Different NGS identification methods of somatic mutation sites in solid tumors impact TMB results

Tumor mutation burden (TMB) is a predictive biomarker for assessing the response of various tumor types to immune checkpoint inhibitors (ICI). TMB is quantified based on somatic mutations identified by next-generation sequencing (NGS) using targeted panel data. This study aimed to investigate whether different NGS methods will affect the results of TMB detection in solid tumors.

In this study, a hybrid capture NGS method was performed to identify Tumor-only (TO) tissue and tumor tissue and white blood cells Tumor Control (TC). The accuracy and specificity of the two employed methods were evaluated by the identification and analysis of standard reference data. Based on the quality control of FFPE samples, 24 pathological and imaging confirmed solid tumor samples were compared to assess the differences between the two methods in identifying and incorporating the mutation sites and the effect on TMB detection.

The data identified 298 common genes in the detection range of TO and TC methods. The detection range of these genes primarily comprised exons and some introns. The coefficient of variation (CV%) between the detected variant and true mutation frequencies was < 10%, confirming their accuracy and specificity. Both methods detected increased mutations of TP53, CDKN2 A, KRAS, PTEN, EGFR, PIK3 CA, BRAF, BRCA2, FGFR2, and NRAS. The consistency rate of TMB was observed as 92% (22/24). The chi-square test indicated a significant difference in TMB results between TO and TC (χ² = 16.667, p = 0.000, p < 0.001). Furthermore, Cohen's kappa analysis showed consistency in the TMB values detected by TO and TC methods, which were good and had high repeatability (kappa = 0.833, p = 0.000, p < 0.001). The Venn analysis revealed that the two methods identified and included different TMB sites, which in turn affected the TMB calculation results.

This study revealed that different algorithms and design panels for mutation filtering affect the TMB test results. When the TMB result is near the 10 mut/Mb threshold, different methods may yield different results. Moreover, a single test result can affect clinical treatment decisions. Therefore, it is recommended to use TO or TC combined with other tests for evaluating somatic mutations.