Functional Genomics Uncover the Biology behind...

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Biology of Human Tumors Functional Genomics Uncover the Biology behind the Responsiveness of Head and Neck Squamous Cell Cancer Patients to Cetuximab Paolo Bossi 1 , Cristiana Bergamini 1 , Marco Siano 1,2 , Maria Cossu Rocca 3 , Andrea P. Sponghini 4 , Federica Favales 1 , Marco Giannoccaro 5 , Edoardo Marchesi 5 , Barbara Cortelazzi 6 , Federica Perrone 6 , Silvana Pilotti 6 , Laura D. Locati 1 , Lisa Licitra 1 , Silvana Canevari 5 , and Loris De Cecco 5 Abstract Purpose: To identify the tumor portrait of the minority of head and neck squamous cell carcinoma (HNSCC) patients with recur- rentmetastatic (RM) disease who upon treatment with plati- num-based chemotherapy plus cetuximab present a long-lasting response. Experimental Design: The gene expression of pretreatment samples from 40 HNSCC-RM patients, divided in two groups [14 long-progression-free survival (PFS) and 26 short-PFS (median ¼ 19 and 3 months, respectively)], was associated with PFS and was challenged against a dataset from metastatic colon cancer patients treated with cetuximab. For biologic analysis, we performed functional and subtype association using gene set enrichment analysis, associated biology across all currently available HNSCC signatures, and inferred drug sensitivity using data from the Cancer Genomic Project. Results: The identied genomic prole exhibited a signicant predictive value that was essentially conrmed in the single publicly available dataset of cetuximab-treated patients. The main divergence between long- and short-PFS groups was based on developmental/differentiation status. The long-PFS patients are characterized by basal subtype traits such as strong EGFR signaling phenotype and hypoxic differentiation, further validated by the signicantly higher association with the hypoxia metagene. The short-PFS patients presented a strong activation of RAS signaling conrmed in an in vitro model of two isogenic HNSCC cell lines sensitive or resistant to cetuximab. The predicted drug sensitivity for all four EGFR inhibitors was higher in long- versus short-PFS patients (P range: <0.00221e07). Conclusions: Our data uncover the biology behind response to platinum-based chemotherapy plus cetuximab in RM- HNSCC cancer and may have translational implications improving treatment selection. Clin Cancer Res; 22(15); 396170. Ó2016 AACR. See related commentary by Chau and Hammerman, p. 3710 Introduction Approximately 55% of head and neck squamous cell carcino- ma (HNSCC) patients presenting with locoregional advanced disease will eventually experience recurrent or metastatic tumor. A study of clinical and pathologic variables, performed before the adoption of cetuximab treatment, showed that weight loss, ECOG performance status, good/moderate tumor cell differentiation, primary tumor in the oral cavity or hypopharynx, and prior radiotherapy (RT) are all associated with an unfavorable outcome (1), and these variables are still valid more than 10 years later. RM-HNSCC patients who are not candidates for local therapies are usually offered platinum-based chemotherapy (CT) plus a targeted therapy with an anti-EGFR mAb, cetuximab, with the goal of prolonging survival and controlling symptoms. This combi- nation, tested in the Extreme trial versus CT alone, resulted in a signicant increase in median overall and progression-free sur- vival (PFS) and increased response rate (2) and entered as rst-line standard treatment in RM-HNSCC setting. However, in the Extreme trial, the median PFS obtained with CT plus cetuximab was 5.6 months and the 5-year follow-up showed that only 5% of the patients in both arms were long-term responders (PFS>12 months). 1 Head and Neck Cancer Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. 2 Cantonal Hospital St Gallen, Department of Internal Medicine, Clinic for Medical Oncology, St Gallen, Switzerland. 3 Division of Medical Oncology, European Insti- tute of Oncology, Milan, Italy. 4 SC of Oncology, AOU Maggiore della Carit a, Novara, Italy. 5 Functional Genomics and Bioinformatics, Department of Experimental Oncology and Molecular Medicine, Fon- dazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. 6 Laboratory of Experimental Molecular Pathology, Department of Diagnostic Pathology and Laboratory, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Authors: Loris De Cecco, Functional Genomics and Bioinfor- matics, IRCCS Istituto Nazionale Tumori, Via Amadeo, 42, 20133 Milan, MI 20133, Italy. Phone: 39-022-390-5130; Fax: 39-022-390-2692; E-mail: loris.decec- [email protected]; Silvana Canevari, Functional Genomics and Bioinfor- matics, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42, 20133 Milan, Italy. Phone: 39-022-390-2567; Fax: 39-022-390-3073; E-mail: [email protected]; and Paolo Bossi, Head and Neck Cancer Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, via Venezian 1, Milan, Italy. Phone: 39-022-390-2810; Fax: 39-022-390-3353; E- mail: [email protected] doi: 10.1158/1078-0432.CCR-15-2547 Ó2016 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 3961 on July 10, 2018. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst February 26, 2016; DOI: 10.1158/1078-0432.CCR-15-2547

Transcript of Functional Genomics Uncover the Biology behind...

Biology of Human Tumors

Functional Genomics Uncover the Biology behindthe Responsiveness of Head and Neck SquamousCell Cancer Patients to CetuximabPaolo Bossi1, Cristiana Bergamini1, Marco Siano1,2, Maria Cossu Rocca3,Andrea P. Sponghini4, Federica Favales1, Marco Giannoccaro5, Edoardo Marchesi5,Barbara Cortelazzi6, Federica Perrone6, Silvana Pilotti6, Laura D. Locati1, Lisa Licitra1,Silvana Canevari5, and Loris De Cecco5

Abstract

Purpose: To identify the tumor portrait of theminority of headand neck squamous cell carcinoma (HNSCC) patients with recur-rent–metastatic (RM) disease who upon treatment with plati-num-based chemotherapy plus cetuximab present a long-lastingresponse.

Experimental Design: The gene expression of pretreatmentsamples from 40 HNSCC-RM patients, divided in two groups [14long-progression-free survival (PFS) and 26 short-PFS (median¼19 and 3months, respectively)], was associated with PFS and waschallenged against a dataset frommetastatic colon cancer patientstreated with cetuximab. For biologic analysis, we performedfunctional and subtype association using gene set enrichmentanalysis, associated biology across all currently available HNSCCsignatures, and inferred drug sensitivity using data from theCancer Genomic Project.

Results: The identified genomic profile exhibited a significantpredictive value that was essentially confirmed in the single

publicly available dataset of cetuximab-treated patients. Themaindivergence between long- and short-PFS groups was based ondevelopmental/differentiation status. The long-PFS patients arecharacterized bybasal subtype traits such as strongEGFR signalingphenotype and hypoxic differentiation, further validated by thesignificantly higher association with the hypoxia metagene. Theshort-PFS patients presented a strong activation of RAS signalingconfirmed in an in vitro model of two isogenic HNSCC cell linessensitive or resistant to cetuximab. The predicted drug sensitivityfor all four EGFR inhibitors was higher in long- versus short-PFSpatients (P range: <0.0022–1e�07).

Conclusions:Our data uncover the biology behind responseto platinum-based chemotherapy plus cetuximab in RM-HNSCC cancer and may have translational implicationsimproving treatment selection. Clin Cancer Res; 22(15); 3961–70.�2016 AACR.

See related commentary by Chau and Hammerman, p. 3710

IntroductionApproximately 55% of head and neck squamous cell carcino-

ma (HNSCC) patients presenting with locoregional advanceddisease will eventually experience recurrent or metastatic tumor.A study of clinical and pathologic variables, performed before theadoption of cetuximab treatment, showed thatweight loss, ECOGperformance status, good/moderate tumor cell differentiation,primary tumor in the oral cavity or hypopharynx, and priorradiotherapy (RT) are all associated with an unfavorable outcome(1), and these variables are still valid more than 10 years later.

RM-HNSCC patients who are not candidates for local therapiesare usually offered platinum-based chemotherapy (CT) plus atargeted therapywith an anti-EGFRmAb, cetuximab,with the goalof prolonging survival and controlling symptoms. This combi-nation, tested in the Extreme trial versus CT alone, resulted in asignificant increase in median overall and progression-free sur-vival (PFS) and increased response rate (2) and entered asfirst-linestandard treatment in RM-HNSCC setting. However, in theExtreme trial, the median PFS obtained with CT plus cetuximabwas 5.6months and the 5-year follow-up showed that only 5% ofthe patients in both arms were long-term responders (PFS>12months).

1Head and Neck Cancer Medical Oncology Department, FondazioneIRCCS Istituto Nazionale dei Tumori, Milan, Italy. 2Cantonal Hospital StGallen, Department of Internal Medicine, Clinic for Medical Oncology,St Gallen, Switzerland. 3Division of Medical Oncology, European Insti-tute of Oncology, Milan, Italy. 4SC of Oncology, AOU Maggiore dellaCarit�a, Novara, Italy. 5Functional Genomics and Bioinformatics,Department of Experimental Oncology and Molecular Medicine, Fon-dazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. 6Laboratoryof Experimental Molecular Pathology, Department of DiagnosticPathology and Laboratory, Fondazione IRCCS Istituto Nazionale deiTumori, Milan, Italy.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Loris De Cecco, Functional Genomics and Bioinfor-matics, IRCCS Istituto Nazionale Tumori, Via Amadeo, 42, 20133 Milan, MI 20133,Italy. Phone: 39-022-390-5130; Fax: 39-022-390-2692; E-mail: [email protected]; Silvana Canevari, Functional Genomics and Bioinfor-matics, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42, 20133Milan, Italy. Phone: 39-022-390-2567; Fax: 39-022-390-3073; E-mail:[email protected]; and Paolo Bossi, Head and Neck CancerMedical OncologyDepartment, Fondazione IRCCS IstitutoNazionale dei Tumori,via Venezian 1, Milan, Italy. Phone: 39-022-390-2810; Fax: 39-022-390-3353; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2547

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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Because after six cycles of CT plus cetuximab, the anti-EGFRantibody is delivered as maintenance, it is likely that long-termeffect could be obtained by cetuximab alone. In this context, toincrease our understanding of the molecular mechanismsinvolved in sensitivity to cetuximab treatment, we compared thegene expression of two series of patients with marked oppositeoutcomes, with the assumption that this selection may enhancesensitivity in discovering relevant molecular pathways associatedwith response to anti-EGFR antibodies in RM-HNSCC.

Materials and MethodsPatients and study design

Each one of the 15 RM-HNSCC patients treated between 2008and 2012 with first-line CT and cetuximab-based combinationand showing a PFS >12 months (long-PFS) was matched for atleast three prognostic factors (1) with two RM-HNSCC patients,similarly treated but with PFS <5.6 months, median PFS reportedin the Extreme trial (short-PFS; see Fig. 1 for further details). Thestudy was approved by the Independent Ethics Committee ofFondazione IRCCS Istituto Nazionale dei Tumori (ApprovalNumber 55/12).

Whole genome gene-expression profilingFormalin-fixed, paraffin-embedded (FFPE) tumor specimens

of the recurrence or, if unavailable, of the primary tumor werecollected and selected areas weremacrodissected. RNA extraction,quality control, and hybridization on a Whole Genome–cDNA-mediated Annealing, Selection, extension, and Ligation (WG-DASL) microarray were performed essentially as previouslydescribed (3). Raw data were deposited with accession numberGSE65021 on GEO repository (http://www.ncbi.nlm.nih.gov/geo/). The WT-Ovation FFPE System Version 2 (NuGEN) wasused for technical validation of the gene expression.

Statistical and bioinformatic analysesStatistical analysis was performed using R (4), version 2.15,

BioConductor (5), release 2.10, and the BrB-ArrayTool developed

by Dr. Richard Simon and the BRB-ArrayTools DevelopmentTeam (v4.2.0; National Cancer Institute, USA). The differentiallyexpressed (DE) genes between long- and short-PFS patients weredefined by imposing an FDR of <15% and a list of 509 genes wasidentified. The probability of finding 509 genes significant bychance at FDR<15% if there were no real differences between theclasses was 0.01, determined by the global test through 1,000permutations as computed by BRB-tool using default parameters.Principal component analysis (PCA) was performed using thePCA module present in the imDEV graphical interface (6).Because 9 cases derive from recurrence or metastasis, we assessedthe influence of tissue origin in our gene signature throughBiasogram R package (7). The method allows the visualizationusing orthogonal projections of a gene expression matrix byvectors quantifying the outcome and the tissue of origin. Forcomparison purposes, a dataset (ID: GSE5851) containing theAffymetrix gene expression profile of 80 pretreatment biopsiesfrom metastatic colon cancer patients who had undergone cetux-imab treatment was analyzed (8). Pathway and oncosignatureanalysis was performed using Gene Set Enrichment Analysis(GSEA). From the gene ontology terms, a total of 556 gene sets(GS) with FDR < 0.15 were included in the pathway analysis;for oncosignatures, 188 GSs were analyzed. The degree of genesets' enrichment in the long- and short-PFS patients was repre-sented by a normalized enrichment score (NES) and NES withFDR < 0.05 and fold change >|1.5| was considered statisticallysignificant. We used single-sample gene set enrichment analysis(ssGSEA) projection (9) through GenePattern (10) to assess geneset activation scores in each sample of our case material. ssGSEAis a variant of GSEA designed for single sample analyses, whichboth transform gene-level expression data into pathway-levelscores. Graphical representation of GSEA findings was providedby GOCircle plot function of GOplot R package (11), displayinginformation about the significance of the enrichment (�log10adjusted P) and the z-score of each gene set. To better disclosethe biology underlying long- and short-PFS, we applied the Pre-diction Analysis for Microarrays (PAM) subtype classifier describ-ed in De Cecco and colleagues (12) and the centroid supergroupclassification described in Keck and colleagues (13). Subsequent-ly, a score was determined according to the Pearson correlationbetween the gene expression profile of each sample of our casematerial and the centroids for Cl3 and Basal subtypes of DeCecco and Keck classification, respectively. In silico confirmationof molecular pathways was achieved by retrieving the geneexpression data of a cetuximab-sensitive cell line (SCC1) and itscetuximab-resistant derivative (1Cc8; GSE21483; refs. 14, 15).The GS, oncosignatures, and subtype classifications as describedabove were applied to the data to support our findings.

Five microarray-based signatures are described as associatedwith outcome in HNSCC: (i) the hypoxia metagene (16); (ii) the13-gene OSCC signature (17); (iii) the radio-sensitivity index(RSI) (18); (iv) the 42-gene Chung's high-risk signature (19); (v)the 172-gene signature (20). We applied the procedures devel-oped in the respective papers by the authors, and a value for all thesamples entering into our dataset was assessed.

For external validation of drug sensitivity, the cetuximabsensitivity signature was assessed on the public data from theCancer Genomic Project (CGP; ref.21) through pRRophetic Rpackage (22). The analysis was performed by selecting 46HNSCC cell lines. ROC curves were estimated by pROC Rpackage (23).

Translational Relevance

The anti-EGFR mAb cetuximab plus a platinum-based che-motherapy is usually offered to head and neck squamous cellcarcinoma (HNSCC) patients with recurrent–metastatic (RM)disease. Despite a proven clinical benefit, only a minority ofpatients present a long-lasting response, and analysis of EGFRor other single markers does not show any correlation withprogression-free survival (PFS). By selection of patients withmarked opposite outcomes after treatment and a thoroughbiologic analysis of genomics data, we identify a signature ableto predict sensitivity to cetuximab-based therapy. The long-PFS patients are characterized by basal subtype traits, such asstrong EGFR signaling phenotype and hypoxic differentiation,while the short-PFS patients presented a strong activation ofRAS signaling. This is the first study specifically investigatingcetuximab/platinum resistance in RM-HNSCC patients andillustrates the feasibility of gene expression profiling of pre-treatment tumor to identify candidate biomarkers of responseto anti-EGFR treatment in this subset of patients.

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ResultsCase material

After the retrieval of matched tumor specimens from RM-HNSCCpatients, 1 and 4 samples from the long-PFS (15 patients)

and the short-PFS (30 patients) groups, respectively, were exclud-ed (see consort diagram, Fig. 1A). Thirty-one samples were fromprimary tumor and 9 from recurrence or metastasis. The twogroupswerewell balanced for prognostic factors, andno statistical

Figure 1.Workflowof the study andmain clinical–pathological characteristics of theanalyzed patients. A, consort diagramand workflow of theanalyses performed. B, mainclinical–pathological characteristics ofthe analyzed patients. T stageat first diagnosis and at recurrence/metastasis according to AJCC 8thedition. Grading was done according toWHO. Performance status according toECOG. Weight loss: percentage atrecurrence/metastatic disease versusdiagnosis. The P values are reportedas c2 tests.

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difference was present for gender; primary tumor stage, site, gradeand HPV status (only oropharynx); age; prior radiotherapy;performance status; weight loss; origin of tissue analyzed for geneexpression; types of drugs associated with cetuximab treatment(Fig. 1B). The group achieving a long PFS had a median PFS of 19months (range, 12–36), with 7 patients maintaining response atthe time of data analysis; the median PFS in the short-PFS groupwas 3 months (range, 1–5).

Identification and technical validation of a gene expressionprofile differentiating long- from short-PFS patients

After profiling by the WG-DASL assay, a supervised classcomparison analysis identified 509 differentially expressed(DE) genes imposing an FDR of <15% (336 and 173 upregu-lated in long- and in short-PFS samples, respectively; Supple-mentary Fig. S1 and Supplementary Table S1) and PCA dis-tributed patients in two main clusters, matching with the long-and short-PFS groups (Fig. 2A). The whole transcriptomeobserved by WG-DASL profiling was essentially confirmed byNuGEN methodology (Supplementary Fig. S2). In order toascertain the degree of influence of the tissue origin on theidentified gene signature, we applied an orthogonal projectionthrough two vectors quantifying the outcome (long vs. shortPFS; Y axes) and the tissue origin (primary vs. recurrence/metastatic; B axes). Because the B vector lied orthogonal tothe Y vector and all 509 genes fall along the Y vector (Supple-mentary Fig. S3), the expression of our gene signature isassociated with outcome and not to the tissue origin.

Challenging the identified signature in a dataset of cetuximab-treated patients

To explorewhether theDE genes fromour selected RM-HNSCCpatients has a predictive value in other studies, we searched forpublicly available datasets of cetuximab-treated patients. Wefound a single available dataset (GSE5051) that is composed ofa metastatic colorectal population after several lines of treatment(1–4), treated with cetuximab monotherapy and not dichoto-mized into long and short responders. When we applied oursignature to these 80 metastatic colorectal cancer patients, weclassified them in 28 and 52 cases expectedwith a long- and short-PFS, respectively. Kaplan–Meier survival analysis confirmed ourprediction (Fig. 2B).The differences between the two studies mayaccount for the lower PFS of colorectal cancer patients with asignature similar to long-PFSHNSCC patients; in spite of that, thesignature of the long PFS group is able to identify somemetastaticcolorectal cancer cases having a durable response in this confir-mation group.

Pathway analysesTo gain further insight into the biologic pathways involved in

response to CT plus cetuximab treatment, we applied GSEAanalysis, and due to the limited number of cases we used verystringent thresholds. Starting analysis from 556 GSs, we found 11(from GS-01 to GS-11) and 7 (from GS-12 to GS-18) of themsignificantly enriched in long- and short-PFS groups, respectively(Supplementary Table S2, ordered for each group according toNES significance); considering the redundancy in GS terms withoverlapping genes, 7 GSs were selected as representative andgraphical representation is displayed by the heatmap of Fig.3A. Themain divergence between the tumors was associated with

their developmental/differentiation status, being those fromlong-PFS patients characterized by genes of "Ectoderm/Epider-mis" (GS-01, -02, and -03) and those from short-PFS patients bygenes of "Muscle" (GS-12, -13, -14, -15, and -18). Furthermore, inthe long-PFS group, GS-06, -07, -08, and -09 were associated with"Defense response," GS-10 and GS-11 characterized the "EGFRSignaling Pathway" and the "Protein catabolism," respectively; inthe short-PFS group, GS-16 and -17were associated with "Cation-transport."

Figure 2.Identification of a signature separating long- versus short-PFS in HNSCCpatients and its challenging in a publicly available dataset of cetuximab-treated patients. A, a signature of 509 genes was found and, as visualized byPCA, was able to separate the samples into two well-defined groupscorresponding to long- and short-PFS cases. Blue bins, short-PFS; red bins,long-PFS. B, challenging the signature in a publicly available dataset ofcetuximab-treated patients (GSE5851). Kaplan–Meier survival analysis for thecases predicted as long-PFS patients (red line) or short-PFS patients(blue line).

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Ability of existing gene expression signatures to predictoutcome following cetuximab treatment

In the last decade, a number of gene expression–basedsignatures have been proposed as prognostic factors in HNSCC,but no data exist on their capability to predict sensitivity/resistance to anti-EGFR/platinum regimens. In order to assesswhether and to what extent five signatures (16–20) are asso-ciated with response to cetuximab/platinum, we applied to ourcohort the algorithms developed by the authors in the respec-tive papers. A score for all the samples entering into our datasetwas assessed and it was compared between long- and short-PFScases. The hypoxia signature, the 13-gene OSCC, and the RSIsignatures displayed a significant difference in relationship to

outcome with the long-PFS cases having higher scores (Sup-plementary Fig. S4).

Then, we extended our analysis on determining to whichmolecular subtype our cases belong. For this aim, two recentmolecular classifications were considered (12, 13). We stratifiedour case material determining to which of the three subtypes (BA,basal; CL, classical; IMS, inflamed/mesenchymal) reported byKeck and colleagues (13) and to which of the EGFR-expressingHNSCC subtypes (Cl2-Mesenchymal and Cl3-Hypoxia)described in De Cecco and colleagues (12) the cases are classified.As reported at the bottomof the heatmap of Fig. 3A, 11 of 14 long-PFS patients could be attributed to the BA (P ¼ 0.0005) and theCl3-Hypoxia (P ¼ 0.0017) subtypes. The centroid scores for BA

Figure 3.Functional analysis of pathwaysderegulated between long- and short-PFSpatients.A, heatmapof the ssGSEApathway enrichment scores (meancentered) for seven selected pathways.Each column represents a sample, andeach row represents a gene signature.Color code: red, high enrichment;green, low enrichment. GS-01,ectoderm development; GS-09, multi-organism process; GS-10, epidermalgrowth factor receptor signalingpathway; GS-11, cellular proteincatabolic process; GS-14, muscledevelopment; GS-15, regulation ofmuscle contraction; GS-16, di-, tri-valent inorganic cation transport (forgene list, see Supplementary Table S2).The bar below the heatmap representsthe membership to Cl2-mesenchymal(yellow) and Cl3-hypoxia (blue)subtypes (12) and IMS (orange), BA(green), and CL (light blue)supergroups (ref.13; P ¼ 0.0017 and0.0005, respectively). B and C,boxplots of centroid correlation inlong- and short-PFS tumors toKeck-BA subtype and De Cecco-Cl3 subtype, respectively. D and E,corresponding ROC curves.

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and Cl3-Hypoxia were able to reflect cetuximab outcome; long-PFS compared with short-PFS cases showed higher correlationscore to BA and Cl3 subtypes (Fig. 3B and C, respectively),reaching a significant accuracy as demonstrated by ROC analysiswith AUC¼ 0.876 for BA and 0.AUC¼ 0.885 for Cl3 (Fig. 3D andE, respectively).

Oncosignature analysesTo further analyze the correspondence of the expression data of

the long-PFS group and our Cl3-hypoxia subtype, starting from188 oncosignatures, we selected those reported enriched by DeCecco and colleagues (12). The pathways related to TGFb, RAS,Cyclin D1, bCAT, p53, WNT, and E2F3 were tested for theirassociation in our cohort of patients. The most significant asso-ciation was recorded with signatures related to cell lines trans-fected with oncogenic forms of KRAS, and the genes upregulatedupon oncogenic transformation are enriched in short-PFS; fur-thermore, gene sets related to bCAT, E2F3, MYC, and p53 onco-pathways were significantly enriched in long-PFS (Fig. 4A). Tosupport our findings on the molecular pathways involved inoutcome following cetuximab treatment, we took into consider-ation an isogenic model of two sensitive and resistant cell lines(15) for whom the gene expression data are publicly available.1CC8 cetuximab-resistant cells confirmed a significant upregula-tion of RAS oncogenic pathway compared with SCC1-sensitivecells along with GS-15, while SCC1 cells showed significantupregulation of GS-1 (Fig. 4B); furthermore, when comparedwith gene expression subtype classification, SCC1 cells havehigher Cl3 scores than 1CC8 (Fig. 4B).

Drug-sensitivity analysisWe searched for a potential link between the expression profiles

of the patients entered in this studywith those of CGP cell lines forwhich sensitivity data to 130 drugs are available. Cetuximab wasnot screened against the cell lines, but data on other four EGFRpathway inhibitors are available and selected; in addition, weselected other drugs of interest for HNSCC treatment (chemother-apeutics and targeted agents in clinical development). The asso-ciation of drug sensitivity for 11 of the 18 compounds selectedretaining a significance threshold is depicted in Fig. 5A and B.Among the four EGFR inhibitors, the long-PFS patients showedthe significantly greater sensitivity to afatinib (Fig. 5C and D).Seven other compounds showed a significance difference in pre-dicted activity between long- and short-PFS patients, and thedrug to which short-PFS patients showed the greater sensitivitywas gemcitabine (Fig. 5E and F).

DiscussionCetuximab, an anti-EGFR monoclonal antibody, has been the

first targeted therapy approved for HNSCC patients, representingthe major systemic treatment improvement in the last decade(24); however, about 40% of patients are responsive, and themedian PFS is limited (2).

The data presented here indicate that tumors with markedopposite treatment outcomes have different molecular pat-terns, with separate specific deregulated pathways. To ourknowledge, this is the first study of RM-HNSCC that specif-ically investigates sensitivity to cetuximab/platinum-basedtherapy by gene expression and biology behind. After manyyears of unsuccessful research for biomarker identification

and in-depth analysis of EGFR (25, 26), our data suggest thata whole-transcriptome approach followed by in silico valida-tion, a thorough functional genomics and drug-sensitivityestimate could uncover the biology behind responsiveness tocetuximab/platinum in RM-HNSCC patients, opening the wayto the definition of a cetuximab-based treatment-sensitivitypredictor.

Our selection of RM-HNSCC cases potentially sensitiveto cetuximab/platinum treatment was based on their PFS

Figure 4.Oncogenic signatures in RM-HNSCC. A, the circle plot depicts the eightoncosignatures enriched in Cl3-hypoxia (12) and found significantlyassociated in long- or short-PFS cases. The height of the bars in the inner ringdisplays the significance of oncosignatures (�log10 adjusted P)and colors correspond to z-scores (yellow, upregulated in short-PFS; blue,upregulated in long-PFS). The outer ring shows the scatterplot of theexpression levels (logFC) for the genes. B, the boxplots display theenrichment ssGSEA scores for GS-1, GS-15, and RAS oncogenic gene set andcentroid Cl3-hypoxia scores for 1CC8 and SCC1 cells, resistant andsensitive to cetuximab, respectively.

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extraordinarily long, as already done with some success onanother cancer type by others (27).

We should note that cetuximab exhibits two distinctmechanisms of action: (i) a competitive inhibition of ligand

binding to EGFR, thereby inhibiting activation of EGFR tyro-sine kinase activity and subsequent signaling; and (ii) anantibody-dependent cellular cytotoxicity mediated by hostimmune cells. Our functional annotation analysis of the

Figure 5.Prediction of drug sensitivity in RM-HNSCC. A, the heatmap depicts predicted sensitivity in each sample of our study. Colors range from blue (sensitive) toyellow (resistant). B, the table summarizes relative P values and differences between IC50 values for long- and short-PFS (selection criteria P < 0.05 and a|log(IC50)| difference between long- and short-PFS cases>0.15).C andD,boxplot of predicted sensitivity to afatinib for long- and short-PFS patients (P¼ 3.47E�09)and ROC curve (AUC ¼ 0.992; 95% CI, 0.897–1.000; P < 0.0001). E and F, boxplot of predicted sensitivity to gemcitabine for long- and short-PFS patients(P ¼ 0.00228) and ROC curve (AUC ¼ 0.788; 95% CI, 0.631–0.901; P ¼ 0.0003).

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identified molecular profile may suggest that the long-PFSpatients are sensitive to the competitive inhibition of cetux-imab. In fact, long-PFS cases showed significant enrichment in(i) genes belonging to EGFR signaling, including moleculeswhose regulation is related to EGFR activation due to itsphysiologic ligands; (ii) processes related to tissue develop-ment, with significant upregulation of genes located in a regionon human chromosome 1q21 known as the "epidermal dif-ferentiation complex" (28) and in many genes of the kallikreinfamily, directly involved in many steps in cancer development,such as regulation of tumor proliferation and invasion (29);(iii) genes involved in intracellular catabolic processes. Alto-gether, these deregulated pathways can partially explain thesuccess of a therapy based on EGFR signaling blocking anddegradation following cetuximab binding.

Several studies, including our recent ones, have indicated therelevance of analysis of whole transcriptome in identifying poten-tially useful prognostic or predictive signatures (16–20) andpatient subtypes (12, 13). These signatures have never been testedin the context of cetuximab treatment. The hypoxiametagene, the13-geneOSCC signature, and RSI display a significant associationwith long-PFS cases having higher scores with aggressive progno-sis compared with short-PFS cases. When our cases were classifiedaccording to the EGFR-expressing subtypes identified in De Ceccoand colleagues (12) and the three supergroups reported by Keckand colleagues (13), long-PFS cases were stratified as Cl3-hypoxiaand Basal subtypes, respectively, characterized by poor prognosis.Noteworthy, the scores associated with Cl3-hypoxia and BA,indicating similarity to those subtypes, might foresee long-PFScases with good accuracy. In addition, a previous work by Chungand colleagues (30) identified a G1 cluster that subsequentlyproved to be associated with basal subtype (12, 31) with detri-mental outcome. Taken together, the signatures and the molec-ular subtype stratification highlight that long-PFS cases are adefined molecular subgroup with poor prognosis that can berescued by cetuximab/platinum treatment.

The oncosignature analysis emphasizes the importance ofconsidering all the signaling pathways linked to the activationof EGFR when evaluating a model for determining outcome. Infact, short-PFS tumors were characterized by strong RAS sig-naling activation. Our data suggest the relevance of the integrityof RAS downstream signaling in conferring sensitivity to cetux-imab/platinum, while overactivation of RAS pathway leads toresistance.

RAS mutations were not analyzed in our case material, butthey are very rare in HNSCC (3% as reported in tumorportal)compared with other cancer types (32). We investigated theactivation of the RAS pathway in an isogenic cetuximab-sensi-tive versus -resistant cellular model having wild-type RAS andconfirmed an aberrant overactivation of RAS signaling in theresistant cell line compared with the sensitive one along withconsistent activation of GS-15 (ectoderm development) anddownregulation of GS-1 (regulation of muscle contraction) andlow Cl3-hypoxia score. Our findings support the idea that theactivation of the RAS pathway even in absence of constitutivegene mutations could lead to cetuximab/platinum resistance.Indeed, we also showed the validity of our predictive signaturein a colon cancer setting (8). Notably, the proteomic profilepredicting for cetuximab efficacy in colon cancer was valid onlyin subjects with RAS WT tumors (33). These observations, ifconfirmed more widely in HNSCC case materials, could open

the way to the selection of the genes most relevant in strong RASsignaling activation as companion diagnostics for anti-EGFRtherapy of RM-HNSCC.

Finally, the association of long-PFS with Cl3-hypoxia (12) is inlinewith the predicted high sensitivity to afatinib of this subgroupof patients. Because Cl3-hypoxia patients are characterized by analtered hypoxia pathway, the use of EGFR-targeted therapiescombined with antioxidant agents and/or additional strategiesexploiting hypoxia, as already suggested (13, 34), deserves to betested in preclinical models recapitulating the gene expressionprofile of long-PFS patients.

The analysis of the link between the expression profiles ofpatients entered in this study with that of cell lines that exhibiteda particular sensitivity to a drug (21) confirmed dependence onthe EGFR pathway signal for the long-PFS tumors. In this group ofpatients, cisplatin and docetaxel gave some hints of activity,although to a far lower extent than EGFR pathway inhibitors. Onthe other hand, it is worth noting how the short-PFS groupsegregates with the activity of other chemotherapeutic drugs,namely, gemcitabine and doxorubicin.

We should accept a limit in defining the predictive value of theidentified gene expression profile because it was not possible tocompare our results using profiles of similar RM-HNSCC patientsnot receiving cetuximab but only CT. However, the importance ofmaintenance cetuximab in the long-PFS group should be empha-sized; in fact, CT was stopped after six cycles (about 5 months),whereas cetuximab administration was continued till progression(range in long-PFS, 7–31 months), suggesting that the predictivevalue of our signature and underlying molecular mechanismscould be related to anti-EGFR treatment.

Our genomic analysis did not take into consideration thecetuximab's second mechanism of action, i.e., ADCC. Thus, theprospective validation of our signature is currently ongoing ontwo phase II trials based on (i) cetuximab combination withcisplatin with/without paclitaxel in a multicentric phase II trialto further define the role of the altered pathways in determinesensitivity to cetuximab independently on the mechanism ofaction of the associated chemotherapeutic agent (2011-002564-24 on www.clinicaltrialsregister.eu); (ii) treatmentwith an anti-EGFR-Mab, panitumumab, showing less ADCCactivity than cetuximab (35) to identify the potential contri-bution of the host immune system in conferring sensitivity/resistance to an anti-EGFR antibody treatment (36). Becausein this study we selected patients with marked opposite out-comes after treatment, these prospective validations in theentire spectrum of patient's outcome could also help in trans-posing our predictor of response into a useful clinical gradeassay.

Finally, because in other cancer settings restricting the use oftargeted therapy to a molecularly selected group of patients hasbeen proven to be cost-effective (37), we believe that theidentification of a gene-expression profile able to potentiallyidentify patients benefiting from anti-EGFR treatment couldreduce the incremental cost-effectiveness ratio and favor a moretailored use of this targeted therapy approach.

Disclosure of Potential Conflicts of InterestP. Bossi is a consultant/advisory board member for Merck Serono. L.

Licitra is a consultant/advisory board member for AstraZeneca, Bayer,Boehringer, Bristol-Myers Squibb, Debiopharm, Eisai, Merck-Serono, MSD,Novartis, Roche and Sobi; reports receiving research funds, through her

Bossi et al.

Clin Cancer Res; 22(15) August 1, 2016 Clinical Cancer Research3968

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institution, from AstraZeneca, Boehringer, Eisai, Merck-Serono, MSD,Novartis and Roche; and reports receiving travel reimbursement from Bayer,Debiopharm, Merck-Serono, and Sobi. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: P. Bossi, M. Siano, M. Cossu Rocca, S. Canevari,L. De CeccoDevelopment of methodology: P. Bossi, M. Cossu Rocca, B. Cortelazzi, F. PerroneAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Bossi, C. Bergamini, M. Siano, M. Cossu Rocca,B. Cortelazzi, F. Perrone, L. De CeccoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Bossi, M. Siano, M. Cossu Rocca, S. Pilotti,S. Canevari, L. De CeccoWriting, review, and/or revision of the manuscript: P. Bossi, M. Siano,M. Cossu Rocca, S. Pilotti, S. Canevari, Loris. De CeccoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.P. Sponghini, F. Favales, M. Giannoccaro,E. Marchesi, B. Cortelazzi

Study supervision: P. Bossi, S. Pilotti, L. Licitra, S. CanevariOther (centralized histopathologic revision and microdissection): S. PilottiOther (contributed to the discussion): L.D. Locati

AcknowledgmentsThe authors thank Dr. Wolfram Jochum (Institute of Pathology, Kantons-

spital St Gallen, St Gallen, Switzerland) for help in case material collection andpathologic review.

Grant SupportThis work was partially supported by Associazione Italiana Ricerca Cancro

(AIRC IG 14750 to S. Canevari).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 25, 2015; revised January 22, 2016; accepted February 4,2016; published OnlineFirst February 26, 2016.

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2016;22:3961-3970. Published OnlineFirst February 26, 2016.Clin Cancer Res   Paolo Bossi, Cristiana Bergamini, Marco Siano, et al.   to CetuximabResponsiveness of Head and Neck Squamous Cell Cancer Patients Functional Genomics Uncover the Biology behind the

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