Abstract 435: Accurate measurement of tumor mutation burden through tumor-only sequencing using a 500-gene panel
Authors: So Alex Steven , Kaplan Shannon , Zhao Chen , Zhang Shile , Liu Li , Le Phillip , Vijayaraghavan Raakhee , Jiang Tingting , Bilke Sven , Pawlowski Traci , Gutekunst Karen 
Published: August 2018

Introduction: Recently, it has been shown by various studies that tumor mutation burden (TMB) can correlate with a cancer patient's responsiveness to checkpoint inhibitor immunotherapy. These findings have stimulated a widespread interest in the development of cost-effective assays that accurately measure TMB. Here we assess whether TMB measured through next-generation sequencing (NGS) of a 2-megabase 500-gene panel correlates with that determined through whole exome sequencing (WES). We further investigate whether sequencing tumor samples alone combined with a germline filtering and background noise removal algorithm can measure TMB accurately compared with sequencing of tumor-normal pairs.

Experimental Method: WES data of tumor and subject-matched normal DNA were either downloaded from The Cancer Genome Atlas (TCGA) or generated in-house. A 500-gene targeted DNA sequencing panel, which includes various cancer-relevant genes and covers two megabases, was also developed in-house. TMB was calculated as mutations (single nucleotide variants, insertions, and deletions) per megabase.

Data Summary: Using downloaded TCGA data from 2,385 subjects across colon, lung and melanoma cancer types, TMB was measured using tumor-normal WES or the in-silico-filtered tumor-only 500-gene panel and the two approaches correlated linearly (R2 = 0.96). Furthermore, we demonstrated that the targeted panel achieved &gt;90% sensitivity and &gt;85% specificity when classifying TMB-high and low tumor samples at the cutoff of 15 compared to the same TCGA data. TMB as determined from tumor-only WES was also analyzed using computational germline-filtering and background noise-removal to demonstrate excellent concordance with WES of tumor-normal pairs. To further validate these findings, by analyzing WES data and 500-gene panel targeted NGS data of &gt;45 subjects generated in house. Through interrogating the 500-gene NGS data, we found that TMB determined using tumor-only sequencing was also similar from that ascertained through tumor-normal pairs. Of interest, further analysis showed that TMB determined from the 500-gene panel data using tumor-only sequencing along with germline filtering and background noise removing was comparable to that calculated from WES of tumor-normal pairs.

Conclusion: Collectively, our data demonstrates that TMB determined from NGS of a two megabase DNA region utilizing a 500 gene panel correlates strongly with TMB measured through WES, with a sensitivity of &gt;90% and a specificity of &gt;85%. In addition, sequencing tumor samples alone combined with bioinformatic germline-filtering and background noise-removal was similar to sequencing subject-matched normal DNA. These findings support the use of a 500 gene panel for accurate measurement of TMB while the addition of bioinformatics enables tumor-only sequencing.

Citation Format: Alex Steven So, Shannon Kaplan, Chen Zhao, Shile Zhang, Li Liu, Phillip Le, Raakhee Vijayaraghavan, Tingting Jiang, Sven Bilke, Traci Pawlowski, Karen Gutekunst. Accurate measurement of tumor mutation burden through tumor-only sequencing using a 500-gene panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 435.