INTRODUCTION Tumor mutational burden (TMB) is an emerging biomarker used to identify patients more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables like tumor cell content and intratumor heterogeneity (ITH) may play an important role in determining TMB. METHODS TMB estimates were determined applying the TruSightTM Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multi-region sequencing (2-6 samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in 5 patients. RESULTS On average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7/24) of the cases (max difference 14.13 mut/Mbp). Lymph node derived TMB was significantly lower (p=0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, ITH and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound. CONCLUSIONS Our data demonstrate that, in addition to technical aspects like germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single sample based TMB estimations in a clinical context.

中文翻译：

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肺腺癌中基于面板的肿瘤突变负荷（TMB）的时空异质性：将生物学与技术伪影分开

介绍 肿瘤突变负荷 (TMB) 是一种新兴的生物标志物，用于识别更有可能从免疫肿瘤治疗中受益的患者。除了各种未解决的技术方面，肿瘤细胞含量和肿瘤内异质性 (ITH) 等生物学变量可能在确定 TMB 方面发挥重要作用。方法 应用 TruSightTM Oncology 500 靶向测序面板确定 TMB 估计值。通过对 24 例肺腺癌进行多区域测序（2-6 个样本）和对一组匹配的原发肿瘤、局部淋巴结转移和 5 名患者的远处转移进行测序，分析了时空异质性。结果 平均而言，一个 1.28 Mbp 的编码区被覆盖，平均读取深度为 609 倍。突变调用的手动验证证实了良好的性能，但在种系过滤过程中发现了明显的错误分类。肿瘤内的不同区域在 30% (7/24) 的病例中显示出相当大的空间 TMB 差异（最大差异为 14.13 mut/Mbp）。淋巴结来源的 TMB 显着降低（p=0.016）。在 13 个案例中，不同的突变谱是肿瘤不同区域独有的，导致模拟聚集的 TMB 值更高。综合起来，ITH 和聚合的 TMB 可能导致 17% 的分析患者出现不同的 TMB 指定。原发肿瘤和远处转移之间存在 TMB 变异，但不明显。结论我们的数据表明，除了生殖系过滤等技术方面外，肿瘤内的肿瘤含量和空间差异突变谱是影响 TMB 估计的相关因素，