Analytic validation of a next generation sequencing assay to identify tumor mutational burden from blood (bTMB) to support investigation of an anti-PD-L1 agent, atezolizumab, in a first line non-small cell lung cancer trial (BFAST)

Background

Recent data suggest that analysis of tumor mutational burden (TMB), a measure of tumor neo-antigenicity derived from tissue biopsies, has shown clinical utility in predicting outcomes for patients treated with anti-PDL1/PD1 therapies across a range of tumor types. Unfortunately, such analyses require quality tumor tissue that in many cases is not available for patients diagnosed in the metastatic setting. As such, there exists a significant unmet medical need for orthogonal diagnostic approaches that enable the analysis of TMB in patient samples without requiring tumor tissue. Herein, we describe the development of an assay to identify TMB from the circulating tumor DNA derived from blood (bTMB), and the analytical validation (AV) that supports its application in a phase III clinical trial in 1L non-small cell lung cancer comparing the anti-PD-L1 agent, atezolizumab, against standard of care platinum-based doublet chemotherapy (BFAST).

Methods

The bTMB assay delivers a count of 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. AV focused on establishing accuracy and precision, as well as the limit of circulating tumor DNA required to make precise and reliable bTMB calls. Accuracy of the two different bTMB cutoffs being evaluated in BFAST was established against an orthogonally validated TMB platform. Precision was evaluated by comparing the reproducibility of bTMB calls across replicate samples.

Results

The average PPA, NPA and PPV across both bTMB cutoffs was 95%, 100% and 100%, respectively. The average precision was 96%, with a coefficient of variation of 17% across all replicates. The assay limit of detection was defined as 1% tumor content in at least 20 ng of cfDNA.

Conclusions

We have developed and analytically validated a blood-based assay to determine bTMB with high accuracy and precision from as little as 1% tumor content in 20 ng of cfDNA. Clinical validation of bTMB will be established in a prospective, randomized phase III clinical trial, BFAST, with a primary endpoint of progression free survival.

Clinical trial identification

Legal entity responsible for the study

Foundation Medicine, Inc.

Funding

Genentech, Inc.

Disclosure

D. Fabrizio, C. Malboeuf, D. Lieber, S. Zhong, J. He, E. White, M. Coyne, J. Silterra, T. Brennan, J. Ma, M. Kennedy, D. Lipson, G. Otto: Employee and stockholder of Foundation Medicine. E. Schleifman, S.M. Paul, Y. Li, D.S. Shames, C.A. Cummings, E. Peters, M. Kowanetz: Employee and stockholder at Genentech.