Enrichment technique to allow early detection and monitor emergence of KRAS mutation in response to treatment

• Published in: Scientific reports Vol. 9; no. 1; pp. 11346 - 9
• Main Authors: Kitagawa, Yoshiyasu, Okumura, Kazuhiro, Watanabe, Takayoshi, Tsukamoto, Kei, Kitano, Shiro, Nankinzan, Rino, Suzuki, Takuto, Hara, Taro, Soda, Hiroaki, Denda, Tadamichi, Yamaguchi, Taketo, Nagase, Hiroki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05.08.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 38/22; 38/47; 631/1647/2217/2018; 631/67/2322; 692/4020/1503/1504/1885/1393; 692/53/2421; Adult; Aged; Aged, 80 and over; Alleles; Cell Line, Tumor; Cell-Free Nucleic Acids - blood; Circulating Tumor DNA - blood; Codon - drug effects; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - blood; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - drug therapy; Colorectal Neoplasms - genetics; Deoxyribonucleic acid; DNA; DNA structure; DNA, B-Form - drug effects; DNA, B-Form - genetics; Early Detection of Cancer; Female; Humanities and Social Sciences; Humans; Imidazole; Imidazoles - chemistry; Imidazoles - pharmacology; K-Ras protein; Male; Metastases; Metastasis; Middle Aged; multidisciplinary; Mutation; Mutation - genetics; Nylons - chemistry; Nylons - pharmacology; Patients; Polyamides; Proto-Oncogene Proteins p21(ras) - blood; Proto-Oncogene Proteins p21(ras) - genetics; Pyrroles - chemistry; Pyrroles - pharmacology; Science; Science (multidisciplinary); Therapeutic applications; Tumors
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-47700-9

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS . After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P  < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P  = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.