Tumor mutational burden (TMB): Assessment of inter- and intra-tumor heterogeneity.

Abstract

27 Background: As an emerging biomarker, a high TMB is associated with response to immune checkpoint inhibitors. The tissue selected for molecular analysis could be critical for accurate TMB assessment and therapeutic selection. We evaluated TMB and the tumor specimen tested to determine the extent tissue selection has on TMB analysis. Methods: TMB profiling was performed on 73 cases representing 8 tumor types. 8 patients with multiple specimens from the same surgical event were evaluated for intra-tumor heterogeneity, and 20 patients with specimens from multiple surgical events spanning up to seven years were tested for inter-tumor heterogeneity. Two to six specimens, including primary, recurrent and metastatic cases, were evaluated per patient. For each patient, TMB values within and between tumors were assessed for concordance using a clinically relevant TMB-high cutoff of ≥ 10 mutations/Mb. Results: TMB values ranged from 0 – 58, with 7 cases scored as TMB-high. One patient with a primary skin melanoma and a lung metastasis biopsied after 34 months was concordant for TMB-high. For 19 patients (76%), all samples were concordant for TMB-low. For intratumor testing, 2 of 8 (25%) patients had discordant specimens (crossed the TMB-cutoff). The first patient had 4 intra-tumor CRC samples with TMB values of 10.6, 10.7, 9.7 and 9.7. The second patient had 5 intra-tumor pancreatic samples with TMB values of 10.7, 4.4, 6.2, 8.8, 7.9. Of the patients with 20 inter-tumor samples, 2 patients (each with melanoma) had discordant TMB-high calls. One had a lymph node metastasis TMB of 12.3 and a liver metastasis collected five months later with a reduced TMB of 0.9. In contrast, the second discordant melanoma patient had a peritoneum metastasis with an elevated TMB of 11.4 collected 14 months after a mesentery metastasis with a TMB of 8.7. Conclusions: TMB variation within tumors and between tumors from different surgical events occurs equally and in 17% of patients. This clinical tumor heterogeneity on the inter- or intra-tumor level may limit the utility and the application of TMB at a cutoff of 10. A careful analysis of tissues to enrich for neoplastic cells may improve assay sensitivity and increase analytical accuracy at this precise cut point.