RT Journal Article SR Electronic T1 Clinical advantage of targeted sequencing for unbiased tumor mutational burden estimation in samples with low tumor purity JF Journal for ImmunoTherapy of Cancer JO J Immunother Cancer FD BMJ Publishing Group Ltd SP e001199 DO 10.1136/jitc-2020-001199 VO 8 IS 2 A1 Hong, Tae Hee A1 Cha, Hongui A1 Shim, Joon Ho A1 Lee, Boram A1 Chung, Jongsuk A1 Lee, Chung A1 Kim, Nayoung K D A1 Choi, Yoon-La A1 Hwang, Soohyun A1 Lee, Yoomi A1 Park, Sehhoon A1 Jung, Hyun Ae A1 Kim, Ji-Yeon A1 Park, Yeon Hee A1 Sun, Jong-Mu A1 Ahn, Jin Seok A1 Ahn, Myung-Ju A1 Park, Keunchil A1 Lee, Se-Hoon A1 Park, Woong-Yang YR 2020 UL http://jitc.bmj.com/content/8/2/e001199.abstract AB Background Tumor mutational burden (TMB) measurement is limited by low tumor purity of samples, which can influence prediction of the immunotherapy response, particularly when using whole-exome sequencing-based TMB (wTMB). This issue could be overcome by targeted panel sequencing-based TMB (pTMB) with higher depth of coverage, which remains unexplored.Methods We comprehensively reanalyzed four public datasets of immune checkpoint inhibitor (ICI)-treated cohorts (adopting pTMB or wTMB) to test each biomarker’s predictive ability for low purity samples (cut-off: 30%). For validation, paired genomic profiling with the same tumor specimens was performed to directly compare wTMB and pTMB in patients with breast cancer (paired-BRCA, n=165) and ICI-treated patients with advanced non-small-cell lung cancer (paired-NSCLC, n=156).Results Low tumor purity was common (range 30%–45%) in real-world samples from ICI-treated patients. In the survival analyzes of public cohorts, wTMB could not predict the clinical benefit of immunotherapy when tumor purity was low (log-rank p=0.874), whereas pTMB could effectively stratify the survival outcome (log-rank p=0.020). In the paired-BRCA and paired-NSCLC cohorts, pTMB was less affected by tumor purity, with significantly more somatic variants identified at low allele frequency (p<0.001). We found that wTMB was significantly underestimated in low purity samples with a large proportion of clonal variants undetected by whole-exome sequencing. Interestingly, pTMB more accurately predicted progression-free survival (PFS) after immunotherapy than wTMB owing to its superior performance in the low tumor purity subgroup (p=0.054 vs p=0.358). Multivariate analysis revealed pTMB (p=0.016), but not wTMB (p=0.32), as an independent predictor of PFS even in low-purity samples. The net reclassification index using pTMB was 21.7% in the low-purity subgroup (p=0.016).Conclusions Our data suggest that TMB characterization with targeted deep sequencing might have potential strength in predicting ICI responsiveness due to its enhanced sensitivity for hard-to-detect variants at low-allele fraction. Therefore, pTMB could act as an invaluable biomarker in the setting of both clinical trials and practice outside of trials based on its reliable performance in mitigating the purity-related bias.