of the head and neck [84].

Novel therapeutic methods like gene therapy are widely used to investigate bystander effects in cancers including head and neck. A number of viral vectors have been developed that are able to transfer genes to therapy of tumours known as gene transduction. The occurrence of a bystander effect after wild-type p53 gene transduction has been investigated for human squamous cell carcinomas of the head and neck [85]. Wild-type p53 gene transduction for apoptosis-inducing molecular therapy has been shown capable of producing a bystander effect in squamous cells *in vitro.* Additionally, it was demonstrated that this phenomenon requires intercellular contact between wild-type p53 transduced and bystander, non-transduced cell populations. The study concluded that other therapies associated with apoptosis (such as radiotherapy or chemotherapy) might also demonstrate bystander effects.

Enhancing gap-junctional intercellular communication in squamous cell carcinomas of the head and neck and understanding the other mechanisms behind cancer cell communication may lead to increased therapeutic efficacy.


**Table 1.** Bystander effects in squamous cell carcinoma of the head and neck

An interesting observation that could have implications in the development of new thera‐ peutical agents for cancer was reported by Cogan *et al*[88]. The group has shown that bystander signals from cancer cells after exposure to chromium VI results in DNA damage in neigh‐ bouring cells that is strongly dependent on telomerase (complex enzyme that maintains the telomere length). Low dose exposure to Cr(VI) was able to induce cancer cells to continuously secrete bystander signals that caused DNA damage in the neighbouring cells. However, these bystander signals were telomerase-dependent, meaning that the status of the telomere (negative or positive) has dictated the affinity of cancer cells for bystander signals.

happens as a consequence of cellular communication when radiation-hit cells direct damage signals through gap junctions to the neighbouring non-targeted cells, which then act as being

Bystander effects have been evidenced in both tumour and normal cells, which implies that such remote effects could have clinical implications. The finding that gamma-ray-induced bystander effects have influence on epithelial cells and not fibroblasts, suggested that tissue architecture and also cell communication play a significant role in this process [83]. Since squamous cell carcinomas originate from epithelial cells, the bystander effect becomes an

It is known that in normal tissues, gap junctions physiologically connect one cell to the adjacent one to enable the transmission of genetic signals between cells. Both metabolic cooperation between cells and the regulation of normal tissue homeostasis requires the involvement of gap junctions. This normal phenotype is usually lost during head and neck carcinogenesis. Although the complete function of gap junctions in head and neck neoplasms is not fully clarified, experimental studies demonstrate that gap-junctional intercellular communication (GJIC) could mediate apoptotic cell death in non-targeted squamous cell carcinoma adjacent

Novel therapeutic methods like gene therapy are widely used to investigate bystander effects in cancers including head and neck. A number of viral vectors have been developed that are able to transfer genes to therapy of tumours known as gene transduction. The occurrence of a bystander effect after wild-type p53 gene transduction has been investigated for human squamous cell carcinomas of the head and neck [85]. Wild-type p53 gene transduction for apoptosis-inducing molecular therapy has been shown capable of producing a bystander effect in squamous cells *in vitro.* Additionally, it was demonstrated that this phenomenon requires intercellular contact between wild-type p53 transduced and bystander, non-transduced cell populations. The study concluded that other therapies associated with apoptosis (such as

Enhancing gap-junctional intercellular communication in squamous cell carcinomas of the head and neck and understanding the other mechanisms behind cancer cell communication

**Bystander effect Mode of cellular communication Reference**

Frank *et al* 1998 [85]

transduced and non-transduced cell population

proliferation arrest Gap-junctional intercellular communication Kucerova *et al* 2013 [87]

Apoptotic cell death Frank *et al* 2005 [84] Genomic instability Cellular interaction influenced by stromal fibroblasts Kamochi *et al* 2008 [86]

important consideration in the treatment of head and neck tumours (table 1).

to individually targeted squamous cell carcinomas of the head and neck [84].

radiotherapy or chemotherapy) might also demonstrate bystander effects.

may lead to increased therapeutic efficacy.

Cell dormancy and

Growth inhibition Intercellular contact between wild-type p53

**Table 1.** Bystander effects in squamous cell carcinoma of the head and neck

hit by radiation.

46 Contemporary Issues in Head and Neck Cancer Management

It is well known that telomere activation has a therapeutic potential for cancer, given that with each cell division, the length of telomeres shortens, fact that triggers cell senescence. In order to survive, cancer cells are required to employ a mechanism to stabilise this process of telomere reduction. There are several reports in the literature showing that telomerase promoter mutations (TERT) are more prevalent in aggressive cancers and they are a major indicator of poor prognosis among head and neck cancer patients [89,90]. The effect of chromium VI on telomerase offers, therefore, a potential anticancer avenue that needs to be further explored.

Bystander effects largely relate to nontargeted effects after exposure to low dose radiation. While tumours are targeted with high doses, the surrounding normal tissue receives much lower doses of radiation and organs that are out-of-field even lower doses. Exposure of nontargeted tissue raises several concerns regarding the risk of second primary cancers. However, it was shown that low dose exposure of healthy cells (2 - 50 mGy) stimulates intercellular induction of apoptosis in the precancerous cell population, via cytokine and reactive oxygen species signalling [91]. This selective elimination of precancerous cells could reduce the incidence of second cancers following radiotherapy.

There is a limited number of studies on the risk of second cancers after primary head and neck carcinoma treatment and they reveal the observation that among these patients second cancers occur, most often, due to smoking [92] and drinking habits [93]. Second cancers are common among long-term survivals, however they are not necessarily second primaries. Recurrences occur very often and they arise mostly in the treatment field [94]. Common sites for second primary cancers are the lung and the esophagus. These occurrences are often linked to the same risk factors as for the primary head and neck cancer, i.e. tobacco smoking and alcohol consumption. The radiation-induced incidence of second primary cancers after head and neck radiotherapy was analysed in a study by Yamamoto *et al* [95]. The group has concluded that radiotherapy of the primary tumour is not thought to be associated with an increased risk of second tumours. Furthermore, it was underlined that a clonal relationship exists between the primary head and neck cancer and second primaries, suggesting that the latter are a result of micrometastatic foci migration from the site of origin.

Beside bystander effects, there is another phenomenon that is linked to low dose cellular exposure, namely the adaptive response. This effect of low dose radiation on cells was first demonstrated by Olivieri *et al* [96] after observing that human lymphocytes growing in radioactive thymidine solution were more resistant to the effect of subsequent high doses of radiation than the control group grown in non-radioactive culture. Cellular radioresistance to subsequent doses was materialised through a reduction of chromosomal aberrations.

*In vitro* experimental studies have shown that the two low-dose phenomena: the bystander effect and the adaptive response basically coexist. However, their effect on cells works in opposition, as bystander effects result in excess cell kill, while the adaptive response confers resistance to subsequent doses of radiation.

According to the adaptive response model, the isoeffect per dose fraction is not a valid theory anymore, as the first dose of radiation should kill a higher percent of the tumour cell population than the subsequent doses. Furthermore, for those patients that undergo pre-treatment diagnostic examinations that employ low doses, the adaptive response might work even for the first treatment dose.

More studies are needed to explain these two effects in head and neck cancer cell lines and to determine their magnitude for the *in vivo* state.

## **4. Conclusions**

The management of locally advanced head and neck cancers is demanding. Tumour hypoxia, accelerated repopulation during treatment and inherent radioresistance are the main culprits for the suboptimal tumour control. The Rs of radiotherapy, as described above, play an important role in treatment design, particularly when it comes to dose fractionation in radiotherapy. Questions then arise as to how fractionated the treatment should be and what other parameters should be taken into consideration in order to achieve a high therapeutic gain.

As demonstrated by clinical trials, conventionally fractionated treatments are not efficient for head and neck cancer patients. Instead, altered fractionation schedules should be employed to overcome the radiobiological challenges. Accelerated fractionation is a rather aggressive protocol that, however, is needed in response to an aggressive tumour. Treatment breaks are often scheduled in these situations to allow time for normal tissue repair. Hyperfractionated radiotherapy is a perfect way to apply the Rs of radiotherapy for rapidly proliferating tumours such as head and neck carcinomas. By giving more than one dose fraction a day, tumour repopulation during treatment is minimised and tumour reoxygenation is stimulated. Hyperfractionated schedules were shown to provide the greatest benefit among these patients.

While hypoxia and repopulation are usual characteristics of head and neck cancers, the extent of hypoxia and the degree of tumour proliferation differ from patient to patient. These pretreatment disparities lead to different post-treatment tumour responses. Decades ago, the idea of predictive assays for tumour oxygenation, proliferation and radioresistance has been embraced with high optimism. However, due to several technical and clinical challenges, the routine implementation of such predictive assays has been hindered and other methods to characterise the metabolic properties of tumours have been designed. Advanced imaging techniques such as BOLD-MRI (Blood Oxygen Level Dependent – Magnetic Resonance Imaging) or PET (Positron Emission Tomography) can give valuable indication regarding oxygenation and proliferation. The goal to gather such information is to design individualised treatments, based on patient-specific parameters. Personalised treatment is therefore the key solution for the management of advanced head and neck cancers. This will be achieved with (figure 1):


*In vitro* experimental studies have shown that the two low-dose phenomena: the bystander effect and the adaptive response basically coexist. However, their effect on cells works in opposition, as bystander effects result in excess cell kill, while the adaptive response confers

According to the adaptive response model, the isoeffect per dose fraction is not a valid theory anymore, as the first dose of radiation should kill a higher percent of the tumour cell population than the subsequent doses. Furthermore, for those patients that undergo pre-treatment diagnostic examinations that employ low doses, the adaptive response might work even for

More studies are needed to explain these two effects in head and neck cancer cell lines and to

The management of locally advanced head and neck cancers is demanding. Tumour hypoxia, accelerated repopulation during treatment and inherent radioresistance are the main culprits for the suboptimal tumour control. The Rs of radiotherapy, as described above, play an important role in treatment design, particularly when it comes to dose fractionation in radiotherapy. Questions then arise as to how fractionated the treatment should be and what other parameters should be taken into consideration in order to achieve a high therapeutic

As demonstrated by clinical trials, conventionally fractionated treatments are not efficient for head and neck cancer patients. Instead, altered fractionation schedules should be employed to overcome the radiobiological challenges. Accelerated fractionation is a rather aggressive protocol that, however, is needed in response to an aggressive tumour. Treatment breaks are often scheduled in these situations to allow time for normal tissue repair. Hyperfractionated radiotherapy is a perfect way to apply the Rs of radiotherapy for rapidly proliferating tumours such as head and neck carcinomas. By giving more than one dose fraction a day, tumour repopulation during treatment is minimised and tumour reoxygenation is stimulated. Hyperfractionated schedules were shown to provide the greatest benefit among these patients.

While hypoxia and repopulation are usual characteristics of head and neck cancers, the extent of hypoxia and the degree of tumour proliferation differ from patient to patient. These pretreatment disparities lead to different post-treatment tumour responses. Decades ago, the idea of predictive assays for tumour oxygenation, proliferation and radioresistance has been embraced with high optimism. However, due to several technical and clinical challenges, the routine implementation of such predictive assays has been hindered and other methods to characterise the metabolic properties of tumours have been designed. Advanced imaging techniques such as BOLD-MRI (Blood Oxygen Level Dependent – Magnetic Resonance Imaging) or PET (Positron Emission Tomography) can give valuable indication regarding oxygenation and proliferation. The goal to gather such information is to design individualised

resistance to subsequent doses of radiation.

48 Contemporary Issues in Head and Neck Cancer Management

determine their magnitude for the *in vivo* state.

the first treatment dose.

**4. Conclusions**

gain.

**•** improved *perfusion* of oxygen inside the tumour to allow for reoxygenation and better chemotherapy delivery.

These P's of head and neck cancer treatment can lead to an enhanced therapeutic ratio by increasing tumour control and decreasing normal tissue toxicity.

**Figure 1.** The Ps and Rs of head and neck cancer management towards Personalised Radiotherapy.

A more accurate patient selection for the administration of chemotherapeutical agents or altered fractionation schedules could lead to a better management of head and neck cancer with personalised treatment planning and delivery.

## **Acknowledgements**

LGM would like to acknowledge the support offered by the Ministry of National Education (Romania), CNCS-UEFISCDI, Project no. PN-II-ID-PCE-2012-4-0067.

ITD and AD acknowledge the support offered by the Cancer Research Funds of Radiumhem‐ met, Stockholm (Sweden), the LiU Cancer research network at Linköping University (Sweden) and the County Council of Östergötland (Sweden).

## **Author details**

Loredana G. Marcu1,2, Iuliana Toma Dasu3 and Alexandru Dasu4

\*Address all correspondence to: loredana@marcunet.com

1 Faculty of Science, University of Oradea, Oradea, Romania

2 School of Chemistry & Physics, University of Adelaide, SA, Australia

3 Medical Radiation Physics, Stockholm University and Karolinska Institutet, Stockholm, Sweden

4 Department of Radiation Physics and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden

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**Author details**

Sweden

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50 Contemporary Issues in Head and Neck Cancer Management

Linköping University, Linköping, Sweden

\*Address all correspondence to: loredana@marcunet.com

1 Faculty of Science, University of Oradea, Oradea, Romania

2 School of Chemistry & Physics, University of Adelaide, SA, Australia

and Alexandru Dasu4

3 Medical Radiation Physics, Stockholm University and Karolinska Institutet, Stockholm,

4 Department of Radiation Physics and Department of Medical and Health Sciences,

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