title: Non-invasive sensitive detection of KRAS and BRAF mutation in circulating tumor cells of colorectal cancer patients.
abstract: Characterization of genetic alterations in tumor biopsies serves as useful biomarkers in prognosis and treatment management. Circulating tumor cells (CTCs) obtained non-invasively from peripheral blood could serve as a tumor proxy. Using a label-free CTC enrichment strategy that we have established, we aimed to develop sensitive assays for qualitative assessment of tumor genotype in patients. Blood consecutively obtained from 44 patients with local and advanced colorectal cancer and 18 healthy donors were enriched for CTCs using a size-based microsieve technology. To screen for CTC mutations, we established high-resolution melt (HRM) and allele-specific PCR (ASPCR) KRAS-codon 12/13- and BRAF-codon 600- specific assays, and compared the performance with pyrosequencing and Sanger sequencing. For each patient, the resulting CTC genotypes were compared with matched tumor and normal tissues. Both HRM and ASPCR could detect as low as 1.25% KRAS- or BRAF-mutant alleles. HRM detected 14/44 (31.8%) patients with KRAS mutation in CTCs and 5/44 (11.3%) patients having BRAF mutation in CTCs. ASPCR detected KRAS and BRAF mutations in CTCs of 10/44 (22.7%) and 1/44 (2.3%) patients respectively. There was an increased detection of mutation in blood using these two methods. Comparing tumor tissues and CTCs mutation status using HRM, we observed 84.1% concordance in KRAS genotype (p = 0.000129, Fishers' exact test; OR = 38.7, 95% CI = 4.05-369) and 90.9% (p = 0.174) concordance in BRAF genotype. Our results demonstrate that CTC enrichment, coupled with sensitive mutation detection methods, may allow rapid, sensitive and non-invasive assessment of tumor genotype.
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Nur-Afidah
authors.initials: NA
authors.lastname: Mohamed Suhaimi
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Yu Miin
authors.initials: YM
authors.lastname: Foong
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Daniel Yoke San
authors.initials: DY
authors.lastname: Lee
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Wai Min
authors.initials: WM
authors.lastname: Phyo
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Igor
authors.initials: I
authors.lastname: Cima
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Esther Xing Wei
authors.initials: EX
authors.lastname: Lee
authors.affiliation: Fortis Surgical Hospital Singapore, Singapore.
authors.forename: Wei Lin
authors.initials: WL
authors.lastname: Goh
authors.affiliation: Saw Swee Hock School of Public Health, National University of Singapore, Singapore.
authors.forename: Wei-Yen
authors.initials: WY
authors.lastname: Lim
authors.affiliation: Saw Swee Hock School of Public Health, National University of Singapore, Singapore.
authors.forename: Kee Seng
authors.initials: KS
authors.lastname: Chia
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Say Li
authors.initials: SL
authors.lastname: Kong
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Min
authors.initials: M
authors.lastname: Gong
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Bing
authors.initials: B
authors.lastname: Lim
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Axel M
authors.initials: AM
authors.lastname: Hillmer
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore; Fortis Surgical Hospital Singapore, Singapore.
authors.forename: Poh Koon
authors.initials: PK
authors.lastname: Koh
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Jackie Y
authors.initials: JY
authors.lastname: Ying
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore; National Cancer Centre Singapore, Singapore. Electronic address: mhtan@ibn.a-star.edu.sg.
authors.forename: Min-Han
authors.initials: MH
authors.lastname: Tan
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D004252:DNA Mutational Analysis; D005260:Female; D060005:Genotyping Techniques; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Circulating tumor cells; Colorectal cancer; Non-invasive; Targeted phenotyping