Analysis of tumor mutational burden: correlation of five large gene panels with whole exome sequencing

Abstract

Outcome of immune checkpoint inhibition in cancer can be predicted by measuring PDL1 expression of tumor cells. Search for additional biomarkers led to tumor mutational burden (TMB) as surrogate marker for neoantigens presented. While TMB was previously determined via whole exome sequencing (WES), there have been approaches with comprehensive gene panels as well. We sequenced samples derived from formalin-fixed tumors, a POLE mutated cell line and standard DNA by WES and five different panels. If available, normal tissue was also exome sequenced. Sequencing data was analyzed by commercial software solutions and an in-house pipeline. A robust Pearson correlation (R = 0.9801 ± 0.0167; mean ± sd; N = 7) was determined for the different panels in a tumor paired normal setting for WES. Expanded analysis on tumor only exome sequenced samples yielded similar correlation (R = 0.9439 ± 0.0632; mean ± sd; N = 14). Remaining germline variants increased TMB in WES by 5.761 ± 1.953 (mean ± sd.; N = 7) variants per megabase (v/mb) for samples including synonymous variants and 3.883 ± 1.38 v/mb for samples without synonymous variants compared to tumor-normal paired calling results. Due to limited sample numbers in this study, additional replication is suggested for a clinical setting. Remaining germline variants in a tumor-only setting and artifacts caused by different library chemistries construction might affect the results. Immune checkpoint inhibitors significantly increased survival across several tumor entities including non-smallcell lung cancer (NSCLC) and melanoma 1,2. However, the response rate is highly variable even within a certain tumor entity, ranging from complete to no response. Thus, there is an urgent need for new predictive biomarkers to identify patients who are most likely to respond 3-5. Currently, two biomarkers are used to select patients: PDL1 expression as measured by immunohistochemistry is approved for companion and complementary testing prior to immunotherapy by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). In 2017, FDA also granted approval for the immunotherapy of solid tumors showing high microsatellite instability. Despite several attempts for standardization it seems that PDL1 immunohistochemistry assays alone remain insufficient as also some patients that are negative for PDL1 expression revealed response to immunotherapy 6-9. Retrospective studies showed the predictive ability of tumor mutational burden (TMB) to discriminate responders from non-responders across several tumor entities 10-12. It is hypothesized that tumors with a higher mutation burden are likely to express and present more neoantigens and thereby induce a stronger immune response 13. Supporting evidence came from the identification of other genome-related markers for response like mutations in genes related to DNA repair 14 and deficiencies in the mismatch repair system 15. In previous clinical studies, whole exome sequencing (WES) was used for the measurement of TMB 16,17. WES tumor versus ...

Panels and models for accurate prediction of tumor mutation burden in tumor samples

“…Several studies have reported the development of methods for TMB calculation based on commercial NGS panels 43 , 44 . However, to the best of our knowledge, there is only one study where “in silico” tools were used to select genes for TMB prediction 45 .…”

Tumor Mutational Burden as a Predictor of Immunotherapy Response: Is More Always Better?

“…The challenge of interpreting TMB scores is further complicated by the availability of multiple diagnostic assays. It is unlikely that a single TMB threshold could uniformly be applied to all assays (11,12). Blood based cfDNA offers a more convenient approach to assess TMB and potentially overcome the problem of tumor heterogeneity, but fewer clinical studies have validated this approach.…”

The value of tumor mutational burden to select patients for immunotherapy

“…High costs, extensive time-consuming analysis, and high amounts of DNA requirements for WES are challenging for daily routine TMB assessment. Therefore, gene panels may be preferable, because they are less cost-intensive, require lower input of DNA, and have an overall shorter turnaround time [12,13]. With increasing interest in TMB as a biomarker for response to ICI therapies, many commercial providers developed their own panel-based TMB assays, to be performed by molecular diagnostic labs in-house [13].…”

“…Therefore, gene panels may be preferable, because they are less cost-intensive, require lower input of DNA, and have an overall shorter turnaround time [12,13]. With increasing interest in TMB as a biomarker for response to ICI therapies, many commercial providers developed their own panel-based TMB assays, to be performed by molecular diagnostic labs in-house [13]. Various challenges arise, impacting TMB determination: In the course of implementation of complex assays for TMB determination, factors like composition, distribution, and size of gene panels, influence of sequencing platforms, genomic coverage, bioinformatics evaluation, and definition of thresholds are becoming increasingly important.…”