Multiplex ddPCR for the detection of tumour-specific mutations in ctDNA

Abstract

Circulating tumour DNA (ctDNA) are short extracellular DNA fragments that have been explored by a number of studies to potentially reflect a more complete representation of tumour burden without utilizing the highly invasive tissue biopsy. Current advances in technology have investigated the use of multiplex droplet digital PCR (ddPCR) in providing a highly sensitive, simultaneous screening for multiple mutations in the ctDNA of limited clinical samples as an alternative to the current molecular detection approaches. This specific detection could potentially serve as a more accurate and cost-effective surveillance tool in clinical applications. Hence, the study was conducted in metastatic colorectal and breast cancer patients to investigate the use of multiplex ddPCR for the detection of tumour-specific mutations in ctDNA. Rare or difficult to detect pathogenic somatic mutations were identified for each cancer patient by sequencing their respective ctDNA and/or FFPE DNA. Patient samples were then subjected to an optimized multiplex ddPCR containing two gene mutation assays in a single ddPCR reaction. The assay was run on ctDNA isolated from several blood collection cycles of patients and the final mutant droplet concentration (copies per mL plasma ± SE) for each cycle was calculated.

Dynamic serial changes in ctDNA were observed to correlate with disease burden, response to administered treatments and overall clinical outcome earlier than scheduled CT imaging. Higher levels of ctDNA were mostly observed in patients with metastasis to the liver compared to those with metastases to lymph nodes, lungs and bones. Multiplex ddPCR assays also successfully detected KRAS, TP53, PIK3CA, BRCA2 and BRAF gene mutations in the ctDNA of metastatic breast and CRC patients. Similar dynamic patterns were observed for each mutation marker and the overall level of ctDNA is comparable to the results acquired from single mutation ddPCR assays. Overall, the results presented in this study have contributed to the establishment of the highly sensitive multiplex ddPCR analysis of ctDNA in serial blood samples as a possible cancer surveillance tool that could help combat the current limitations of the cancer healthcare system and hopefully reduce the continually increasing mortality rates of cancer in New Zealand.