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  • 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-rentmetastatic (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 (median19 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: 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 dellaCarita, 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: loris.decec-co@istitutotumori.mi.it; 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:silvana.canevari@istitutotumori.mi.it; 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: paolo.bossi@istitutotumori.mi.it

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

    2016 American Association for Cancer Research.

    ClinicalCancerResearch

    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

    http://clincancerres.aacrjournals.org/

  • 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

  • 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 clinicalpathological characteristics of theanalyzed patients. A, consort diagramand workflow of theanalyses performed. B, mainclinicalpathological 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.

    Responsiveness of HNSCC to Platinum/Cetuximab

    www.aacrjournals.org Clin Cancer Res; 22(15) August 1, 2016 3963

    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

    http://clincancerres.aacrjournals.org/

  • 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, 1236), with 7 patients maintaining response atthe time of data analysis; the median PFS in the short-PFS groupwas 3 months (range, 15).

    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

  • Ability of existing gene expression signatures to predictoutcome following cetuximab treatment

    In the last decade, a number of gene expressionbasedsignatures 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 (1620) 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 0.0005) and theCl3-Hypoxia (P 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 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.

    Responsiveness of HNSCC to Platinum/Cetuximab

    www.aacrjournals.org Clin Cancer Res; 22(15) August 1, 2016 3965

    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

    http://clincancerres.aacrjournals.org/

  • and Cl3-Hypoxia were able to reflect cetuximab outcome; long-PFS compared with