RNA-seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancer With and Without Defective DNA Mismatch Repair

Tumor mutation burden (TMB) is an evolving biomarker for predicting response to immune checkpoint inhibitors (ICI’s). The goal of this study was to evaluate the use of RNA-seq for determining TMB in patients with defective DNA MMR (dMMR) due to MMR mutations or MLH1 promoter hypermethylation (HM) and tumors without dMMR. We included tumors and paired normal tissue from 58 patients for TMB analysis using RNA-seq. Forty-six tumors were MSI-H (29 with a DNA MMR germline mutation and 17 with MLH1 promoter HM) and 12 were MSS. TMB was measured using the expressed somatic nucleotide variants (eTMB). We developed a method that leverages mutational signatures to remove FFPE derived artifact from total eTMB and thereby accurately measure the eTMB. There was a significant difference in the eTMB observed between MSI-H and MSS cases; MSI-H cases had a median of 27.3 mutations/Mb compared to 6.7 mutations/Mb for MSS cases (p=3.5e-9). Among tumors with dMMR the tumors with DNA MMR mutations had a significantly higher eTMB (p=0.037) than tumors with MLH1 promoter HM: a median of 28.1 mutations/Mb vs. a median of 17.5 mutations/Mb. Furthermore, through multivariate analysis we found that MSI status, tissue type (endometrial or colorectal) and patient ages are significantly associated with eTMB. These results demonstrate that RNA-seq analysis can be used to measure eTMB in FFPE tumor specimens. Further studies are needed to determine how eTMB as determined by RNA-seq compares to TMB as determined by DNA-based NGS assays RNA-seq was perfomed on 91 tumors, with 19 MSS, 19 MSI-H cases due to HM and the remaining 53 were putative Lynch Syndrome cases. For 85 out of the 91 tumors, RNA-seq was performed adjacent normal tissue

肿瘤突变负荷（Tumor mutation burden, TMB）是一类用于预测免疫检查点抑制剂（immune checkpoint inhibitors, ICI）治疗响应的新兴生物标志物。本研究旨在评估RNA测序（RNA-seq）在因DNA错配修复（DNA Mismatch Repair, MMR）突变或MLH1启动子高甲基化（MLH1 promoter hypermethylation, HM）导致错配修复缺陷（defective DNA MMR, dMMR）的患者，以及无错配修复缺陷肿瘤患者中，用于测定TMB的应用价值。我们纳入了58例患者的肿瘤及配对正常组织，采用RNA-seq开展TMB分析，其中46例肿瘤为微卫星高度不稳定（Microsatellite Instability-High, MSI-H）：29例存在DNA MMR生殖系突变，17例存在MLH1启动子高甲基化；剩余12例为微卫星稳定（Microsatellite Stable, MSS）。本研究通过表达体细胞核苷酸变异（expressed somatic nucleotide variants, eTMB）来测定TMB，并开发了一种利用突变特征去除福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）样本来源的伪迹的方法，从而实现eTMB的精准测定。MSI-H组与MSS组的eTMB存在显著差异：MSI-H组的中位eTMB为27.3突变/Mb，而MSS组仅为6.7突变/Mb（p=3.5e-9）。在错配修复缺陷的肿瘤中，存在DNA MMR突变的肿瘤的eTMB显著高于MLH1启动子高甲基化的肿瘤（p=0.037），前者中位eTMB为28.1突变/Mb，后者为17.5突变/Mb。此外，通过多变量分析我们发现，微卫星状态、组织类型（子宫内膜或结直肠）以及患者年龄与eTMB显著相关。本研究结果表明，RNA-seq分析可用于测定福尔马林固定石蜡包埋肿瘤样本的eTMB，后续仍需开展进一步研究，以明确RNA-seq测定的eTMB与基于DNA的下一代测序（Next-Generation Sequencing, NGS）检测所得TMB之间的对比关系。本研究对91例肿瘤进行了RNA-seq检测，其中19例为MSS，19例为因MLH1启动子高甲基化导致的MSI-H病例，剩余53例为疑似林奇综合征（Lynch Syndrome）病例；91例肿瘤中，85例还完成了配对正常组织的RNA-seq检测。