Assessment of tissue and blood tumor mutational burden in patients with melanoma using a 523-gene clinical assay.

Abstract

e21570 Background: High tumor mutation burden (TMB) predicts improved efficacy of immune checkpoint inhibitors (ICIs) across a range of malignancies, including melanoma. Circulating tumor DNA (ctDNA) has emerged as a powerful complement to tumor biopsies for the diagnosis and clinical management of cancer patients. The TruSight Oncology 500 (TSO500) clinical assay interrogates 523 cancer-related genes and has shown good concordance between tissue and ctDNA using a high throughput sequencer such as NovaSeq. There are no studies looking at concordance and utility of this assay using more affordable mid throughput platforms. Here we present a pilot study comparing TSO500 sequencing results from matched tumor and plasma specimens and concordance of selected actionable gene variants investigated by ctDNA digital PCR. Methods: Samples from 18 metastatic cutaneous melanoma patients with matched formalin-fixed, paraffin-embedded (FFPE) tumor tissue and plasma collected in Streck tubes had DNA extracted for library preparation using the Illumina TSO500 NGS assay with inputs of ≥40ng of FFPE DNA and ≥10ng of ctDNA. Libraries were sequenced on a NextSeq2000 P3 flow cell to achieve recommended sequencing depth of 800 million reads per sample. A TMB cut off ≥10 mut/Mb was used as high TMB. Droplet digital PCR (ddPCR) was performed for orthogonal validation of selected clinically significant somatic mutations. Results: Sequencing on the NextSeq2000 achieved an average of 797 million pass filter reads per sample and somatic variant allele frequency (VAF) sensitivity down to 0.12%, which exceeded vendor recommendations. There was 75% concordance on the TMB classification (low and high values, Cohen’s k = 0.714) based on tumor and plasma (r = 0.86, p < 0.01) on 8 paired samples (extended analysis will be presented). Some high TMB cases carried POLE or POLD1 variants. Actionable variants identified by ctTSO500 in 15 cases were confirmed by ddPCR, with an outstanding concordance of their VAF (R2= 0.99, p < 0.0001). Diagnosis of tier I/II actionable variants (21 variants; 81% accuracy) was achieved in 7 matched tumor tissue and blood samples. Some variants found in tumor tissue were not detected in the blood. Conclusions: This pilot study demonstrates the clinical utility of the TSO500 ctDNA panel using an affordable mid throughput sequencing platform to identify actionable variants and TMB from blood specimens with high sensitivity and specificity. Testing of a larger cohort of matched tumor and plasma samples will serve to evaluate the clinical utility of the TSO500 ctDNA panel as a complement or alternative strategy to tissue biopsy for the genomic profiling of melanoma patients.