Droplet digital PCR of circulating tumor cells from colorectal cancer patients can predict KRAS mutations before surgery

Abstract

In colorectal cancer (CRC), KRAS mutations are a strong negative predictor for treatment with the EGFR-targeted antibodies cetuximab and panitumumab. Since it can be difficult to obtain appropriate tumor tissues for KRAS genotyping, alternative methods are required. Circulating tumor cells (CTCs) are believed to be representative of the tumor in real time. In this study we explored the capacity of a size-based device for capturing CTCs coupled with a multiplex KRAS screening assay using droplet digital PCR (ddPCR). We showed that it is possible to detect a mutant ratio of 0.05% and less than one KRAS mutant cell per mL total blood with ddPCR compared to about 0.5% and 50-75 cells for TaqMeltPCR and HRM. Next, CTCs were isolated from the blood of 35 patients with CRC at various stage of the disease. KRAS genotyping was successful for 86% (30/35) of samples with a KRAS codon 12/13 mutant ratio of 57% (17/30). In contrast, only one patient was identified as KRAS mutant when size-based isolation was combined with HRM or TaqMeltPCR. KRAS status was then determined for the 26 available formalin-fixed paraffin-embedded tumors using standard procedures. The concordance between the CTCs and the corresponding tumor tissues was 77% with a sensitivity of 83%. Taken together, the data presented here suggest that is feasible to detect KRAS mutations in CTCs from blood samples of CRC patients which are predictive for those found in the tumor. The minimal invasive nature of this procedure in combination with the high sensitivity of ddPCR might provide in the future an opportunity to monitor patients throughout the course of disease on multiple levels including early detection, prognosis, treatment and relapse as well as to obtain mechanistic insight with respect to tumor invasion and metastasis.

Multiple KRAS mutations in the non‐mucinous epithelial lining in the majority of mucinous cystic neoplasms of the pancreas

“…This higher rate of positive cases is probably the result of the mutation detection methods used. In previous studies, Sanger sequencing, PCR‐based restriction fragment length polymorphism, whole‐exome sequencing and next‐generation sequencing have been used to detect KRAS mutations; however, droplet digital PCR is one of the most sensitive methods for the detection of mutations …”

“…The other KRAS gene included the wild‐type gene in all normal cases (G12R, G12C, G12A, and G12V). Allowing a mean + 3 standard deviations, the percentage of each type of KRAS mutation in MCN was normalised on thresholds of 1.1% for G12R, 0.22% for G12D, 0.25% for G13D, and 0% for others, based on previous studies …”

Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs

“…Therefore, the detection of the plasma EGFR ctDNA through the ARMS PCR technology appears to be a highly sensitive method. At the same time, as a non-invasive liquid biopsy, the ctDNA in plasma has been proposed to detect genetic alterations without additional burden and risk to patients[19–21], which also can provide information on molecular evolution of the tumors.…”

High‐grade PanIN presenting with localised stricture of the main pancreatic duct: A clinicopathological and molecular study of 10 cases suggests a clue for the early detection of pancreatic cancer

“…In our preliminary study to optimise the assay, the sum of the KRAS mutation value (%) in three samples of unremarkable pancreatic tissue (the background parenchyma of benign pancreatic tumours) was 0.74 ± 0.38%. Therefore, allowing a mean plus 3 standard deviations, more than 1.88% of KRAS mutations were considered to be significant, according to a previous study …”