Inflation of tumor mutation burden by tumor-only sequencing in under-represented groups

Abstract

With the recent FDA approval of tumor mutational burden-high (TMB-H) status as a biomarker for treatment with a PD-1 inhibitor regardless of tumor type, accurate assessment of patient-specific TMB is more critical now more than ever. Using paired tumor and germline exome sequencing data from 701 patients newly diagnosed with multiple myeloma, including 575 self-reported White patients and 126 self-reported Black patients, we observed that compared to the gold standard of filtering germline variants with patient-paired germline sequencing data, TMB estimates were significantly higher in both Black and White patients when using public databases for filtering non-somatic mutations; however, TMB was more significantly inflated in Black patients compared to White patients. TMB as a biomarker for patient selection to receive immune checkpoint inhibitors (ICIs) therapy without patient-paired germline sequencing may introduce racial bias due to the under-representation of minority groups in public databases.

Fig. 1. Impact of variant filtering criteria on TMB calculation.

The TMB values were calculated as number of nonsynonymous mutations per Mb of coding regions. Four criteria were applied to identify patient-specific somatic mutations: (1) TMB_Germline: excluding variants in patient-matched germline exome; (2) TMB_DB0: excluding all variants reported by 1000G or ExAC; (3) TMB_DB0.001: excluding variants with MAF ≥ 0.1% in 1000G or ExAC; and (4) TMB_DB0.01: excluding variants with minor allele frequency (MAF) ≥ 1% in 1000G or ExAC DBs. The violin and box plots in red are TMB values from Black patients, and blue are from White Patients. a Comparisons of TMBs calculated from all protein-coding genes in Black and White patients from four variant filtering criteria. b Comparisons of TMB calculated from 1059 cancer genes in Black and White patients from four variant filtering criteria.

Fig. 2. Pair-wise visualization of the differences in tumor mutational burdens between race and variant filtering criteria.

This is a 5 × 5 matrix of pair-wise comparison. Red colors are data from Black patients, and blue colors are data from White patients. (1) The diagonal: density plots showing the increased separation of TMB distributions while relaxing the non-somatic variant filtering thresholds from paired germline (TMB_Germline), to ascending variant MAFs in 1000G and ExAC (TMB_DB0: MAF = 0; TMB_DB0.001: MAF ≤ 0.1%; TMB_DB0.01: MAF ≤ 1%). The last diagonal plot (bottom right) is the bar plot of the counts of 126 Black and 575 White patients included in the analyses. (2) The upper right panels of correlation values: the Pearson Correlation r values of TMBs across patients. The black numbers are the r values of all 701 patients. The last columns of the upper right panels are the box plots of the TMBs from different filtering criteria. (3) The lower left panels of dot plots illustrate the individual TMB values per patient; and the last row of the lower left panels are the jittered-point bar plots of individual TMB values.