Abstract LB-398: Detection of copy number alterations in 124 head and neck squamous cell carcinomas using the Nanostring nCounter assay
Authors: Keck Michaela Kristina , Zuo Zhixiang , Khattri Arun , Braegelmann Johannes , Lingen Mark , Rieke Damian , Geiss Gary , Gerlach Jay , Vokes Everett , Seiwert Tanguy Y. 
Published: August 2012

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Little is known about changes in copy number (CN) in many cancer associated genes in this tumor type, which may contribute to carcinogenesis and could become useful treatment targets in the future. Many analyses of CN are complicated by the need to use formalin-fixed paraffin-embedded (FFPE) tissues posing technical challenges. We determined copy number alterations (CNA) using a novel, medium-throughput technology (NanoString nCounter) in order to determine common cancer associated CNAs and assess its performance in FFPE tissues. Results were compared with more established technologies such as SNP array, aCGH, and qPCR. Methods: We investigated CNA in 124 tumor specimens and 22 cell lines for 100 literature curated cancer genes using the NanoString nCounter. Most samples were OCT frozen tissues, with a small subset having both OCT frozen, and FFPE tissues. Slides were assessed for tumor content by a HNC pathologist and samples with at least 60% tumor content selected. DNA was extracted using standard column-based methods (Qiagen). We performed CN analysis in 124 frozen (+4 matching FFPE) HNSCC specimens and cell lines (Nanostring nCounter assay) focusing on a selection of cancer associated genes. Furthermore we used aCGH and SNP-CHIP to analyse 20 and 4 cell lines respectively two of which were covered by all three methods. FGFR1 was assessed by qPCR. For FFPE samples a special Nanostring probeset was used with 3-5 probes per gene to provide redundancy with degraded DNA samples. HPV status of samples was assessed by a nested PCR for E6. Results Copy number changes detected by Nanostring and aCGH correlated well. The Nanostring nCounter assay appeared more accurate in calling deletions, which were detected in MST1R, PBRM1, PTPRD for instance. We found amplifications in multiple samples and genes, e.g. CCND1, EGFR, MDM4, MYC, VEGFA, PAX9, ITGB4, SSND1, CTTN, FADD, FGF19, ORAOV1, PPFIA1, some of which were frequently (n&gt;50 samples) or highly amplified (&gt;30 copies). Some of these amplifications such as ORAOV1 and PPFIA1 seemed higher/more frequent in HPV(-) compared to HPV(+) samples. Samples with FGFR1 amplification were validated using qPCR and correlated very closely. FFPE sample processing was uncomplicated using the FFPE probeset. While some probes failed, using degraded FFPE derived DNA, the redundancy of probes allowed accurate calling of CNA that closely correlated with frozen sample results. Conclusions Copy number alterations are frequent in HNSCC and involve many cancer associated genes, including potentially targetable genes such as EGFR, MDM4, and PIK3CA. Most of the CN changes are recurrent. Amplifications and deletions to some extent differed depending on HPV status. The role and implications of these CN aberrations in a clinical setting need to be further elucidated and validated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-398. doi:1538-7445.AM2012-LB-398