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Predicting Recurrence AfterRadiotherapy in Head and Neck CancerAdrian C. Begg, PhD

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancerworldwide. Radiotherapy is a mainstay of treatment, either alone for early stage tumors orcombined with chemotherapy for late stage tumors. An overall 5-year survival rate ofaround 50% for HNSCC demonstrates that treatment is often unsuccessful. Prediction ofoutcome is, therefore, aimed at sparing patients from ineffective and toxic treatments onthe one hand, and indicating more successful treatment modalities on the other. Bothfunctional and genetic assays have been developed to predict intrinsic radiosensitivity,hypoxia, and repopulation rate. Few, however, have shown consistent correlations withoutcome across multiple studies. Messenger RNA and microRNA profiling show promisefor predicting hypoxia, whereas epidermal growth factor receptor expression combinedwith other measures of tumor differentiation grade shows promise for predicting repopu-lation rate. Intrinsic radiosensitivity assays have not proven useful to date, althoughdevelopment of repair protein foci assays indicates promise from preclinical studies.Assays for cancer stem cell content have shown promise in several clinical studies. Inaddition, 2 assays showing robustness as predictors for outcome in HNSCC are humanpapilloma virus status and epidermal growth factor receptor expression. Neither these norstem cell assays, however, can as yet reliably indicate alternative and better treatments forpoor prognosis patients. It would be of great value to have assays that predict the benefitfor an individual from combining new molecularly targeted agents with radiotherapy toincrease response, in particular those that exploit tumor mutations to provide tumorspecificity. Predictive assays are being developed for detecting defects in repair pathwaysfor single- and double-strand DNA breaks, which should allow selection of drugs targetingthe appropriate backup pathway, thus exploiting the concept of synthetic lethality. This isone of the most promising areas for prediction, both currently and in the future.

Semin Radiat Oncol 22:108-118 © 2012 Published by Elsevier Inc.

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Head and neck squamous cell carcinoma (HNSCC) com-prises cancers of the oral cavity, oropharynx, larynx,

nd hypopharynx. It is the sixth most common cancer world-ide,1 with an incidence of around 600,000 cases per year.

The overall 5-year survival rate for such patients is around50% or less. Thus, there is considerable room for improve-ment in the treatment of HNSCC. Present treatment optionsinclude radiotherapy with or without chemotherapy, surgerywith or without radiotherapy or chemotherapy, and morerecently radiotherapy with molecularly targeted agents suchas cetuximab. The choice of treatment will depend on clinicalfactors, such as site, stage, and size of the tumor. The staging

Division of Experimental Therapy, The Netherlands Cancer Institute, Am-sterdam, The Netherlands.

Address reprint requests to Adrian C. Begg, PhD, Division of ExperimentalTherapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX

sAmsterdam, The Netherlands. E-mail: a.begg@nki.nl

108 1053-4296/12/$-see front matter © 2012 Published by Elsevier Inc.doi:10.1016/j.semradonc.2011.12.002

systems currently used for HNSCC account for �30% of thevariation observed in the survival rates.2,3 In addition to stag-ng systems (tumor, nodes, metastases classification), the

ost important clinical factor predicting outcome is tumorolume.4-6 However, there is considerable variation betweenatients in the chance of recurrence and survival even afterccounting for stage and tumor volume. Given the inherentenomic instability of cancers, including those of the headnd neck, it is likely that the large remaining variation can bexplained by biological factors that differ markedly betweenumors.

It would, therefore, be useful to predict beforehand whichatients will benefit from radiotherapy. It would be evenore useful to be able to offer patients who are predicted toave radioresistant tumors an alternative or additional treat-ent. Prediction of response to chemotherapeutic agents that

an be combined with radiation would, therefore, be of sub-

tantial benefit. With the current development of biological

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Radiotherapy in head and neck cancer 109

modifiers that increase the effects of radiation, prediction ofefficacy of such modifiers in an individual tumor would alsogreatly aid the clinician in the choice of treatment. Methodsto predict the chance of recurrence should, therefore, ideallybe developed for a range of possible treatment options.

Based on a large number of studies, 3 major biologicalfactors have been defined that determine the response ofHNSCC to fractionated radiotherapy, namely intrinsic radi-osensitivity of the tumor cells,7 the extent of tumor hyp-oxia,8,9 and the capacity of surviving tumor cells to repopu-ate during gaps in treatment.10,11 More recently, humanpapilloma virus (HPV) infection in the tumor has been shownto be a significant outcome predictor, particularly in oropha-ryngeal cancer (12and refs therein). In addition, host factorsan influence tumor behavior, such as tumor vascularity andost cell infiltrates. It is likely that both HPV and host factorsffect tumor response to radiotherapy by modifying 1 orore of the 3 major biological processes (intrinsic radiosen-

itivity, hypoxia, repopulation capacity).For advanced tumors, radiotherapy is usually combined

ith chemotherapy, often cisplatin or taxanes.13 Becausehere are usually unwanted side effects of varying severityith the use of these agents and because they can also in-

rease radiation-induced morbidity, the availability of robustredictors of their efficacy would aid the decision as to theirse. Sparing patients unwanted morbidity from drugs of littler limited efficacy in their particular tumor would also rep-esent a step forward in patient management.

In addition to classic chemotherapeutic agents, the lastecade has seen the development of many molecularly tar-eted agents, designed to be more specific and less toxic. Therchetypical example in head and neck cancer is the epider-al growth factor receptor (EGFR) inhibitor cetuximab,hich has shown improved response in a randomized radio-

herapy trial.14 Many other agents targeting genes or path-ays often found to be deregulated in cancer are being de-eloped, and many of these are being tested in the clinic.ome examples of targeted pathways are phosphoinositide-kinase (PI3K), nuclear factor kappa light polypeptide genenhancer in B cells (NF-KB1), mitogen-activated protein ki-ase (MAPK), and angiogensis.15 Such agents are also notffective in every tumor and are also not innocuous but usu-lly (almost always) associated with side effects. Thus, foroth efficacy and toxicity of such agents, having good pre-ictors of their efficacy in individual tumors would be ofreat benefit.

Lastly, the most promising anticancer agents are those thatre tumor specific, having little, or a far smaller, effect onormal tissues. Developing such agents has been the aim ofuch research over decades and represents the holy grail of

ancer research and treatment. The concept of synthetic le-hality has emerged in recent years as one that can be appliedo cancer therapy with precisely the goal of tumor-specificill. The concept is that 2 pathways must be inactivated to be

ethal to the cell, whereas inactivation of any one pathway isot. This can occur when 1 pathway serves as a backup for

nother, so that the backup takes over function if the primary c

athway is defective, thus saving the cell from lethal conse-uences.Tumors are by definition genetically unstable, harboring

p to thousands of mutations.16,17 Thus, many tumors haveenetic defects affecting damage response pathways. Suchumor-specific mutations can be exploited by applying arug that inactivates the backup pathway. Such an approachill have little effect on normal tissues because they are pro-cient in the primary pathway, and thus resistant to knock-

ng out the backup. The most promising example to date ishe use of poly ADP ribose polymerase (PARP) inhibitors inatients with breast-cancer-early-onset (BRCA) gene-defi-ient tumors.18-20 Here, unrepaired single-strand DNA

breaks (SSB) are converted to double-strand breaks (DSB)during replication attempts and are subsequently repaired bythe DSB repair pathway of homologous recombination (HR).The breast cancer susceptibility genes BRCA1 and BRCA2function in the HR pathway. PARP inhibitors reduce SSBrepair, leading to reliance on HR to repair the DNA damage.Tumors with HR defects such as in BRCA patients are thushighly sensitive to this drug treatment and represent a goodexample of synthetic lethality, that is, drug inhibition of onepathway combined with an inactivating mutation in theother. A new and potentially valuable aspect of prediction is,therefore, to define in an individual tumor which pathway(s)are defective, and then exploit this by treating with an appro-priate drug targeting backup pathway.

These various aspects of prediction are discussed later,specifically in relation to HNSCC, with treatments involvingradiotherapy. Hypothesis-driven approaches will be dis-cussed first, in which predictors have been sought for biolog-ical processes known to influence outcome. These includeintrinsic radiosensitivity, hypoxia, repopulation rate, andothers. Data-driven approaches will then be discussed, usu-ally involving large-scale, often genome-wide, genetic screen-ing where any genes or their combinations are sought, whichbest correlate with outcome. This can provide information onhitherto unconsidered genes or processes that can determineoutcome.

Intrinsic RadiosensitivityFunctional assays were first developed in which cell suspen-sions were made from pretreatment tumor biopsies and thenassayed for radiosensitivity in vitro using clonogenic assays.This was first carried out in cervix tumors,21 and then appliedto head neck tumors.7 In both cases, patients with tumorshat were more radioresistant than average when assayed initro had a worse outcome than those with more radiosensi-ive tumors. This provides proof-of-principle that intrinsicadiosensitivity is a significant factor in determining outcomen HNSCC. However, the technique cannot be routinely ap-lied because it takes too long and requires specialized lab-ratories and personnel not available in most hospitals.A number of shorter term surrogate assays for radiosensi-

ivity were then sought, including DNA breaks, chromosomeberrations, and apoptosis.22 Results of these assays as out-

ome predictors in the clinic have been variable, often ham-

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pered by difficult reproducibility and the disadvantage thatthe correlation between these surrogate assays and direct as-says of radiosensitivity in cell lines has often been poor. Inwhat could be regarded as an extension of a chromosome orDNA break approach, genomic amplifications and deletionson a genome-wide scale have been measured using the nowrobust technique of comparative genomic hybridization(CGH).23 Specific genomic alterations have been reportedhat correlate with radiosensitivity in HNSCC cell lines.24

Applying CGH to HNSCC biopsies, van den Broek et al25

found significant correlations of particular chromosome re-gion gains and losses with outcome after combined radiationand cisplatin treatment. As with many predictor studies, thishas not been independently validated and suffers the disad-vantage that no mechanistic information is provided, and sothat indications of how to treat resistant patients are lacking.Further studies are needed to confirm the result and to findwhich genes on the altered regions are causal.

A more recent functional assay has arisen from molecularstudies of the DNA damage response. The most lethal DNAlesion induced by ionizing radiation is the DSB. Within min-utes after DSB induction, a subtype of histone called H2AX, achromatin protein, is locally phosphorylated, usually byataxia telangiectasia mutated kinase (ATM, the gene mutatedin ataxia telangiectasia patients). The phosphorylation is ex-tensive, extending to a megabase around each DSB. Antibod-ies have been developed that specifically recognize phos-phorylated H2AX (called gamma-H2AX or �-H2AX). Theseantibodies reveal the presence of discrete foci of �-H2AXfter irradiation, each focus representing (with a few excep-ions) a DSB. This allows the sensitive measurement of induc-ion and repair of DSB, one of the most important factors inetermining radiosensitivity. In a number of studies, goodorrelations have been found between residual �-H2AX foci

24 hours after irradiation and intrinsic radiosensitivity.26 Theechnique is now also being applied in clinical studies. Thisnd other foci methods represent a promising approach toadiosensitivity prediction. To date, only one fairly smalllinical study has been carried out where no significant cor-elation was found between residual �-H2AX foci and out-ome in cervix cancer patients given radiotherapy.27 How-

ever, a recent preclinical study suggests that foci need to bestudied in well-perfused areas of the tumor only,28 which was

ot done in the clinical study. More and larger clinical studiesre needed.

In recent years, there has been a logical transition fromunctional studies to powerful genetic assays. In particular,enome-wide gene expression assays using microarray tech-ology have been used to find gene sets that correlate with,nd thus predict, radiosensitivity. Two such gene signatureredictors have been reported by analyzing all or part of theational Cancer Institute panel of 60 tumor cell lines.29,30

Unfortunately, the cell line panel does not contain anyHNSCC cell lines. In our laboratory, we therefore attemptedto find a radiosensitivity gene signature in a panel of 33HNSCC cell lines, although only weak and insignificantlycorrelating gene sets could be found (de Jong M and Begg AC,

unpublished data). The Torres-Roca et al29 signature was not

found to correlate with outcome in a series of patients treatedwith radiotherapy plus cisplatin,31 although a modified sig-nature was found to be marginally significant.32 The Amund-son et al30 signature derived from the full National CancerInstitute panel of 60 tumor cell lines was not found to bepredictive in a series of laryngeal carcinomas treated withradiotherapy alone.33

The lack of strong predictive power of these signaturescould be because of several reasons. Intrinsic radiosensitivitymay not be a significant factor determining outcome, al-though this is unlikely given the large variation in radiosen-sitivity of HNSCC cell lines and the functional (clonogenic)assay data.7 A more likely cause is limitations of the in vitrocell line models, which have usually undergone many pas-sages in the artificial in vitro growth conditions and whichmay, therefore, not accurately represent the in vivo situation.A recent study found �2200 significant promoter methyl-ation differences between HNSCC cancer cell lines and pri-mary tumors but no significant differences between primarytumor xenografts and primary tumors in the patient,34 illus-rating the extent of changes that can occur during the culturef cell lines. Better in vitro models are necessary and are noweing developed by several groups.

HypoxiaAs with radiosensitivity predictors, the first proof-of-princi-ple studies showing that tumor hypoxia is an important out-come determinant came from functional assays, in particulardirect measurements of oxygen tension by electrodes in-serted into the tumor.35,36 These studies showed that hypoxia

as a negative prognostic factor not only for radiotherapy butlso for surgery and chemotherapy. For radiotherapy, therobable explanation is that hypoxic cells are 2-3-fold moreadioresistant than normoxic cells. For chemotherapy, hy-oxic cells often reside in effective pharmacological sanctu-ries, far removed from the blood (and thus drug) supply. Inddition, hypoxic cells are often non- or slowly cycling, aug-enting drug resistance. For surgery, hypoxia has been

hown to lead to selection of more apoptotic resistant cells37

and to a more metastatic phenotype.38 For HNSCC, severaltudies using oxygen electrodes have shown that high hyp-xia is associated with worse outcome after radiotherapy.8,39

The technique is, however, limited to accessible tumors andis by nature invasive. Much effort has, therefore, been ex-pended to find robust surrogate markers for tumor hypoxia.

Several gene signatures for hypoxia have been definedfrom cell culture studies in which changes in gene expressionhave been monitored after exposure of the cells to differenttimes and degrees of hypoxia.40,41 The in vitro derived hyp-xia signature proved to be predictive in breast cancer.40 In

larynx cancer treated with radiotherapy alone, it also showeda correlation with local control in a univariate analysis, al-though significance was lost after correcting for multiple test-ing.33 An alternative approach by Winter et al42 was to definea hypoxic metagene, comprising genes that were coregulatedwith 10 “seed” genes known to be hypoxia responsive (be-

cause they belong to the hypoxia inducible factor (HIF) path-

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Radiotherapy in head and neck cancer 111

way) across a series of head and neck tumors. Thus, thederived 99 gene set represents an in vivo hypoxia signature.This proved a significant predictor of outcome after surgeryin HNSCC.42 However, it was not predictive after combinedradiotherapy and cisplatin for HNSCC,33 although it showeda nonsignificant trend with outcome after radiotherapy forearly larynx cancer.33

MicroRNAs (miRNAs) have been shown to regulate theexpression of up to 50% of human genes, including thoseinvolved in the response to radiation,43-45 and are often de-egulated in cancer.46 Expression profiling of miRNAs is

therefore a valuable additional tool in searching for outcomepredictors, and it gives additional and independent informa-tion than messenger RNA (mRNA) profiling. Some miRNAshave been shown to be hypoxia inducible and one of thesehas been shown to have prognostic significance in HNSCCtreated with postoperative radiotherapy.47 This may prove tobe a good and relatively simple surrogate marker for hypoxia,applicable in most centers, although this result needs confir-mation in independent trials.

The transcriptional, translational, and posttranslational re-sponse to hypoxia has received considerable attention, lead-ing to detailed depiction of the HIF pathway. Briefly, HIF-1�is upregulated under hypoxia through inhibition of degrada-tion, binds to its nonhypoxia responsive partner HIF-1�which together function as a transcription factor, switchingon several target genes with hypoxia-responsive elements intheir promoters.48 Such HIF-responsive genes include car-bonic anhydrase (CA9), glucose transporters such as GLUT1,and vascular endothelial growth factor, among others. Con-sequently, expression of HIF-1� itself as well as several ofhese downstream target genes has been studied as possibleurrogates for hypoxia using standard immunohistochemicalethods. However, correlations with outcome have been

ariable,49 one confounding factor being that almost all suchenes are also regulated by factors other than hypoxia, andhese factors can vary between tumors resulting from theirenomic instability. For example, this can result in all cells intumor being HIF-1� positive despite only a minor hypoxic

fraction, or no positive cells despite the presence of hyp-oxia.50 However, given that several studies have shown cor-elations of HIF-1� and the related HIF-2� expression with

outcome, this gene family may also influence response inde-pendently of hypoxia. CA9 shows one of the highest extentsof upregulation under hypoxia, but was not predictive foroutcome in a large randomized radiotherapy trial forHNSCC.51 This is probably due its lack of specificity as ahypoxia marker, as in similar patient cohorts, hypoxia wasshown to be a limiting factor (the hypoxic cell radiosensitizernimorazole significantly increases response to radiother-apy.52) It is possible that combining more than one HIF-

ependent marker may increase hypoxia specificity, as wasound in a study of CA9 and GLUT1.53 This also needs inde-endent confirmation.Finally, one determinant of the extent of tumor hypoxia is

he vasculature, which is often chaotic and insufficient inroviding adequate perfusion to all tumor regions but shows

onsiderable variation between tumors. Therefore, several

tudies have attempted to use vascular parameters as out-ome predictors, with mixed success. One of the largest suchtudies in a randomized radiotherapy trial involving �400NSCC patients showed that microvascular density was not

ignificantly correlated with outcome.54 Such parameters areherefore unlikely to provide robust predictors in HNSCC.

Repopulation RateIt is clear from many radiotherapy trials for HNSCC in whichthe overall time has been varied, that shorter treatment (suchas accelerated fractionation) gives increased local control.55

This is likely because of the rapid repopulating capacity ofHNSCC cells during gaps in treatment. Shortening the over-all time reduces the opportunity for such repopulation, re-sulting in increased cell kill for a given delivered radiationdose. However, although late responding normal tissues (bydefinition slowly proliferating) will not be affected bychanges in the overall time, early responding (proliferating)normal tissues, such as buccal and intestinal mucosa, willsuffer extra damage by limiting recovery through shorteningthe treatment. It would therefore be valuable to have a pre-dictor of tumor repopulation rate, so that patients withslowly repopulating tumors can be spared the extra toxicityassociated with accelerated fractionation schedules or anti-proliferative drugs.

Early functional assays involved administration of tracerdoses of thymidine analogs that are incorporated by prolifer-ating cells and detected using immunohistochemistry or flowcytometry. Thus, they provide direct measures of tumor pro-liferation. Such studies in HNSCC showed that the fraction oflabeled cells (labeling index, LI) was only marginally signifi-cant as a predictor of local control, whereas the potentialdoubling time of the tumor was not.56

Because these measurements require administration of atracer and did not provide robust predictors, surrogate andalternative markers for repopulation rate have been sought.These include expression of proliferation dependent genes,such as various cyclins (A, D, E), Ki-67 (MKI67), and prolif-erating cell nuclear antigen. In one of the few studies ofreasonable size of radiotherapy alone for HNSCC, Couture etal57 found that low Ki-67 expression, as assessed by immu-nohistochemistry, was a bad prognostic indicator. Two otherstudies came to the same conclusion.58,59 This is contrary to

hat would be expected based on the hypothesis that theore rapidly proliferating tumors (high Ki-67) would be as-

ociated with a worse outcome. One factor that could providepartial explanation is that pretreatment proliferation ratesay not reflect repopulation rates during treatment, whichay accelerate in response to damage.10 Slowly proliferating

umors before treatment may have the capacity for rapidepopulation during treatment.

Support for this hypothesis comes from 2 studies of accel-rated fractionation.60,61 Both studies showed that patients

with high EGFR-expressing tumors benefitted from acceler-ated fractionation schedules. They also showed significantcorrelations between EGFR expression and differentiation

grade (higher EGFR, more differentiated). The suggestion is,

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therefore, that differentiated tumors are more similar to nor-mal epithelial tissues in their capacity to respond to damageby accelerated repopulation. Further support derives from astudy showing that tumors with negative TP53 and BCL2 andlow and organized pattern of Ki-67 staining benefit signifi-cantly from accelerated schedules.62 These characteristics aregain consistent with a greater differentiation. It thus appearshat the response capacity, rather than undisturbed prolifer-tion rate, could be the important factor and a predictor ofepopulating ability.

Several gene signatures specific for proliferating cells haveeen reported, some of which have been tested as predictors

n the clinic. In general, high proliferation rate has been a badrognostic factor in several cancer types.63 However, in HN-CC patients treated with combined radiotherapy and cispla-in31 or with radiotherapy alone,33 proliferation signatures

were not predictive. Some proliferation signatures have beenshown to correlate with the thymidine analog LI in humantumor xenografts.64 However, LI has not been proven to be aobust outcome predictor in HNSCC,56 consistent with the

failure of gene signatures. The explanation again may lie inthe fact that pretreatment proliferation rate is not a goodmonitor of repopulating ability.

Signal TransductionVarious signal transduction pathways have been shown toaffect radiosensitivity, including the PI3K/AKT (murine thy-moma viral oncogene homolog 1), NFKB1, MAPK, and trans-forming growth factor beta pathways.15 The PI3K/AKT path-way in particular has received much attention. Activation ofthis pathway is associated with radioresistance, and its inhi-bition can radiosensitize cells.65 In addition, AKT activationn HNSCC tumors, as indicated by phosphorylated AKT de-ected immunohistochemically, has been reported to be as-ociated with a worse outcome after radiotherapy.66 Phos-

phorylated AKT was also a bad prognostic factor in a series ofHNSCC treated with surgery with or without radiotherapy orchemotherapy.67 Both of these studies were relatively smallnd need confirmation, but are consistent with a consider-ble body of preclinical data.

One of the most important membrane receptors that driveshe PI3K/AKT pathway is the EGFR, which is overexpressedn many HNSCC tumors and is correlated with a poor re-ponse68,69 (Fig. 1). Measurement of EGFR expression can,herefore, provide prognostic information relevant to radio-herapy. Realization of the important role of EGFR in tumorehavior and treatment resistance has also led to develop-ent of EGFR inhibitors, either antibodies against the recep-

or or drugs that inhibit the tyrosine kinase activity of thectivated receptor. This, in turn, led to the testing of cetux-mab in combination with radiotherapy, which resulted in anmproved response.14 It would be valuable to be able to pre-dict which patients benefit from cetuximab or related anti-EGFR agents. However, the exact determinants of responseto cetuximab and other agents in HNSCC are unclear,70 andthus, robust predictors of response to these molecularly tar-

geted agents still need to be found. g

There are a considerable number of preclinical studies linkingthe NF-KB, MAPK, and transforming growth factor beta path-ways with radioresistance, including studies showing that path-way inhibition can radiosensitize.15 However, as far as this au-hor is aware, no clinical studies in HNSCC of sufficient sizeave been carried out correlating activation of these pathwaysith outcome after radiotherapy. Therefore, it is not possible atresent to judge the potential of such activation measurementsor these pathways as predictors in HNSCC.

HPV StatusIn the last few years, it has become increasingly clear thatHPV infection in HNSCC, and in oropharyngeal tumors inparticular, is a good prognostic marker.12,71 This is also truefor HNSCC patients treated with radiotherapy (Fig. 2).72,73

Several methods are available for testing HPV status, includ-ing detection of viral genomic integration with polymerasechain reaction or fluorescence in situ hybridization, detec-tion of viral gene expression, particularly E6 and E7, and theexpression of p16 (cyclin-dependent kinase inhibitor 2A).Immunostaining for p16 expression has proven to be a reli-able predictor of HPV infection status, improved further bycombining with genomic polymerase chain reaction for viralintegration.74 Both these techniques are routinely available in

ost centers. Gene expression signatures have also been de-eloped for distinguishing HPV-positive and HPV-negativeumors.75,76 These have also proved predictive in HNSCC,

such that tumors exhibiting high expression of genes associ-ated with HPV positivity have a better outcome.77

The reason why patients with HPV-positive tumors do betteris not clear. However, 2 studies suggest it may be related tohypoxia. Lassen et al78 showed that only patients with HPV-egative tumors benefitted from the hypoxic cell radiosensitizerimorazole, whereas HPV-positive patients did not. This sug-

Figure 1 Predictive value of epidermal growth factor receptor ex-pression in head and neck cancer treated by conventionally fraction-ated radiotherapy. High epidermal growth factor receptor predicts aworse outcome. (Redrawn from Ang et al68) (Color version of figures available online.)

ests that HPV positivity is associated with less hypoxia, and

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Radiotherapy in head and neck cancer 113

thus a better response to radiotherapy. Brockton et al79 showedhat HPV-negative tumors had high expression of the hypoxiaarker CA9 in stromal regions, consistent with the Lassen

tudy. Of potential interest is that one would normally expecthe stroma to be well oxygenated because it contains the vascu-ature. However, if some blood vessels have temporary low or noow, this would lead to high CA9 staining. Intermittent or acuteypoxia can occur in such situations, and so one could speculatehat HPV-negative tumors have a particular problem with acuteypoxia, probably a more dangerous type (cells are more viable)han chronic hypoxia.9

Cancer-Initiating (Stem) CellsIt has become apparent that only a minor fraction of tumor

Figure 2 Two independent studies showing that human papillomavirus-related p16 expression is a strong predictor of outcome inhead and neck squamous cell carcinoma. (A) A total of 316 patientsgiven conventional or accelerated fractioned radiotherapy, eachcombined with cisplatin. (Redrawn from Ang et al.)73 (B) A total of156 patients treated with conventional radiotherapy alone in thecontext of a randomized trial. (Redrawn from Lassen et al72) (Colorersion of figure is available online.)

cells in many cancer types is capable of regrowing the tumor,

giving rise to the concept of cancer stem cells or cancer-initiating cells.80 This concept can also have an impact onadiotherapy and outcome prediction.81,82 Tumors with aigher fraction of stem cells will be more difficult to cure withadiotherapy. This is not dependent on whether the stemells are inherently more resistant to radiation but simply thator a given tumor volume, there will be more cells to kill if themportant stem cell fraction is higher. There is, as yet, insuf-cient evidence to answer the question of whether stem cellsre more resistant to radiation than bulk tumor cells, al-hough stem cells have been reported to reside in a hypoxiciche, implying radioresistance.82 One way or another, the

stem cell concept implies that assays to monitor stem cellnumber would help in predicting outcome after radiother-apy.

In early stage laryngeal cancer treated with radiotherapyalone, expression of the putative stem cell marker CD44 wasshown to predict local control using both microarray tech-nology to monitor mRNA expression and, in a separate clin-ical series, immunohistochemistry to monitor protein ex-pression (Fig. 3A).33 Pramana et al31 also reported that bothCD44 and another stem cell signature83 showed a strong but

onsignificant trend with outcome after combined radiother-py plus cisplatin in HNSCC. Wu et al84 showed that coex-

pression of the 2 stem cell markers GRP78 (heat shock 70kDaprotein 5) and NANOG correlated with a worse prognosis inHNSCC treated with surgery with or without radiotherapy orchemotherapy. Thus, high stem cell content appears to be afactor determining treatment outcome after radiotherapy inHNSCC, and these data suggest ways to monitor it.

Epithelial Mesenchymal TransitionTumor cells can undergo a change in morphology from anepithelial to a mesenchymal phenotype, called epithelialmesenchymal transition (EMT), which influences the cell’sinvasive and metastatic behavior.85,86 More recently, associa-tions between EMT and response to radiation have beenfound. Chung et al87 defined a high-risk gene signature pre-dicting HNSCC outcome after surgery with or without radio-therapy or chemotherapy. This signature contained severalEMT genes. Pramana et al31 subsequently showed that thissignature was predictive in HNSCC treated with radiother-apy plus cisplatin (Fig. 3B). In our laboratory, we showedthat forcing EMT in HNSCC cells by expressing relevant tran-scription factors resulted in increased radioresistance (deJong M and Begg AC, unpublished data). Theys et al88 re-cently reported that E-cadherin loss associated with EMTpromotes radioresistance in human tumor cells. However, ina study of 340 HNSCC patients, Marsit et al89 showed thathypermethylation of the E-cadherin promoter, thus down-regulating E-cadherin expression, was an independent pre-dictor of improved survival in HNSCC. This is contrary towhat would be expected if E-cadherin loss, and thus EMT,promotes radioresistance in vivo. The importance and role ofEMT in determining outcome, and its use as a predictor,

therefore remains uncertain at this time.

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Synthetic LethalityAs described in the introduction, one of the most promisingapproaches to cancer treatment that has emerged in the lastdecade is the exploitation of synthetic lethality, its great ad-vantage being tumor-specific cell kill. The archetypical exam-ple of the large increase in efficacy of PARP inhibitors inBRCA-deficient tumors has been translated successfully fromthe laboratory18,19 to the clinic, with promising early resultsin breast cancer.90 The 2 pathways involved are the basexcision repair (BER) pathway for repairing SSB and the HRathway for repairing DSB, the latter providing a backup forhe former. Therefore, prediction of HR deficiency would be

Figure 3 Two studies showing gene expression predicting outcomein head and neck cancer. (A) A total of 52 patients with early stagelarynx cancer given radiotherapy alone. High expression of the pu-tative stem cell marker CD44 predicted poor outcome (From thestudy of de Jong et al.33) (B) A total of 96 patients with advanced

ead and neck cancer given radiotherapy combined with cisplatin.igh expression of the Chung high-risk signature genes87 predicted

poor outcome (Redrawn from Pramana et al.31) In both studies,ene expression at the messenger RNA level was determined inretreatment biopsies using microarray technology (Color versionf figure is available online.).

aluable for predicting the efficacy of PARP inhibitors. l

BRCA status is an obvious predictor choice, which can bessessed by mutational analysis using DNA sequencing. Inddition, methylation of the BRCA1 promoter, with conse-uent reduced gene expression, has been shown to predictensitivity to PARP inhibition.91 A further possibility is these of CGH, where specific patterns of amplifications andeletions can define BRCA1 and BRCA1-associated deficien-ies.92 This will pick up more relevant deficiencies thanCRA1 analysis alone.Genes other than BRCA1 and BRCA2 are also involved in

R, deficiencies that will affect PARP efficacy. Willers et al93

used functional radiation-induced RAD51, Fanconi anemiagroup D2 protein, and BRCA2 foci assays in tumor biopsiesto show a higher proportion of tumors with HR deficienciesthan expected from BRCA deficiency alone. The Fanconi ane-mia pathway genes are also on the HR pathway. Two studieshave shown promoter methylation and downregulation ofFanconi anemia genes in a proportion of HNSCC,94,95 pro-viding an additional possible predictor. Further, Mendes-Pereira et al96 showed that phosphatase and tensin homologPTEN) mutations correlated with reduced HR capacity, re-ulting in cells with PTEN-deficient cells being more sensitiveo PARP inhibitors. Therefore, monitoring PTEN mutationaltatus potentially provides an additional predictor of syn-hetic lethality with PARP inhibitors.

Some tumors have deficiencies in the BER pathway, andARP inhibition should have little benefit in these.97 In such

tumors, it would be valuable to inhibit the HR backup path-way. This represents an example of synthetic lethality, whichis the inverse of the earlier example, namely mutation in BERplus chemical inhibition of HR, as opposed to mutation in HRplus chemical inhibition of BER.97-99 Predicting which tu-mors will benefit from HR inhibition will therefore requiremonitoring BER deficiencies by methods such as DNA se-quencing, gene expression, or promoter methylation.

Several other examples of synthetic lethality for cancertreatment, particularly with drugs, have been reported,100

although few have yet been tested in the context of radiother-apy.

Data-Driven FindingsMost of the earlier discussions concern what can be regardedas hypothesis-driven approaches, involving searching forpredictors of biological processes known or suspected to in-fluence the response of tumors to fractionated radiotherapy.A few other potential predictors have been found from data-driven approaches, in which genome-wide screening hasbeen used to find any genes or genetic alterations that corre-late with outcome. Wreesmann et al,101 using CGH, found 5pecific amplifications and deletions that correlated with out-ome in HNSCC patients treated with surgery with or with-ut radiotherapy, or combination radiotherapy and chemo-herapy. In a follow-up study, van den Broek et al25 usedGH to study HNSCC patients treated with radiotherapylus cisplatin. Several recurrent amplifications and deletionsere found that correlated with outcome, although there was

ittle overlap with the Wreesmann results. At present, these

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Radiotherapy in head and neck cancer 115

cannot be regarded as robust predictors. Furthermore, suchchanges will only prove useful with confirmation and bytracing which genes on which chromosome regions arecausal for failure.

In some cases, genes associated with recurrent genomicalterations in HNSCC have been found and have proven to bepotentially useful predictors. Such examples include cy-clinD1 and cortactin,102,103 both residing on the often ampli-fied region 11q13. Of these 2, cortactin appears to be thestronger predictor. The reason why expression of this gene isassociated with poor prognosis in not clear, although it maybe related to stimulation of the EGFR pathway and down-stream signaling.104 Another genomic change, namely loss of

eterozygosity of 18q21, led to the finding that low SER-INB13 at this locus correlates with poor outcome in HNSCCreated primarily with radiotherapy.105 SERPINB13 is in-olved in suppression of angiogenesis,106 and it can be spec-lated that it influences outcome through effects on hypoxia.

Discussion and ConclusionsMany genes and genomic alterations have been found thatcorrelate with response to radiation-based treatments inHNSCC, giving hope that some of these could be developedinto reliable predictors of response to radiotherapy. Only afew predictors have withstood tests in multiple studies; ex-amples would include EGFR expression and HPV status.However, as stated in the introduction, even more importantthan predictors of outcome for radiotherapy are predictors ofresponse to a range of agents that can enhance the effects ofradiotherapy in resistant tumors. Only this will lead to indi-vidualized therapy and improved survival. Even EGFR andHPV provide insufficient information at present on how totreat patients with poor prognosis. Additional knowledgeand predictors for these biological processes are, therefore,still needed. The same is true for some of the gene expressionpredictors, such as the Chung high-risk signature31,87 and

D44 expression.33 Both have been validated at least once inadditional clinical series, although neither provides clear in-dications on the best treatment for the poor prognosisgroups.

The 3 classic determining factors of outcome after radio-therapy are intrinsic radiosensitivity, hypoxia, and repopula-tion rate. For radiosensitivity, gene signatures have so far notproved robust or strong predictors, possibly because of inad-equacies of the in vitro models used to generate them. Func-tional assays, such as residual foci of repair proteins in biop-sies cultured ex vivo, are interesting and promising, buttechnically difficult and need further validation. Hypoxiagene signatures were prognostic in several clinical series, al-though for patients treated with primary radiotherapy theyhave not yet proved reliable predictors. No single hypoxiaupregulated gene, when protein levels are measured by im-munostaining, has shown consistent predictions for HNSCCpatients given primary radiotherapy. Expression of microR-NAs that are upregulated under hypoxia looks promising,although further validation is needed. For repopulation rate,

it appears that pretreatment proliferation measurements of

classic cell cycle-related genes by immunostaining or geneexpression profiling are not adequate in predicting the pro-liferative response to treatment. Better predictions may comefrom EGFR expression or from factors related to differentia-tion. It should be noted here that gains in local control fromaltered fractionation schedules are relatively modest,55 andherefore, accurate prediction of which patients should re-eive accelerated fractionation will only result in a modestain.

Many new molecularly targeted agents are being discov-red and are entering the clinic, and more of these agents areeing combined with radiotherapy. An obvious way forward

s to incorporate genetic studies into as many of these trials asossible, especially to those including randomization withnd without the new agent. This will allow predictors to beeveloped that are specific to each agent. Many powerfulssays are currently available for such prospective studies,ncluding second-generation (deep) DNA or mRNA sequenc-ng, mRNA expression, miRNA expression, promoter meth-lation, and CGH, all on a genome-wide or near genome-ide scale. Application in new trials can serve to confirm and

hus validate previous predictors and to discover new andetter ones.Prediction of outcome for a particular treatment such as

adiotherapy, without having alternative treatments avail-ble, is of limited use. Progress in prediction should thereforee closely linked to new agent development. Tumor specific-

ty should be a priority for such new agents, which syntheticethality approaches can provide. Predicting which syntheticethality approach could be applied in an individual tumorhould be a priority for the future.

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