Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

Abstract

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.

Detection and clinical significance of circulating tumor cells in colorectal cancer

“…Using a qPCR method, KRAS exon 2 mutations were successfullydetected in CTCs of patients with RAS-WT mCRC who treated with first-linechemotherapy and monoclonal antibodies. [ 116 ] 61 CellSearch ASONE Cell PickingSystem PCR/WGA EpCAM ≥1CTC/7.5 ml 44.3 CTC heterozygosity and heterogeneity exist in KRAS status among CTCs within apatient and between CTCs and tumor tissues. [ 117 ] 50 CellSearch Multiplex-PCR EpCAM ≥3CTC/7.5 ml 46 CTCs counts ≥3/7.5 mL at baseline and day 21 after initiation of regorafenib wereassociated decreased PFS and OS.…”

“…CTCs reflect the real-time status of the tumor genotype, and the molecular profiles of CTCs can help guide treatment plans. Alexios et al successfully detected KRAS exon 2 mutations in single CTCs from wild-type RAS CRC patients [ 116 ]. Yuurin et al analyzed the mutations in codons 12 and 13 of the KRAS gene in single CTCs and corresponding tumor tissue samples from 7 CRC patients.…”

A novel 3D microdevice for the in vivo capture of cancer‐associated cells

“…14 This system has demonstrated its functionality with preclinical validation in liver, lung, prostate, colorectal and other carcinomas. [15][16][17][18][19] Other devices have optimized filtration in terms of capture efficiency adjusting the size, shape, density, and arrangement of micro-pores. [20][21][22][23][24][25][26][27][28] However, relatively high working pressures and mild fixatives such as buffered paraformaldehyde are necessary for these studies to achieve high-throughput filtration in a short processing time.…”

“…The proposed approach is not limited to the isolation of prostate cancer cells, PC3, and can be extended to other types of carcinomas as demonstrated by other technologies based on CTC physical isolation. [15][16][17][18][19] Following this reasoning, the design of the microdevice(s) could also be adjusted in terms of pore size, to address any variability in size and deformability among various types of CTCs present in blood circulation. Thus, we foresee combining microdevices with different pore size.…”

The importance of circulating tumor products as „liquid biopsies” in colorectal cancer

“…The study tesified to the development of KRAS mutations in the plasma during treatment even before radiological indication of progression. More recent applications have now accepted the ctDNA system of attained resistance in the evolution of KRAS mutations (39,40).…”