Effective Biomarkers and Radiation Treatment in Head and Neck Cancer

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Jun 5, 2015 - Context.—Radiation is a key arm in the multidisciplin- ary treatment of patients with head and neck squamous cell carcinoma. During the past 2 ...
Effective Biomarkers and Radiation Treatment in Head and Neck Cancer Thomas J. Ow, MD, MS; Casey E. Pitts, BA; Rafi Kabarriti, MD; Madhur K. Garg, MD, MB, BS

 Context.—Radiation is a key arm in the multidisciplinary treatment of patients with head and neck squamous cell carcinoma. During the past 2 decades, significant changes in the way radiation therapy is planned and delivered have improved efficacy and decreased toxicity. Refined approaches in the application of radiation and chemoradiation have led to organ-sparing treatment regimens for laryngeal and pharyngeal cancers and have improved local and regional control rates in the postoperative, adjuvant setting. The molecular and genetic determinants of tumor cell response to radiation have been studied, and several potential biomarkers are emerging that could further improve application and

efficacy of radiation treatment in head and neck squamous cell carcinoma. Objective.—To discuss the current understanding of potential biomarkers related to radiation response in head and neck squamous cell carcinoma. Data Sources.—Existing published literature. Conclusions.—Several potential biomarkers are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat head and neck squamous cell carcinoma. Several promising candidates have been defined, and new markers are on the horizon. (Arch Pathol Lab Med. 2015;139:1379–1388; doi: 10.5858/arpa.2014-0574-RA)

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also resulted in increased acute and late toxicities.14,15 Figure 1 summarizes the current applications for radiation in the treatment of HNSCC. Given the significant role that radiation therapy has in most locally advanced HNSCCs, novel strategies are needed to optimize radiation treatments to achieve maximal tumor control while minimizing toxicity. Recent findings from the Radiation Therapy Oncology Group (RTOG) 0129 trial demonstrated that patients with oropharyngeal squamous cell carcinoma (OPSCC) that is positive for human papillomavirus (HPV) had significantly improved overall survival and progression-free survival, whereas HPV-negative OPSCCs treated with primary radiation therapy and cisplatin had significantly reduced locoregional control and overall survival.16 These findings have opened the window for personalized medicine based on the molecular signature of the tumor. In fact, 2 RTOG trials were conceived based on this concept, and those trials were designed to evaluate the role of treatment deintensification in HPVþ OPSCCs (RTOG 1016) and the role of treatment intensification in HPVnegative OPSCCs (RTOG 1221). Thus, HPV positivity or expression of the p16 protein, a surrogate marker for HPV infection in OPSCC, appears to be a prognostic biomarker and is a potential predictive biomarker in HNSCC. This would represent the first molecular biomarker employed clinically for both prognostication and management of this disease. This review will discuss the current understanding of molecular determinants of response to radiation therapy in HNSCC. At present, only clinical and pathologic parameters, such as those of the American Joint Committee on Cancer’s tumor, lymph node, distant metastasis (TNM) staging17–19 and tumor volume,20–23 are used to make treatment decisions. None of these parameters, however,

adiation has a significant role in the management of head and neck squamous cell carcinomas (HNSCCs). Strategies to use radiation for the treatment of HNSCC have improved significantly during the past 2 decades. Radiation delivered postoperatively in the adjuvant setting significantly improves eradication of locoregional disease, and organsparing approaches for pharyngeal and laryngeal cancers that rely on radiation as the primary treatment modality yield high rates of disease control.1 Advances in the delivery of radiation therapy, including image guidance and intensity-modulated radiation therapy, as well as advancements in diagnostic imaging, including high-resolution computed tomography scanning, magnetic resonance imaging, and positron emission tomography (PET), have allowed for accurate targeting and delivery of high doses of radiation to the tumor while minimizing the dose to the normal surrounding structures.2–7 Furthermore, the application of concurrent systemic therapies have been shown to improve the efficacy of radiation in both the primary and adjuvant settings.8–13 However, the addition of chemotherapy has Accepted for publication January 13, 2015. Published as an Early Online Release June 5, 2015. From the Departments of Otorhinolaryngology-Head and Neck Surgery (Drs Ow and Garg), Pathology (Dr Ow), Radiation Oncology (Drs Kabarriti and Garg), and Urology (Dr Garg) Montefiore Medical Center, Bronx, New York; and the Albert Einstein College of Medicine (Drs Ow, Kabarriti, and Garg, and Ms Pitts), Bronx. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Thomas J. Ow, MD, MS, Department of Otorhinolaryngology-Head and Neck Surgery, Montefiore Medical Center, Albert Einstein College of Medicine, 3400 Bainbridge Ave, Third Floor Greene Medical Arts Pavilion, Bronx, NY 10467 (e-mail: THOW@ montefiore.org). Arch Pathol Lab Med—Vol 139, November 2015

Radiation Biomarkers in Head and Neck Cancer—Ow et al 1379

Figure 1. Summary of the multiple roles of radiation treatment in the multidisciplinary approach to treatment for primary and recurrent head and neck squamous cell carcinoma. Abbreviation: ECS, extracapsular lymph node spread.

fully account for the variability in response and toxicity seen among individual patients receiving radiation treatment. This leaves a significant number of patients whose treatment is potentially inadequate, ineffective, or unnecessary. Even though many patient characteristics, such as smoking status,24,25 age,26 and nutritional status,26,27 are known to influence response to radiation and patient outcome, it is still very difficult to predict which patients will have a complete response and which will not. TUMOR CHARACTERISTICS Aside from the primary tumor site and American Joint Committee on Cancer’s TNM staging criteria, additional clinical and pathologic characteristics have been proposed to predict response of individual tumors to radiotherapy. These include metabolic tumor volume, assessed through 2[18F]fluoro-2-deoxy-D-glucose (FDG) uptake measured by PET (FDG-PET), intrinsic radiosensitivity of tumor cells, repopulation, and hypoxia.28 Tumor FDG uptake, using both the standardized uptake value and the metabolic tumor volume as determined by PET scanning, has been reported to predict treatment failure. In patients with head and neck cancer, increased metabolic tumor volume29,30 and metabolic tumor volume velocity31 have been shown to be associated with disease recurrence. In 120 patients with head and neck cancer who received either radiation therapy or surgery, higher standardized uptake values were associated with worse local control for both treatment types.32 However, others6,29,31 have not found standardized uptake values on FDG-PET imaging to be significantly associated with disease progression or other clinical outcomes. Other major limitations of FDG-PET prediction include technical variability, potential lack of consistency in region delineation, and inherent patient characteristics that affect FDG uptake and contribute to variability.31 The intrinsic tumor radiosensitivity in vitro has been examined as a potential predictor of treatment response. Bjork-Eriksson and colleagues33 demonstrated that the ¨ surviving fraction at 2 Gy (SF2) of patient specimens propagated in vitro, obtained using a soft-agar clonogenic assay, were greater in patients with head and neck cancer 1380 Arch Pathol Lab Med—Vol 139, November 2015

who developed local recurrence compared with those who did not. However, a similar study34 did not find SF2 to be predictive, although the experimental methods of the 2 studies were very different. Repopulation capacity has been implicated as an effector of radiation response, although the evidence is not promising. Because decreased time between treatments has been shown to improve local control, it was hypothesized that was due to lessened time for cells to recover and to proliferate between dosing fractions.28 Therefore, researchers proposed that potential doubling time may affect treatment outcomes. However, neither data on potential doubling time nor other cell kinetics seem to be associated with response.18,35,36 Clinically, radiation fractionation strategies have attempted to overcome tumor repopulation via hyperfractionation, in which smaller doses are given more frequently, or via accelerated fractionation protocols, in which the dosing schedule occurs at shorter intervals to achieve the total dose more quickly. Data do suggest these strategies offer some improvement in patient outcome,37,38 but that advantage may be lost in the setting of concurrent chemotherapy.15 Efficacy of radiation treatment relies on DNA damage, either via direct damage of DNA by ionizing radiation or by free radicals that are generated and subsequently react with DNA, which is the more frequent mechanism of DNA damage. Oxygen free radicals are highly reactive and are the primary source of radiation-induced DNA damage, and oxygen is, therefore, a potent radiosensitizer. Hypoxia, is known to be an important factor influencing response to radiation. Hypoxic HNSCC tumors, measured by pretreatment tumor PO2, have been shown to be significantly more likely to persist or recur locoregionally.39 This was consistent with data from 67 patients with head and neck cancer studied by Nordsmark and colleagues,40 for whom hypoxia was also associated with decreased 2-year locoregional control. Hypoxia-inducible factor 1 a (HIF1a) is an intracellular transcription factor that undergoes degradation under well-oxygenated conditions but is stabilized under hypoxic conditions. When HIF1a is stabilized, it regulates the expression of proangiogenic genes, such as vascular endothelial growth factor (VEGF). In a study41 of 75 HNSCC Radiation Biomarkers in Head and Neck Cancer—Ow et al

Tumor Characteristics Contributing to Radiation Resistance Characteristic

Description

Metabolic activity Repopulation Hypoxia

FDG avidity on PET imaging may be indicative of metabolic activity and perhaps proliferation rate in HNSCC. These factors may be predictive of radiation response or have prognostic value. The ability of tumor cells to recover and grow between radiation fractions may influence response and prognosis. Hyperfractionation and accelerated fractionation protocols may improve response among tumors with high repopulation rates. Oxygen is a potent radiosensitizer because oxygen free radicals are generated after radiation is delivered and cause DNA damage. Hypoxic tumors are, therefore, generally more resistant to radiation treatment than oxygen-rich tumors.

Abbreviations: FDG, fluorodeoxyglucose; HNSCC, head and neck squamous cell carcinoma; PET, positron emission tomography.

specimens, overexpression of HIF1a, assessed by immunohistochemistry (IHC) was associated with locally aggressive behavior. Hypoxia-inducible factor 1 a was also significantly associated with poor locoregional control in 67 patients with head and neck cancers.40 Other studies28 have not shown HIF1a to be associated with recurrence after radiation, but there is wide heterogeneity and confounders among available reports. The tumor characteristics described above are summarized in the Table. These characteristics may have predictive value in determining response to radiation, but significant advancements in our current understanding of the molecular and genetic underpinnings of HNSCC have led to more-specific potential biomarkers. MOLECULAR BIOMARKERS TO PREDICT RADIATION RESPONSE In current standard-treatment algorithms for head and neck cancers, only clinical and pathologic factors are weighed in the decision to administer radiation, and no biomarkers are used to further tailor treatment to the patient. For example, when employing an organ-sparing approach using chemoradiation for advanced larynx cancer, approximately one-third of patients will not completely respond and will require salvage laryngectomy.9 Currently, there are no predictive biomarkers available that change our treatment decisions, but several potential candidates are actively being studied. Human Papillomavirus As highlighted earlier, HPV infection is a significant factor in a growing population of patients with OPSCC. These cancers are epidemiologically, molecularly, and clinically distinct from their HPV counterparts. The HPV infection appears to be associated with approximately 70% of OPSCCs in contemporary cohorts.42 The oncogenic properties of HPV are conveyed largely through the E6 and E7 oncoproteins, which drive carcinogenesis by abrogating the p53 and pRb tumor suppressors, respectively, which results in dysregulation of various important cell processes, including cell proliferation and genomic integrity.43–45 Although all HPV types contain these oncogenes, only a few types are considered high risk for the potential to promote cancer (namely, HPV-16 and HPV-18), and among those, HPV-16 is responsible for most HPV-associated cancers.46 Compared with patients with ‘‘traditional’’ HNSCC, patients with HPV-associated OPSCC are often younger, are less likely to have a history of tobacco use, and tend to present with lymph node metastases and small primary tumors.44 An HPVþ OPSCC carries a better prognosis and tends to demonstrate a very favorable response to radiation therapy. Arch Pathol Lab Med—Vol 139, November 2015

Research has only recently begun to shed light on the molecular characteristics responsible for this phenomenon. The HPVþ OPSCC tumors are associated with better treatment response after chemoradiotherapy compared with HPV-negative tumors.47–49 In 82 patients with HNSCC, the presence of HPV was found to be associated with decreased disease progression,50 and HPVþ/p16þ expression was significantly associated with better locoregional control following radiation therapy.51 Increased expression of the cell cycle regulator p16 is very common in HPVþ oropharyngeal cancer and has been advocated as a surrogate marker for the presence of HPV in OPSCCs. Evaluation of p16 and HPV in tumor specimens from patients enrolled in RTOG-0129 (a study evaluating chemoradiation in patients with advanced OPSCC) demonstrated that both markers were associated with improved survival and that additional factors, such as advanced T stage and tobacco history, could stratify patients into low, medium, and high-risk groups.16 In vitro data among several studies support the clinical finding that HPVþ tumors respond better to radiotherapy. The HPVþ cells have been shown to have greater radiosensitivity than HPV-negative cells do, which is associated with a greater delay in the G2/M phase52–56 and a decreased ability to repair double-stranded DNA breaks.55 The different response of HPVþ OPSCC to chemoradiotherapy compared with HPV-negative HNSCC offers the possibility to adjust the treatment strategy based on HPV status, and indeed, several active clinical trials are examining approaches toward treatment de-escalation for patients with HPVþ oropharynx cancer,57 specifically, strategies to either reduce radiation dosage or limit the use of cytotoxic chemotherapy. However, until those studies demonstrate a clear benefit for de-escalation protocols with no detriment to recurrence and survival rates, HPV status will remain a prognostic marker, rather than a predictive marker, which could potentially influence treatment recommendations. Tumor Protein p53 TP53 is the most commonly altered gene among head and neck cancers and is mutated in approximately 50% of cases and perhaps 70% to 80% of HPV-negative cases.58–60 The gene codes for the p53 protein, a tumor suppressor that largely acts as a transcription factor, activated by cellular stress (eg, DNA damage, oncogenic stress, hypoxia, etc), resulting in the initiation of cellular programs that ultimately regulate and protect genome integrity. By affecting more than 100 genes, p53 activation results in cell cycle arrest, DNA repair, and can induce apoptosis, if required. When p53 function is lost, these protective mechanisms are disabled, leading to a higher propensity for the cell to accumulate and propagate genomic insults.61,62 Radiation Biomarkers in Head and Neck Cancer—Ow et al 1381

Although TP53 mutations are common, studies examining its value as a prognosticator and as a predictive biomarker have produced conflicting results. Some have found that mutated TP53 is associated with decreased locoregional control following radiation therapy,63,64 whereas others have not found such an association65–69 or have found an association only when p53 is altered in the context of expression of other markers, such as the B-cell lymphoma 2 protein (BCL2).70 The existing evidence is hampered largely by reports that have examined cohorts of patients who varied significantly, both clinically and pathologically and by treatment rendered. Furthermore, studies have examined p53 using differing experimental techniques, including methods that have sequenced only a few exons in the TP53 gene, and reports that use expression of p53 by IHC as a marker, which does not accurately reflect the TP53 mutation and/or functional status.64,71–73 It has been difficult to establish TP53 mutation as a biomarker because of the complexity of the molecule’s structure and function and the interesting mutation patterns demonstrated in human cancers. The TP53 gene is encoded by 11 exons; the transcription and translation of which produce a protein that complexes with 3 other p53 proteins to form a functional tetramer. The main functional activity of the molecule is dependent on an intact DNA-binding domain, encoded by exons 5 to 9. Mutations identified in TP53 tend to be point mutations and mostly accumulate in the DNA-binding domain, with some specific codons that appear to be targeted preferentially. Mutant protein is often overexpressed in cancers, rather than lost. This has led to several reports74,75 demonstrating that some p53 mutants have functional and oncogenic properties. This complex mutation pattern has led to several proposed systems for classifying TP53 mutations and attempts to use these classification systems of TP53 mutations as prognostic variables. p53 has a key role in the response to DNA damage. Because most head and neck cancers are treated with radiation, it is feasible that a mutation in TP53 would correlate with treatment response and outcome. As stated, several mutation classification systems have been examined. One early study64 examined 110 patients with HNSCC, all of whom received radiation as a component of therapy, and exons 5 to 9 of TP53 were sequenced from tumor specimens. Mutations were identified in 44% of tumors, and TP53 mutation was associated with locoregional failure, which remained significant after multivariate analysis.64 In another study by Lindbergh-van der Plas and colleagues,76 141 patients with HNSCC were treated with surgery followed by radiotherapy, and TP53 mutations were identified in 62% of specimens. Mutations that resulted in a truncation of the molecule were associated with decreased progression-free survival, which remained significant after multivariate analysis, which included HPV status as a variable.76 The largest study of TP53 mutations in HNSCC, to our knowledge, was published by Poeta et al,77 in which TP53 was evaluated in 420 HNSCC tumors. This study77 classified mutation as either disruptive or nondisruptive, based on the presence or absence of a significant amino acid change in the core DNA-binding region, and demonstrated that disruptive mutations were associated with worse overall survival, which remained a significant factor after multivariate analysis.77 The disruptive classification was supported by an in vivo study in which cell lines with disruptive TP53 mutations demonstrated aggressive growth and metastasis 1382 Arch Pathol Lab Med—Vol 139, November 2015

in an orthotopic mouse model.78 Another study, by Skinner et al,79 showed that TP53 disruptive mutations were associated with decreased locoregional control among 74 patients treated with surgery and postoperative radiation. The study79 also demonstrated that accelerated senescence was reduced among HNSCC cell lines with disruptive TP53 mutations, suggesting a possible mechanism for decreased locoregional control after radiation. Because p53 is a tumor suppressor, there is no therapeutic strategy currently active that is used to specifically target tumors with p53 mutations. Some studies have examined delivery of functional p53 via gene therapy using an adenoviral vector.80 Another drug, nutlin, targets the MDM2 proto-oncogene, E3 ubiquitin protein ligase (MDM2), which is the major cellular regulator of functional p53, and in vitro studies have shown some promising activity of this drug in combination with radiation.81 However this drug would not likely be useful in tumors harboring mutant p53. No current strategies seek to enhance the radiosensitivity of TP53 mutant cancers, specifically, but strategies to use the TP53 mutation and its sequelae for prognostic and therapeutic advantage are ongoing. Epidermal Growth Factor Receptor EGFR is a transmembrane protein receptor that has a role in several important biologic pathways, which include cell proliferation, inhibition of apoptosis, and angiogenesis, among others.82 EGFR is commonly overexpressed in head and neck cancers.83 Overexpression is thought to represent or correlate with constitutive signaling, contributing to increased proliferation and migration, resulting in cancer cells exhibiting increased growth and metastatic potential.84 EGFR expression has been shown to be associated with poor outcomes following radiation therapy in several studies. The IHC expression of EGFR in 155 patients with HNSCC was a strong, independent predictor of locoregional recurrence,85 which was validated in a larger set of 533 patients86 and was consistent with results demonstrated among patients with early glottic cancer.87,88 In a study89 of patients with advanced nasopharyngeal cancer, the extent of EGFR expression (25%) was also an independent predictor of locoregional failure following radiation therapy after induction chemotherapy. Similarly, high EGFR expression in laryngeal squamous cell carcinoma (SCC) or hypopharyngeal SCC, along with induction chemotherapy and radiotherapy, was associated with a shorter disease-free interval.90 These results are consistent with those from in vitro studies as well. In irradiated cells taken from 24 patients with HNSCC, high EGFR expression was strongly associated with the radioresistant group, compared with those who were radiosensitive.91 Not all studies have consistently demonstrated the association between EGFR expression and radioresistance. For example, 25 patients with laryngeal SCC who received postoperative radiation therapy showed no association between EGFR expression and risk of recurrence.92 The high expression of EGFR noted in HNSCC led to approaches to target EGFR therapeutically in patients with HNSCC. Cetuximab, a monoclonal antibody against the EGFR ligand-binding domain, has been evaluated therapeutically in HNSCC. As a single agent, cetuximab demonstrated a 13% response rate,93 and cetuximab was approved by the US Food and Drug Administration for use in HNSCC as an adjuvant therapy combined with primary radiotherapy after that regimen was shown in a large phase Radiation Biomarkers in Head and Neck Cancer—Ow et al

III trial8 to improve the duration of locoregional control in patients with HNSCC who received radiation therapy compared with those who received radiation alone. The addition of cetuximab to cisplatin-based chemoradiation was not, however, shown to provide benefit.15 Expression of the EGFR receptor in HNSCC is common, and this marker has not been shown to be of value as a biomarker for response to cetuximab.8,93 How cetuximab improves response of patients with HNSCC to radiation is not clear. A recent publication94 demonstrated that cetuximab reduces expression of HIF1a, leading to increased sensitivity to radiation in the HNSCC cell lines. In colorectal cancer, a KRAS mutation, which results in constitutive activation of pathways just downstream of EGFR, is a predictive biomarker for cetuximab response (ie, tumors with wildtype Kirsten rat sarcoma viral oncogene homolog [KRAS] are sensitive to cetuximab).95 A KRAS mutation is not common in HNSCC, but Harvey rat sarcoma viral oncogene homolog (HRAS) mutations have been described.58,60 Whether HRAS mutations or lack thereof are predictive of response to EGFR pathway inhibition has yet to be determined. EGFR is not the only target in the EGFR signaling pathway, and a number of other monoclonal antibodies and tyrosine kinase inhibitors that inhibit EGFR (eg, gefitinib and erlotinib) are currently under investigation as therapeutic options in HNSCC.96,97 MARKERS RELATED TO THE DNA DAMAGE RESPONSE Human cells have evolved a very complex and intricate system for responding to and repairing DNA damage. The details of the molecular signaling pathways involved in the DNA damage response (DDR) are beyond the scope of this review, but because response to radiation is inherently related to the cellular response to DNA damage, a brief summary of this process follows. Generally, when a cell experiences significant DNA damage, molecular sensors identify the damage, triggering steps to halt the cell cycle so that errors are not propagated. Then either repair of the DNA defect is initiated or processes lead to termination of the cell, often via apoptosis. Several different types of DNA damage can occur, and each type has a specific repair system with several key molecules involved. For example, single-strand breaks can be repaired by the base excision repair pathway or the single-strand break pathway; DNA adducts, such as those caused by ultraviolet radiation, can be repaired by the nucleotide excision repair pathway; doublestrand breaks are typically repaired by homologous recombination or via the nonhomologous end joining repair system. As described earlier, ionizing radiation generally leads to the production of reactive oxygen species that cause double-strand breaks. The first step in the DDR is recognition of the damage, and several canonic pathways have been described. When a double-strand break occurs in DNA, the defect is recognized by a complex formed by MRE11 meiotic recombination 11 homolog A (MRE11A), RAD50 homolog (RAD50), and nibrin (NBN)—the MRN complex—which recruits the ATM serine/threonine kinase (ATM), leading to subsequent phosphorylation of H2A histone family, member X (H2AX). A signaling cascade is then initiated via checkpoint kinase 2 (CHK2) activation, which halts the cell cycle. ATM also phosphorylates p53, leading to activation of this tumor suppressor, triggering transcriptional programs that halt the cell cycle, lead to DNA repair, or initiate apoptosis. SingleArch Pathol Lab Med—Vol 139, November 2015

stranded DNA breaks are also recognized via a similar, but alternative, system. The ATR serine/threonine kinase (ATR) is recruited to sites of single-stranded DNA breaks, and this initiates a signaling cascade via activation of checkpoint kinase 1 (CHK1), again leading to cell cycle inhibition and DNA repair. Several other well-described tumor suppressors are involved in these DDR pathways, including breast cancer 1, early onset (BRCA1), breast cancer 2, early onset (BRCA2), and poly (ADP-ribose) polymerase 1 (PARP1). Protein kinase, DNA-activated, catalytic polypeptide (DNAPKcs) and the Ku70/Ku80 complex are other important molecules in the response complex formed when doublestranded DNA breaks occur. Double-stranded break recognition and signaling molecules, such as those described above, lead to a complex series of processes that repair and maintain the integrity of DNA. The homologous recombination process repairs DNA by exchanging similar regions of chromosomes between sister chromatids, requiring a normal template to repair the damaged strand. There are 2 models described by which this occurs: the double-strand break repair model and the synthesis-dependent strand annealing pathway. Nonhomologous end joining repairs double-stranded DNA breaks without necessitating an unaltered template, and several key molecules are involved in tethering the broken ends, processing the ends, and ligating the DNA back together. These mechanisms represent the primary methods by which cells repair the damage caused by ionizing radiation. We direct readers to works by Moding and colleagues98 and Curtin99 for detailed reviews of DNA damage repair pathways and methods for exploiting those processes to treat cancer. In some tumor types, knowledge of alterations in the DDR has been exploited to create potentially beneficial therapeutics. For example, PARP1 inhibitors are being studied in patients with BRCA mutation-positive breast and ovarian cancers.100 The DDR pathways have been explored in HNSCC, but the role of alterations in DDR in HNSCC is still not well understood, and therapies based on the specific alterations in the DDR are in their infancy. Although the BRCA mutation in HNSCC is rare, targeting of the DNA damage pathway, specifically disruption of the MRN complex101 and PARP inhibition,102 has been explored as a therapeutic strategy for HNSCC in preclinical models. Moeller and colleagues103 examined DNA double-stranded break repair proteins using IHC analysis on a tissue microarray among a series of 89 HNSCC specimens, all from patients treated with intensity-modulated radiation therapy. Overexpression of Ku80 was associated with locoregional failure and mortality.103 In a recent work published by Dok et al,104 results showed an association of p16Ink4A expression and decreased recruitment of RAD51 recombinase (RAD51) to sites of DNA damage. Results suggested decreased homologous recombination events with increased nonhomologous end joining activity—the repair process that is more error prone—thus suggesting a potential mechanism for the increased sensitivity to ionizing radiation demonstrated by HPVþ OPSCCs. Most recently, work by Banerjee and colleauges105 demonstrated that thyroid hormone receptor interactor 13 (TRIP13) was commonly overexpressed and amplified in HNSCC tumors. They showed that TRIP13 enhanced nonhomologous end joining and reduced sensitivity to cisplatin. Furthermore, cells expressing TRIP13 were more sensitive to DNA-PKcs inhibitors.105 Radiation Biomarkers in Head and Neck Cancer—Ow et al 1383

The DDR is inherently linked to sensitivity and resistance to radiation therapy. It is hoped that the continued discovery of alterations in the DDR among HNSCC will lead to improvements in therapeutics that can potentially enhance response to radiation. OTHER POTENTIAL MOLECULAR BIOMARKERS There are a myriad of potential biomarkers that have been studied in HNSCC, and with the advent of modern genomics, epigenomics, and transciptomics, and the rapidly evolving field of molecular therapeutics, the list is growing. The BCL2 family is a class of proteins involved in the regulation of apoptosis, which is essential to cell development, integrity, and protection, and a pathway leading to tumor cell death after radiation therapy.106 BCL2 and BCL2like 1 (BCL2L1) inhibit apoptosis by inhibiting mitochondrial release of cytochrome c,107 and both have been implicated in radioresistance of head and neck tumors. Multiple studies have shown a relationship between radiation resistance and expression of BCL2 and BCL2L1. The IHC expression of BCL2L1 was associated with tumor recurrence following radiation therapy in patients with oropharyngeal SCC108 and laryngeal SCC.109 Another study examining IHC expression of BCL2L1 found no association between BCL2L1 expression and event-free survival.110 However, this endpoint included progression, tumor recurrence, or death from any cause, which may have confounded the results. The remaining studies examining BCL2 yielded conflicting results. In patients with HNSCC, BCL2þ tumors, determined by IHC expression, were associated with a lower risk of local failure compared with BCL2negative tumors.68,111 In a study of 400 patients with head and neck cancer, BCL2 expression was associated with decreased locoregional recurrence.112 In contrast, BCL2 overexpression was significantly associated with radioresistance in HNSCC113 and laryngeal SCC69,109 and predicted radiotherapy outcome with 71% accuracy.109 In an alternative study, BCL2 was not found to be associated with treatment response in either HNSCC114 or OPSCC when that factor was considered in a multivariate model with other standard prognosticators.108 Overall, these studies varied largely in their included tumor sites, which make comparison and application difficult. Further examination is warranted to solidify the role of these markers as prognosticators among HNSCC tumors treated with radiation. Transforming growth factor b (TGF-b) family of cytokines are involved in several cellular processes, and TGF-b signaling can have tumor-suppressive effects inhibiting cell growth and proliferation, oncogenic effects enhancing epithelial-to-mesenchymal transition and immune suppression, and is an important factor in processes leading to fibrosis, such as idiopathic pulmonary fibrosis and radiationinduced fibrosis.115 In addition, TGF-b has been shown to be involved in the DDR, and TGF-b loss appears to lead to reduced ATM activity and the inhibition of the DDR, leading to increased genomic instability.116 Therefore, TGF-b represents an interesting therapeutic target, which could enhance radiation sensitivity in tumors while reducing unwanted fibrotic reaction in normal tissues after therapy.115,117 The TGF-b cell membrane receptors signal through the SMAD family of molecules, and SMAD family member 4 (SMAD4) is frequently deleted in HNSCC.118 1384 Arch Pathol Lab Med—Vol 139, November 2015

Several other candidate biomarkers are actively being studied and have shown some associations with radiation response in HNSCC. For example, nuclear factor jb (NFjB) is a protein complex that acts as a DNA transcription regulator and is ubiquitously involved in programs that control cell motility, repair, and survival. Overexpression of NF-jB has been shown to be associated with local tumor recurrence among patients with larynx cancer treated with radiation.119 In another study, nuclear expression of NF-jB/ p65 was associated with poor overall survival, poor progression-free survival, and reduced distant metastasisfree survival among a cohort of patients with locally advanced HNSCC treated with chemoradiation.120 MET proto-oncogene (c-MET), an oncogene that encodes the hepatocyte growth factor/scatter factor receptor, has been implicated in cell invasion. Expression of hepatocyte growth factor/scatter factor has been shown to be an independent predictor of local failure in patients with HNSCC, and has also been shown to be associated with incomplete response to radiotherapy.108 The phosphoinositide 3-kinase (PI3K)/ protein kinase B (PKB or Akt) pathway has also been implicated as a predictor of response to radiation. This pathway is commonly upregulated in human cancers, and PI3K/Akt signaling has been shown to be involved in epithelial-to-mesenchymal transition and inhibition of apoptosis. Studies have demonstrated an association between activation of this pathway and radioresistance in HNSCC.121,122 The PI3K signaling pathway can be activated via several mechanisms, and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit a (PIK3CA) activating mutations, PI3K amplification, and phosphatase and tensin homolog (PTEN) deletion have all been observed in studies of HNSCC.58,60 GENE EXPRESSION ANALYSIS Thus far, this review has largely discussed single genes or individual signaling pathways as molecular biomarkers. A candidate gene or pathway approach explores markers that one hypothesizes could contribute to treatment response or outcome based on the known function of the studied markers or pathways. An alternative approach is to explore large descriptive molecular data sets to identify molecular markers in an unbiased manner. Gene expression microarray technology and newer high-throughput methods for evaluating the transcriptome, namely RNA-Seq, allows researchers to examine the expression of thousands of genes at once to discover novel genes or gene networks that may potentially contribute to a cancer cell phenotype.123 Gene expression microarrays measure the relative abundance of messenger ribonucleic acid (mRNA) transcripts from thousands of genes isolated in individual samples. Because mRNA transcription is a key level of gene regulation, relative expression of a given gene can be indicative of a gene’s activation or quiescence. Therefore, identification of differentially expressed genes between 2 defined groups (eg, responders versus nonresponders to radiation) is a potential method for discovering the underlying molecular mechanisms responsible for a cancer cell phenotype and represent potential prognostic or predictive biomarkers.124 In fact, in breast cancer, this approach has had relative success, and currently, 3 assays based on gene expression have been developed that are able to quantify a patient’s risk of recurrence.125 Similar success, Radiation Biomarkers in Head and Neck Cancer—Ow et al

Figure 2. Opportunities for molecular biomarkers to improve the use and efficacy of radiation for patients with head and neck squamous cell carcinoma (HNSCC). Note that all biomarkers and proposed strategies are potential applications and that currently all molecular biomarkers for head and neck squamous cancer require further validation, and applications require evaluation in well-designed clinical trials. Abbreviations: DNA, deoxyribonucleic acid; EGFR, epithelial growth factor receptor; HPV, human papilloma virus; OPSCC, oropharyngeal squamous cell carcinoma; PI3K, phosphoinositide-3 kinase.

however, has not yet been developed for head and neck cancers. Very few studies have looked at predictive biomarkers of radiation response in head and neck cancer, and those that do are limited by their small sample sizes and lack of validation. This could be due to HNSCC being molecularly heterogeneous. A study by Belbin and colleagues126 reported that there are a base set of genes that are consistently altered in all HNSCC tumors, but additional differentially expressed genes unique to the HNSCC subsite were best able to distinguish aggressive from less-aggressive tumors. There are few studies specifically evaluating radiation response in HNSCC. In one study,127 a 142-probe set signature was found to distinguish between 14 patients with HNSCC who responded to radiation, compared with those who did not respond. This signature was then found to accurately predict response by 100% in 5 subsequent tumor samples.127 In another study evaluating 35 patients with laryngopharyngeal cancer who received chemoradiotherapy, 17 of 277 genes were significantly associated with locoregional response. Using IHC, 7 of the 17 genes were validated on a separate set of 62 patient samples. Of those, 2 genes— MDM2 and erb-b2 receptor tyrosine kinase 2 (ERBB2)— were proposed to be associated with locoregional response.128 In the report by Akervall et al,129 gene expression analysis was performed on samples from 38 patients with HNSCC, and a 72-gene set was found to be significantly associated with radiation response. From this, the 5 strongest gene candidates were validated against a second group of patients. Those genes included VEGF, BCL2, claudin 4 (CLDN4), Yes-associated protein 1 (YAP1), and c-MET, and each was found to significantly predict recurrence-free survival following radiotherapy or chemoradiotherapy. A major limitation of this study, however, besides the small sample size, was that patients were not stratified based on HPV status.129 Only one gene set to date is known to have been validated across several sample sets. This 75-gene signature, which includes genes that are involved in epithelial-to-mesenchymal transition, NF-jB, and cell adhesion pathways, was Arch Pathol Lab Med—Vol 139, November 2015

found to predict worse recurrence-free survival in patients with HNSCC.130,131 In subsequent validation, this panel significantly predicted locoregional recurrence in 92 patients with HNSCC treated with chemoradiation, independent of other clinical prognostic factors, including HPV status.49,132 BIOMARKER APPLICATION The potential molecular biomarkers described above have not yet been incorporated into diagnostic, treatment, and surveillance practices for patients with HNSCC. The potential strategies to apply these markers are summarized in Figure 2. As genetic and molecular biomarkers become better understood, clinical application will require creative and rigorous evaluation with the goals of refining treatment to be more efficacious and less toxic and to improve survival and quality of life. CONCLUSION This review has highlighted several biomarkers that are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat HNSCC. Several promising candidates have been defined, and new markers are surely on the horizon. Biomarkers that lead to significant improvements in patient outcome will require rigorous validation and prospective testing. References 1. Wang X, Hu C, Eisbruch A. Organ-sparing radiation therapy for head and neck cancer. Nat Rev Clin Oncol. 2011;8(11):639–648. 2. Chao KS, Low DA, Perez CA, Purdy JA. Intensity-modulated radiation therapy in head and neck cancers: the Mallinckrodt experience. Int J Cancer. 2000;90(2):92–103. 3. Eisbruch A. Intensity-modulated radiation therapy in the treatment of head and neck cancer. Nat Clin Pract Oncol. 2005;2(1):34–39. 4. Braam PM, Terhaard CH, Roesink JM, Raaijmakers CP. Intensity-modulated radiotherapy significantly reduces xerostomia compared with conventional radiotherapy. Int J Radiat Oncol Biol Phys. 2006;66(4):975–980. 5. Bangalore M, Matthews S, Suntharalingam M. Recent advances in radiation therapy for head and neck cancer. ORL J Otorhinolaryngol Relat Spec. 2007;69(1):1–12. 6. Zundel MT, Michel MA, Schultz CJ, et al. Comparison of physical examination and fluorodeoxyglucose positron emission tomography/computed tomography 4–6 months after radiotherapy to assess residual head-and-neck cancer. Int J Radiat Oncol Biol Phys. 2011;81(5):e825–e832. doi:10.1016/j. ijrobp.2010.11.072.

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Radiation Biomarkers in Head and Neck Cancer—Ow et al