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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.

SUBMITTER: Heydt C

PROVIDER: S-EPMC7347536 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Heydt Carina C Rehker Jan J Pappesch Roberto R Buhl Theresa T Ball Markus M Siebolts Udo U Haak Anja A Lohneis Philipp P Büttner Reinhard R Hillmer Axel M AM Merkelbach-Bruse Sabine S

Scientific reports 20200709 1

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. I ...[more]

PMID: 32647293

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Project description:BackgroundTumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated.MethodWe established a series of reference standard DNA samples derived from 11 pairs of tumor-normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass-to-mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole-exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes.ResultsLinear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content.ConclusionsWe developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy.

Dataset Information

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

Publications

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

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