Background: The need to identify biomarkers that predict benefit to checkpoint inhibitor therapies has led to the discovery and development of tumor mutational burden (TMB), a measure of potential tumor neoantigenicity derived from tissue biopsies that has shown clinical utility across a range of tumor types. A significant fraction of patients, however, are not candidates for tissue biopsies, presenting the need for blood-based methods to determine TMB. Here we describe the development of an assay to identify TMB from cell-free DNA derived from blood (bTMB). We present the analytic validation and clinical feasibility data that support the application of bTMB in a prospective clinical trial, BFAST (NCT03178552), evaluating the anti-PD-L1 agent atezolizumab in patients with non-small cell lung cancer (NSCLC).

Methods: The bTMB assay surveys somatic base substitutions down to 0.5% allele frequency across 394 genes from as little as 1% tumor content in a cell free DNA (cfDNA) sample derived from blood. Analytic validation was focused on establishing accuracy and precision of the bTMB measurement, as well as the minimum amount of cell-free and circulating tumor DNA required to make precise and reliable bTMB calls. The accuracy of two bTMB cutoffs was established against TMB derived from FoundationOne, an analytically validated TMB platform. Precision was evaluated by comparing the reproducibility of bTMB calls across replicate samples. We also retrospectively analyzed plasma samples from the OAK (NCT02008227) and POPLAR (NCT01903993) trials with the bTMB assay to determine the association of bTMB with atezolizumab clinical activity. The biomarker evaluable population (BEP) included 211 patients in POPLAR (intention-to-treat [ITT] =287) and 583 patients in OAK (excludes patients with known EGFR/ALK mutations; ITT=850), with blood samples available for targeted genomic sequencing. Assay positivity was defined as the presence of a number of somatic base substitutions greater than or equal to the bTMB cutoffs.

Results: The average positive percent agreement (PPA), negative percent agreement (NPA) and positive predictive value (PPV) across the bTMB cutoffs were 95%, 100% and 100%, respectively. The average precision was 96%, with a coefficient of variation of 7%. The assay limit of detection was defined as 1% tumor content in at least 20 ng of cfDNA. In POPLAR, improved progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) with atezolizumab vs docetaxel were observed for patients with bTMB at or above a range of bTMB thresholds compared with the ITT and BEP populations. In OAK, PFS benefit with atezolizumab vs docetaxel was observed at bTMB thresholds ≥10 (cut point ≥10: HR 0.73; n=251) compared with BEP (HR 0.87, 95% CI 0.73-1.04; n=585). bTMB did not correlate with PD-L1 expression as measured by VENTANA SP142 immunohistochemistry.

Conclusions: We have developed and analytically validated a blood-based assay to determine TMB with high accuracy and precision, using as little as 1% tumor content in a sample with 20 ng of cfDNA. Retrospective analyses from POPLAR and OAK data provide the first demonstrations that blood-based measurement of TMB may be associated with atezolizumab clinical efficacy in second-line NSCLC. Thus, the bTMB assay may provide a non-invasive biomarker to identify patients who derive clinical benefit from single agent PD-1/PD-L1 inhibition. Prospective studies using bTMB are currently ongoing in patients with first-line NSCLC, including BFAST and B-F1RST (NCT02848651).

Citation Format: Daniel S. Lieber, Emily White, Jacob Silterra, Shan Zhong, Tina Brennan, Michael Coyne, Mark Kennedy, David R. Gandara, Marcin Kowanetz, Sarah M. Paul, Erica Schleifman, Yan Li, Achim Rittmeyer, Louis Fehrenbacher, Lukas Amler, Todd Riehl, Craig Cummings, Priti S. Hegde, Wei Zou, Alan Sandler, Marcus Ballinger, Tony Mok, David S. Shames, Doron Lipson, Christine Malboeuf, David Fabrizio. Analytic validation and clinical feasibility of a next-generation sequencing assay to assess tumor mutational burden from blood (bTMB) as a biomarker for anti-PD-L1 response in NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A41.