title: Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.
abstract: OBJECTIVE
  Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC.
  
  
  METHODS
  In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC.
  
  
  RESULTS
  The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases.
  
  
  CONCLUSIONS
  Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.
authors.affiliation: Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Hospital, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore.
authors.forename: Meggie Mo Chao
authors.initials: MM
authors.lastname: Huang
authors.forename: Sai Mun
authors.initials: SM
authors.lastname: Leong
authors.forename: Hui Wen
authors.initials: HW
authors.lastname: Chua
authors.forename: Steven
authors.initials: S
authors.lastname: Tucker
authors.forename: Wai Chye
authors.initials: WC
authors.lastname: Cheong
authors.forename: Lily
authors.initials: L
authors.lastname: Chiu
authors.forename: Mo-Huang
authors.initials: MH
authors.lastname: Li
authors.forename: Evelyn Siew-Chuan
authors.initials: ES
authors.lastname: Koay
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D009154:Mutation; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D010232:Paraffin; D016612:Paraffin Embedding; D018631:ras Proteins / Q000235:genetics*
chemical_list: D010232:Paraffin; D018631:ras Proteins