Companion Diagnostics and Cancer Biomarkers The FA/BRCA Pathway Identified as the Major Predictor of Cisplatin Response in Head and Neck Cancer by Functional Genomics Sanne R. Martens-de Kemp 1 , Arjen Brink 1 , Ida H. van der Meulen 2 , Ren ee X. de Menezes 3 , Dennis E. te Beest 3 , C. Ren e Leemans 1 , Victor W. van Beusechem 2 , Boudewijn J.M. Braakhuis 1 , and Ruud H. Brakenhoff 1 Abstract Patients with advanced stage head and neck squamous cell carcinoma (HNSCC) are often treated with cisplatin-containing chemoradiation protocols. Although cisplatin is an effective radi- ation sensitizer, it causes severe toxicity and not all patients benefit from the combination treatment. HNSCCs expectedly not responding to cisplatin may better be treated with surgery and postoperative radiation or cetuximab and radiation, but biomar- kers to personalize chemoradiotherapy are not available. We performed an unbiased genome-wide functional genetic screen in vitro to identify genes that influence the response to cisplatin in HNSCC cells. By siRNA-mediated knockdown, we identified the Fanconi anemia/BRCA pathway as the predominant pathway for cisplatin response in HNSCC cells. We also identified the involve- ment of the SHFM1 gene in the process of DNA cross-link repair. Furthermore, expression profiles based on these genes predict the prognosis of radiation- and chemoradiation-treated head and neck cancer patients. This genome-wide functional analysis des- ignated the genes that are important in the response of HNSCC to cisplatin and may guide further biomarker validation. Cisplatin imaging as well as biomarkers that indicate the activity of the Fanconi anemia/BRCA pathway in the tumors are the prime candidates. Mol Cancer Ther; 16(3); 540–50. Ó2016 AACR. Introduction Cancer of the head and neck is the eighth most commonly diagnosed cancer worldwide (1). Over 90% of the head and neck cancers are squamous cell carcinomas that arise in the mucosal linings of the upper aerodigestive tract. Approximately 60% of head and neck squamous cell carcinoma (HNSCC) patients present with advanced stage disease (stage III and IV), which relates to a poor prognosis (2). Treatment options for this group are surgery followed by radiotherapy or cisplatin-containing chemoradiation protocols with salvage surgery if needed. Although chemoradiation is effective (3), not all tumors respond well to this combination of cisplatin and radiotherapy. Cisplatin is an effective and inexpensive addendum to radiation protocols, but only between 5% and 10% of the patients benefit from the combination at the expense of severe toxicity. Patients need to be hospitalized during cisplatin infusion and acute and long term toxicities are frequent. It would be ideal to personalize the application of chemoradiation and to treat only those patients whose tumors are likely to respond to the combination therapy. Major research efforts aimed to find clinical and biomolec- ular markers that predict chemoradiation response of HNSCC. However, the only clinical factor that shows some predictive value for chemoradiotherapy outcome in HNSCC proved to be primary tumor volume (4–11), and not even all studies could confirm this (12). Using a candidate gene or protein approach, several biological and genetic markers have been studied to predict chemoradiation outcome in head and neck cancer patients but none found its way to the clinic (13–20). Other groups have exploited microarray technology to determine expression profiles that might predict treatment response in head and neck cancer (12, 21–28), but a predictive profile has not been validated. There are several explanations why these approaches have not led to breakthroughs. First, tumor biopsies may contain mixed cell populations, and particularly the small subpopulations of treat- ment-resistant cells (e.g., cancer stem cells) may not be analyzed by expression profiling, while they might be highly relevant for treatment outcome (29). Second, genes harboring an activating or inactivating mutation, but that are expressed at close to normal levels, will not be identified as predictors of treatment outcome. We therefore aimed to identify all genes that have an important role in the response to cisplatin using a functional genomics approach. Unveiling these genes might enable us to find biomar- kers that can be used to predict cisplatin-based chemoradiation outcome and to personalize HNSCC treatment by only treating those patients who would benefit from the therapy. The application of loss-of-function high-throughput RNA interference screens has become an important genomic tool in 1 Department of Otolaryngology-Head and Neck Surgery, VU University Medical Center, Amsterdam, the Netherlands. 2 RNA Interference Functional Oncoge- nomics Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands. 3 Department of Clinical Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Ruud H. Brakenhoff, VU University Medical Center, PO Box 7057, Amsterdam 1007 MB, the Netherlands. Phone: 312-0444-0953; Fax: 312-0444-3688; E-mail: rh.brakenhoff@vumc.nl doi: 10.1158/1535-7163.MCT-16-0457 Ó2016 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 16(3) March 2017 540 on June 3, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst December 15, 2016; DOI: 10.1158/1535-7163.MCT-16-0457