Tumor-informed ctDNA monitoring and relapse detection in patients with advanced malignant melanoma

5 Institute of Medical Genetics and Applied Genomics, Tübingen, Germany

Abstract:
Cell-free circulating tumour-DNA (ctDNA) is an emerging biomarker for cancer patients. ctDNA is extracted from plasma of melanoma patients and time series can be used to monitor treatment response and detect relapse. We established GeneBits, an ultra-sensitive approach to detect ctDNA based with a tumour-informed hybridisation capture panel. Up to 100 somatic SNVs are enriched during library preparation and subsequently analysed using NextGeneration-Sequencing. The libraries are sequenced to an ultra-high depth of > 50,000x and subsequent data analysis employs the umiVar tool utilizing molecular barcodes for error correction and deduplication. The sensitivity and specificity of GeneBits was evaluated with commercial reference standards and healthy controls. The detected error rate was found to be between 7.4 × 10⁻⁷ bis 7.5 × 10⁻⁵ for reads with at least four duplicates. The limit of detection for variants was less than 0.002%. No false-positive variants were found in healthy controls. GeneBits was used in the PET/LIT study to monitor treatment response to immunotherapy of 65 advanced melanoma patients as well as detection of early relapse in a cohort of 22 adjuvant melanoma patients. While ctDNA levels correlated with established tumour markers as well as imaging in our treatment cohort, we found increases of ctDNA to be prognostic. Moreover, we were able to detect relapse in 10/13 patients as early as 133 days before the appearance of clinical symptoms. All patients without relapse (n=9) remained ctDNA-negative throughout follow-up. In summary, we show that GeneBits is a highly accurate method for the detection of ctDNA in liquid biopsies of cancer patients. The data analysis pipeline umiVar is implemented in our local genetic decision support system GSvar and publicly available on GitHub (https://github.com/imgag/umiVar).