Development and validation of blood tumor mutational burden reference standards

Abstract

Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.

Keywords: genetic markers; immunotherapy; translational medical research; tumor biomarkers.