Abstract B16: Validation and clinical feasibility of a Foundation Medicine assay to identify immunotherapy response potential through tumor mutational burden (TMB)
Authors: Lieber Daniel S. , Kennedy Mark R. , Johnson Douglas B. , Greenbowe Joel R. , Frampton Garrett M. , Schrock Alexa B. , Ross Jeffrey S. , Stephens Phillip J. , Ali Siraj M. , Miller Vincent A. , Fabrizio David A. 
Published: March 2017

Abstract
Background: The ability of tumors to evade immune surveillance by overexpressing immune checkpoint proteins has been exploited for therapeutic intervention through antibodies designed to interrupt their signaling. A number of patients across a range of disease types, including melanoma, lung, renal and bladder cancer, have demonstrated robust and durable responses using checkpoint inhibitor therapies (CPITs). Still, identifying the most likely responders remains an urgent need for proper clinical management. Tumor mutational burden (TMB) measures the overall number of somatic protein coding mutations per area of sequence counted occurring in a tumor specimen. This measure has been associated with both response and survival for multiple CPITs across an array of indications. It is hypothesized that immunotherapies are more effective for tumors with high TMB because these cells are more likely to express immune-reactive neoantigens. In this study we describe Foundation Medicine's (FMI) work to develop and validate a TMB result as part of the current FoundationOne (F1) and FoundationOne Heme (F1H) comprehensive genomic profiling assays.
Methods: We developed an analysis method to determine TMB based on data from both the F1 and F1H comprehensive genomic profiling assays. TMB is calculated by counting all synonymous and non-synonymous somatic variants across 315 or 405 genes. Germline alterations and known and likely driver alterations are excluded to avoid sample bias, as both F1 and F1H specifically target genes with cancer associations. The resulting mutation count is normalized by expressing the number as a mutation density with units of mutations per megabase (mut/Mb) of coding target territory. Analytic validation of TMB focused on accuracy, precision and sensitivity, while initial clinical feasibility was assessed in a cohort of 65 metastatic melanoma patients receiving immunotherapy. To determine accuracy, we compared the TMB values generated from F1 against a CLIA validated whole-exome sequencing (WES) method on 29 patients with TMB values ranging from &lt;1 mut/Mb up to 600 mut/Mb. Precision was defined as the reliability of the TMB metric when determined from 10 clinical samples replicated 4-6 times. Sensitivity was evaluated by determining the lower limit of sample tumor purity at which a TMB value could be reliably assessed through a dilution series of tumor/normal pairs ranging from 80% to 5% tumor. We also assessed the clinical feasibility of the F1 TMB result by examining its ability to predict clinical response to anti-PD1 or PD-L1 immunotherapy in a cohort of 65 metastatic melanoma patients. The patients were evaluated for best response per RECIST criteria, progression free survival (PFS) and overall survival (OS).
Results: Foundation Medicine's TMB measure provides accurate and precise results across a range of tumor mutational burden values on samples with as little as 20% tumor purity. In a cohort of 65 metastatic melanoma patients, the median TMB value was 37.9 mut/Mb in the responder group and 6.6 mut/Mb in the non-responder group (p&lt;0.0001, Mann-Whitney test). Additionally, TMB-high (≥20 mut/Mb) patients demonstrated superior PFS and OS compared to TMB non-high patients (median PFS and OS not reached for TMB-High through 66 months vs. medians of 3 months PFS and 12 months OS for TMB non-high, p-value &lt;0.001).
Conclusions: We have developed and validated a TMB result as part of the FoundationOne and FoundationOne Heme platforms. Initial clinical feasibility results demonstrate that the FoundationOne TMB value can be used to predict the likely response of metastatic melanoma patients to anti-PD1/PD-L1 checkpoint inhibitors, while feasibility in NSCLC and bladder cancer have been presented elsewhere.
Citation Format: Daniel S. Lieber, Mark R. Kennedy, Douglas B. Johnson, Joel R. Greenbowe, Garrett M. Frampton, Alexa B. Schrock, Jeffrey S. Ross, Phillip J. Stephens, Siraj M. Ali, Vincent A. Miller, David A. Fabrizio. Validation and clinical feasibility of a Foundation Medicine assay to identify immunotherapy response potential through tumor mutational burden (TMB). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B16.