Abstract 537: Development of blood TMB (bTMB) reference materials for validation of cfDNA-based targeted NGS assays that measure tumor mutational burden (TMB) in patient blood samples
Authors: Lowe Dana Ruminski , Forson Benedicta , Butler Matthew G. , Konigshofer Yves , Huang Catherine , Clement Omoshile , Garlick Russell K. , Anekella Bharathi 
Published: June 2022

TMB is a biomarker with potential for predicting positive patient response to immune checkpoint inhibitors. TMB measurements can be determined using genomic DNA extracted from FFPE-preserved tissue biopsy samples. However, assessment of TMB from a liquid biopsy, or blood TMB (bTMB), is an attractive alternative clinical diagnostic tool that would allow clinicians and patients to avoid the invasive challenge of tissue biopsies. Accurately measuring bTMB with targeted gene panels has been problematic, thus we have developed the new Seraseq® bTMB reference materials at various mutational burden levels from genomic DNA extracted from tumor-derived and SNP-matched normal cell lines. Somatic variants were identified in each cell line by whole exome sequencing (WES). TMB assessments were made by an in-house developed TMB bioinformatics pipeline based on recommendations by the Friends of Cancer Research (FoCR) TMB Harmonization Consortium. DNA from tumor and normal cell lines were blended at 0%, 0.5%, and 2% tumor fractions and fragmented to approximate the size of cell free DNA (cfDNA). The resulting bTMB mixes were enriched with the TruSight™ Oncology 500 ctDNA Assay (2x151 bp), in triplicate, and sequenced on a NovaSeq™ 6000. Blood TMB scores were determined using the DRAGEN™ TruSight Oncology 500 ctDNA Analysis Software v1.1. The observed bTMB scores for the tumor-containing materials were slightly lower in the TF 0.5% mix versus the TF 2% mix due to variants moving below the limit of detection of the TSO500 ctDNA assay (Table 1). As is the case with patient samples and clonal hematopoiesis, there are background variants in 0% tumor content material that may elevate apparent bTMB scores, thus adjusted bTMB scores are shown. The Seraseq Blood TMB Score reference materials provide a tumor-normal matched blood TMB control to aid development, validation and QC of cfDNA NGS assays for accurate determination of bTMB Scores.

Table 1. Average bTMB scores and adjusted bTMB scores for each mix. Mix Average bTMB Score (mutations/megabase ± 1 SD) Adjusted bTMB Score (mutations/megabase ± 1 SD) Score 7 0% 7.5 ± 1.7 0 Score 7 0.5% 13.1 ± 2.6 5.6 ± 3.1 Score 7 2% 17.9 ± 1.3 10.4 ± 2.1 Score 13 0% 4.6 ± 0.5 0 Score 13 0.5% 18.7 ± 2.1 14.1 ± 2.2 Score 13 2% 24.6 ± 0.8 20.0 ± 0.9 Score 20 0% 7.5 ± 1.4 0 Score 20 0.5% 26.0 ± 2.3 18.5 ± 2.7 Score 20 2% 35.6 ± 1.0 28.1 ± 1.7 Score 26 0% 6.0 ± 0.5 0 Score 26 0.5% 20.7 ± 5.5 14.7 ± 5.5 Score 26 2% 30.4 ± 1.5 24.4 ± 1.9

Citation Format: Dana Ruminski Lowe, Benedicta Forson, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Omoshile Clement, Russell K. Garlick, Bharathi Anekella. Development of blood TMB (bTMB) reference materials for validation of cfDNA-based targeted NGS assays that measure tumor mutational burden (TMB) in patient blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 537.