Abstract
ObjectivesTumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. Materials and MethodsWe analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%). ResultsAt 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. ConclusionOur results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.
Highlights
Tumor mutational burden (TMB), defined as the total number of somatic mutations per tumor genome, is a known predictive biomarker for immune checkpoint inhibitor (ICI) therapy outcome, following the observation that high TMB associates with positive treatment response [1,2,3,4,5,6]
Our results revealed that cytological samples are well suited for TMB measurement and that formalin fixation artifacts impact variant calling and TMB esti mation in formalin-fixed paraffin-embedded (FFPE) samples even after uracil-DNA glycosylase (UDG) treatment
We included 12 non-small cell lung cancer (NSCLC) patients (7 males, 5 females), who were diagnosed with adenocarcinoma (n = 6), squamous cell carcinoma (n = 5), or a mixed large cell neuroendocrine carcinoma – adenocarcinoma (n = 1) of the lung (Supplementary Table 2)
Summary
Tumor mutational burden (TMB), defined as the total number of somatic mutations per tumor genome, is a known predictive biomarker for immune checkpoint inhibitor (ICI) therapy outcome, following the observation that high TMB associates with positive treatment response [1,2,3,4,5,6]. One additional chal lenge is to understand how the use of different sample types as a source of DNA affects mutation calling and TMB estimation. This knowledge is relevant in lung cancer, where diagnoses are made from formalin-fixed paraffin-embedded (FFPE) tissue, but frequently from cytological specimens obtained by bronchial washing, brushing, and needle aspiration [10]. In this study, we assessed TMB through a targeted NGS-based gene panel on matched histological and ethanol-fixed cytological sam ples obtained from lung cancer patients (n = 12). Our data indicate that cytological specimens provide a reliable source for TMB estimation, and are well suited for ICI therapy stratification
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