Heads Up! Predictive Gene Signatures in Head and Neck Cancer May Be Coming Soon

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1 Heads Up! Predictive Gene Signatures in Head and Neck Cancer May Be Coming Soon Nicole G. Chau 1 and Peter S. Hammerman 1 Affiliations: 1 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, and Harvard Medical School, Boston, MA Corresponding Author: Peter S. Hammerman, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA Phone: ; Fax: ; Peter_Hammerman@dfci.harvard.edu Running Title: Predictive Gene Signatures for HNSCC Cetuximab-Chemotherapy Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed. 1

2 SUMMARY: Cetuximab-platinum chemotherapy is used for recurrent-metastatic head and neck squamous cell carcinoma (HNSCC), however a minority of patients benefit. Gene expression profiling (GEP) of HNSCCs with prolonged responses to cetuximab-chemotherapy demonstrate basal subtype traits including signatures of EGFR signaling and hypoxic differentiation. GEP of short-response patients show RAS activation. 2

3 In this issue of Clinical Cancer Research, Bossi and colleagues (1) report that pretreatment tumor gene expression profiles (GEP) of recurrent/metastatic head and neck squamous cell carcinomas (HNSCC) from patients who experience prolonged response (median progression-free-survival (PFS) of 19 months) to treatment with palliative cetuximabchemotherapy are characterized by basal subtype traits, and significantly differ from GEP of patients with short responses, thus highlighting the potential opportunity for the future development of a predictive gene signature to select HNSCC patients for cetuximabchemotherapy. Cetuximab, a chimeric immunoglobulin G1 antibody to the Epidermal Growth Factor Receptor (EGFR) is the only molecularly targeted therapy in HNSCC. It was FDA approved in 2006 for locally advanced HNSCC in combination with radiation (2), or as monotherapy in the platinum-refractory, recurrent or metastatic setting. In 2011, cetuximab was FDA approved in combination with platinum-based chemotherapy for the treatment of recurrent/metastatic (R/M) HNSCC, based on a 2.7 month overall survival (OS) benefit in the EXTREME phase III trial of first-line treatment of recurrent/metastatic HNSCC with the addition of cetuximab to cisplatin or carboplatin plus infusional fluorouracil (median OS 10.1 vs. 7.4 months; HR 0.8; p=0.04) (3). The addition of cetuximab to platinum plus fluorouracil resulted in an objective response rate of 36%, and median PFS of 5.6 months; however, only 5% of patients had a PFS over 12 months. Unfortunately, since the approval of cetuximab, there has been modest progress over the past decade toward the development of more targeted therapies for HNSCC in the metastatic setting, and no additional targeted therapies have been FDA-approved. The EXTREME regimen is still a benchmark for comparison in large phase III studies in R/M HNSCC, and cetuximab is offered to virtually all advanced HNSCC patients at some point in their care regardless of underlying tumor biology, or patient risk factors. This modest therapeutic progress in HNSCC can be attributed in part to the lack of validated prognostic and therapeutic biomarkers, and it remains the case that despite substantial effort in this area it remains unclear which HNSCC patients are likely to benefit from cetuximab therapy. In the past decade, several prognostic gene expression based signatures have been proposed in HNSCC; however, none have been prospectively validated or examined to predict response to cetuximab-platinum chemotherapy. 3

4 In this study, Bossi and colleagues (1) retrospectively compared gene-expression profiles of pre-treatment archival formalin-fixed, paraffin-embedded tumor specimens obtained from 14 HNSCC patients with prolonged response (median PFS 19 months) and 26 HNSCC patients with short response (median PFS 3 months) to first line palliative cetuximab-chemotherapy. Supervised class comparison analysis identified 509 differentially expressed genes and PCA distributed patients in two main clusters, distinguishing the prolonged responders from the shortterm responders. Tumor tissue origin, either from the primary (n=31) or recurrence or metastasis (n=9), did not appear to confound the association of the gene signature with clinical outcomes. The gene signature identified by Bossi and colleagues (1) was then compared to other datasets including: 1) a publically available dataset of 80 metastatic colorectal cancer patients who had received several lines of treatment followed by cetuximab monotherapy, and 2) five separate microarray based signatures associated with outcome in HNSCC patients who had not received cetuximab. The gene-expression signature by Bossi and colleagues was able to classify metastatic colorectal cancer cases according to PFS, and among HNSCC patients, long cetuximab-chemotherapy-pfs correlated with high scores in 3 out of 5 of the HNSCC prognostic signatures. Further molecular subtype analysis revealed that gene expression patterns derived from the long cetuximab-chemotherapy-pfs patients appeared to be correlated with previously described basal and hypoxia molecular subtypes of HNSCC (4, 5), and enriched for gene-sets related to βcat, E2F3, MYC and p53. Additional drug sensitivity studies in this report showed that the gene expression profile of long cetuximab-chemotherapy-pfs patients appeared to select for cell lines sensitive to EGFR tyrosine kinase inhibitors (afatinib, gefitinib, erlotinib, lapatinib), whereas gene expression profile of short cetuximab-chemotherapy-pfs patients selected for gemcitabine sensitivity. This is the first study to specifically utilize expression profiling to evaluate response/sensitivity to cetuximab-platinum chemotherapy in HNSCC patients, and revealed enrichment for previously identified molecular subtypes with prolonged response to anti-egfr therapy. In contrast, further analysis of the expression data of short cetuximab-chemotherapy- PFS patients revealed enrichment for RAS activation (Figure 1). Indeed, activation of the RAS- 4

5 RAF-ERK pathway, as a result of activating mutations in KRAS, is a clearly established mechanism of resistance to cetuximab therapy in metastatic colorectal cancer. This study highlights the utility of gene expression profiling to uncover underlying biologic differences and candidate predictive biomarkers when evaluating response to anti-egfr and platinum therapy in HNSCC, as prior predictive biomarker studies utilizing less comprehensive methodologies evaluating only one or a few biomarkers in HNSCC, have not led to further validation of a biomarker. Although EGFR is overexpressed by immunohistochemistry (IHC) in the majority of HNSCC, multiple studies have failed to identify EGFR gene copy number or IHC as a predictive biomarker of response to cetuximab (6, 7). In other tumor types with high EGFR expression such as non-small cell lung cancer, studies evaluating EGFR IHC as a biomarker to predict response to cetuximab have also yielded variable results and cetuximab does not yet have an established role in NSCLC (8, 9). As absence of RAS mutation is a validated predictor of response to cetuximab-chemotherapy regimens in metastatic colorectal cancer (10), other biomarker studies in HNSCC patients treated with cetuximab have evaluated RAS pathway activation as a potential prognostic and/or predictive biomarker of cetuximab response. ERK1/2 expression was significantly associated with poorer OS and PFS in locally advanced patients treated with cetuximab-chemotherapy in a phase 2 trial (11). In addition, the germline mutation in the 3 -untranslated region of KRAS (rs , KRAS-variant: TG/GG), which has been associated with drug resistance/sensitivity in various cancers, has also been associated with worse prognosis and cisplatin resistance in retrospective analysis of recurrent or metastatic HNSCC patients (12). Interestingly, KRAS-variant HNSCC patients appeared to have improved disease control with the addition of cetuximab to platinum chemotherapy in a small subset of patients, suggesting KRAS-variant mutation may be a predictive biomarker of treatment response. Predictive gene expression signatures can be a powerful tool for precision medicine; however, few have been validated in prospective studies of any cancer type. The study by Bossi and colleagues is an important first step in the identification of candidate predictive biomarkers of response to cetuximab-platinum therapy in R/M HNSCC and efforts to prospectively validate this signature in HNSCC are underway and include a European phase 2 trial of cetuximab- 5

6 chemotherapy in R/M HNSCC. Despite these efforts it may be difficult to distinguish the relative contribution of predictors of cetuximab activity from EGFR targeting versus antibody-dependent cell mediated cytoxicity (ADCC), and further, it may also be challenging to predict response to chemotherapy versus cetuximab. Recent genomic characterization and classification of molecular subtypes in HNSCC has improved our understanding of underlying disease biology and identified candidate prognostic biomarkers for the development of novel personalized biomarker driven therapeutic approaches for HNSCC. Taken together with findings from this study, there is suggestion that EGFR-directed therapies should be considered for patients with basal molecular subtypes and that patients with RAS-activation may be more suitable for other molecularly targeted agents or chemotherapy. 6

7 References: 1. Bossi P, Bergamini C, Siano M, Cossu Rocca M, Sponghini AP, Favales F, et al. Functional genomics uncover the biology behind the responsiveness of head and neck squamous cell cancer patients to cetuximab. Clin Cancer Res 2016 Feb 26. [Epub ahead of print]. 2. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006;354: Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki A, Rottey S, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med 2008;359: De Cecco L, Nicolau M, Giannoccaro M, Daidone MG, Bossi P, Locati L, et al. Head and neck cancer subtypes with biological and clinical relevance: meta-analysis of gene-expression data. Oncotarget 2015;6: Keck MK, Zuo Z, Khattri A, Stricker TP, Brown CD, Imanguli M, et al. Integrative analysis of head and neck cancer identifies two biologically distinct HPV and three non-hpv subtypes. Clin Cancer Res 2015;21: Licitra L, Mesia R, Rivera F, Remenar E, Hitt R, Erfan J, et al. Evaluation of EGFR gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck: EXTREME study. Ann Oncol 2011;22: Licitra L, Storkel S, Kerr KM, Van Cutsem E, Pirker R, Hirsch FR, et al. Predictive value of epidermal growth factor receptor expression for first-line chemotherapy plus cetuximab in patients with head and neck and colorectal cancer: analysis of data from the EXTREME and CRYSTAL studies. Eur J Cancer 2013;49: Khambata-Ford S, Harbison CT, Hart LL, Awad M, Xu LA, Horak CE, et al. Analysis of potential predictive markers of cetuximab benefit in BMS099, a phase III study of cetuximab and firstline taxane/carboplatin in advanced non-small-cell lung cancer. J Clin Oncol 2010;28: Pirker R, Pereira JR, von Pawel J, Krzakowski M, Ramlau R, Park K, et al. EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol 2012;13: Allegra CJ, Rumble RB, Schilsky RL. Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary. J Oncol Pract 2016;12: Psyrri A, Lee J-W, Pectasides E, Vassilakopoulou M, Burtness B, Rimm D, et al. Predictors for response to cetuximab in a prospective clinical trial (E2303) of patients with head and neck squamous cell carcinoma (HNSCC). J Clin Oncol 30, 2012 (suppl; abstr 5576). 12. Chung CH, Lee JW, Slebos RJ, Howard JD, Perez J, Kang H, et al. A 3'-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma. Ann Oncol 2014;25:

8 Figure Legend Figure 1. Gene expression profiles differ between long-responders versus short-responders in HNSCC patients treated with cetuximab-chemotherapy. Heatmap of the ssgsea pathway enrichment scores for seven selected pathways. Each column represents a sample; each row represents a gene signature. Color code: red = high enrichment, green = low enrichment. GS-01 = ectoderm development; GS-09 = multiorganism process; GS-10 = epidermal growth factor receptor signaling pathway; GS-11 = cellular protein catabolic process; GS-14 = muscle development; GS-15 = regulation of muscle contraction; GS-16 = di tri valent inorganic cation transport. The bar below the heatmap represents the membership to mesenchymal (yellow), hypoxia (blue) subtypes [De Cecco et al. (4)], and Inflamed/mesenchymal (orange), Basal (green), Classical (light blue) supergroups [Keck et al. (5)]. 8

9 Figure 1: R/M HNSCC Cetuximab-chemotherapy long responders Cetuximab-chemotherapy short responders Basal subtype EGFR signaling phenotype Hypoxic differentiation phenotype RAS activation phenotype GS-16 GS-14 GS-15 GS-09 GS-10 GS-01 GS-11 Mesenchymal Hypoxia Subtypes [De Cecco et al. (4)] Subtypes [Keck et al. (5)] Inflamed/mesenchymal Basal Classical 2016 American Association for Cancer Research

10 Heads Up! Predictive Gene Signatures in Head and Neck Cancer May Be Coming Soon Nicole G. Chau and Peter S. Hammerman Clin Cancer Res Published OnlineFirst April 29, Updated version Author Manuscript Access the most recent version of this article at: doi: / ccr Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.