Spatial and Temporal Heterogeneity of Panel-Based Tumor Mutational Burden in Pulmonary Adenocarcinoma: Separating Biology From Technical Artifacts

Abstract

Background: Tumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB. Methods: TMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five 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 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node-derived TMB was significantly lower (p ¼ 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, intratumor heterogeneity 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 show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of singlesample-based TMB estimations in a clinical context.

Tumour mutational burden: primary versus metastatic tissue creates systematic bias

“…This has been emphasized by previous studies which compared TMB values across multiple regions from the same tumour. In 12.5% (3/24) of pulmonary adenocarcinomas [28], 20% (20/100) of NSCLC and 52% (12/23) of urothelial carcinomas [29], the TMB classification was inconsistent across multiple regions. The current study was not powered for adequate subclonality analysis due to its single biopsy sampling approach and panel dataset alone; therefore, subclonal deconstruction is not feasible.…”

The Promises and Challenges of Tumor Mutation Burden as an Immunotherapy Biomarker: A Perspective from the International Association for the Study of Lung Cancer Pathology Committee

“…The authors suggested that heterogeneity across tumor compartments might impact our interpretation of the tumor genome, stressing the fundamental technical differences in computational pipelines and sample characteristics between tTMB and bTMB. 39 Retrospective analysis of samples from the POPLAR study determined that a bTMB cut point of greater than or equal to 16 mutations/sample had the strongest PFS treatment effect and an overall prevalence of 30%. Similarly, data from the OAK study on the groups with bTMB greater than or equal to 16 revealed a significant PFS benefit (HR 0.65 95% CI: 0.47-0.92) from atezolizumab versus docetaxel.…”

“…58 Indel burden, in particular, may be informative in high-TMB cases. 39 Need for Normal Matched Sample. Sequencing of paired tumor-normal matched samples with germline variants removed from the TMB count is regarded as the definitive standard for TMB analysis.…”

Targetable ERBB2 mutations identified in neurofibroma/schwannoma hybrid nerve sheath tumors

“…DNA was extracted using a Maxwell 16 Research System (Promega), followed by quantification using the QuBit 2.0 DNA High Sensitivity Kit (Thermo Fisher Scientific). Library preparation for the capture-based TruSight Oncology 500 panel (Illumina) (Supplemental Table 3)was performed as previously described (31). DNA integrity was assessed using the Genomic DNA ScreenTape Analysis on a 4150 TapeStation System (Agilent).…”