Abstract 137: Development and application of a 62-gene panel for assessment of somatic sequence and structural variants in tumor DNA derived from non-Hodgkin lymphoma patients treated in a phase 1 clinical trial with the EZH2 inhibitor tazemetostat

Abstract

Abstract Tazemetostat is a small molecule inhibitor of the histone methyltransferase EZH2 and is currently in phase 2 clinical trials in relapsed refractory Non-Hodgkin's Lymphomas (RR-NHL) including diffuse large B cell and follicular lymphoma. We report the development and application of a NHL targeted sequencing panel designed to identify molecular variants, including specific somatic sequence mutations (single base and insertion/deletion), amplifications and translocations in both tumor and cell free circulating tumor DNA (ctDNA). Analysis of somatic alterations in both tissue and ctDNA collected pre-dose enables determination of molecular variants that may correspond with clinical activity, while longitudinal analysis of ctDNA sampled on therapy could help monitor patients for minimal residual disease and emergence of acquired resistance. In collaboration with Personal Genome Diagnostics (PGDx) a panel of 62 NHL specific genes was designed to selectively analyze regions of the genome previously identified as somatically altered in NHL. DNA derived from matched tumor tissue and plasma were screened utilizing this panel using the Illumina HiSeq 2500 platform with 100 bp paired-end reads. Average target coverage for the tissue panel was 1,250-fold while coverage for the ctDNA was approximately 20,000-fold and 3,700-fold for sequence mutations and structural alterations, respectively. Data were analyzed using PGDx's validated cancer genome analysis algorithms that allow for reliable identification of mutations with high sensitivity and specificity. Validation of both the tumor and ctDNA panels was performed using tumor and plasma specimens previously characterized for sequence mutations, amplifications, translocations, and microsatellite instability. For archive tumor, analyses of cell line specimens with an experimental tumor purity of 20-100% using 50-100ng of DNA yielded sensitivity and specificity of 100% for detection of 358 previously characterized sequence mutations and structural variants. Similar ctDNA analyses using fragmented cell line and plasma derived DNA with an experimental tumor purity of 0.10%-25.0% using 9-167ng of DNA yielded a sensitivity of 100% for detection of over 100 genetic variants. Following successful validation of the panel we proceeded to sequence tumor tissue from 11 patients and ctDNA from 16 NHL patients enrolled in the tazemetostat phase 1 clinical trial. Tumor tissue from these patient samples had been previously sequenced using a smaller 39 gene panel. We observed high concordance with 100% of variants detected within the shared gene set of 33 genes between the historic data and our new 62 gene panel. We will report the landscape and concordance of genetic alterations identified through next-generation sequencing analyses of tumor and cell-free DNA. Citation Format: Scott R. Daigle, Samuel Angiuoli, Sian Jones, Scott Ribich, Heike Keilhack, Mark Sausen, Peter T. Ho, Stephen J. Blakemore. Development and application of a 62-gene panel for assessment of somatic sequence and structural variants in tumor DNA derived from non-Hodgkin lymphoma patients treated in a phase 1 clinical trial with the EZH2 inhibitor tazemetostat. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 137.