Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing

Abstract

Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45–63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR

“…Those patients with KRAS mutation clearance at follow-up had better disease control and most notably better OS and PFS [ 111 ]. The same results have been observed by Holm et al in patients included in the AXOAXI trial (NCT01531595 and EudraCT 2011-003137-33), treated with bevacizumab in combination with altering capecitabine and oxaliplatin or irinotecan [ 132 ], or by Kelin-Scory et al where KRAS mutations cleared precipitously independently of type and intensity of chemotherapy and regardless of bevacizumab anti- VEGF treatment [ 112 ]. KRAS mutations have been detected 10 months earlier than radiographic confirmation of disease progression [ 113 ].…”

Testing EGFR with Idylla on Cytological Specimens of Lung Cancer: A Review

“…Because the bioptic material is often scant, it is convenient to be able to use whatever is available, or even to repurpose samples that would otherwise have been wasted (e.g., needle rinse, supernatant) [ 71 , 72 , 73 ]. The Idylla EGFR test has been performed, in different studies, on the whole range of cytological specimens: cell-block sections (unstained or stained with H&E), scraped smears (stained with Papanicolaou, Diff-Quik, H&E, or even immunocytochemistry), suspensions of the freshly aspirated material (fixed in CytoLit or unfixed, in PBS), and finally extracted DNA from sources as diverse as cell-block sections, cell pellets, pre-capture NGS libraries and stained smears ( Table 1 ).…”

Clinical Applications of Minimal Residual Disease Assessments by Tumor-Informed and Tumor-Uninformed Circulating Tumor DNA in Colorectal Cancer

“…Longitudinal sampling of ctDNA in this population was more sensitive than CEA for radiographic progression events where rises in ctDNA occurred significantly earlier than CEA [ 116 ]. Many groups have shown ctDNA-based genotyping of KRAS to have value for prognosticating survival to combination chemotherapy and predicting early emergence of resistance during chemotherapy with lead times as early as 51 days before radiographic progression [ 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 ].…”