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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

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Abstract

Background and Objectives

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 104 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.

Conclusion

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.

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Acknowledgments and Disclosures

This work was funded by the Public Sector Research Funding from the Science and Engineering Research Council (grant number NR11AOT141), Agency for Science, Technology and Research (A-STAR), Singapore, and the National University Cancer Institute (NCIS) Centre Grant, Singapore (grant number NR12NMR044OM). The authors have no conflicts of interest that are directly relevant to the content of this article.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Hospital, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore

    Meggie Mo Chao Huang, Sai Mun Leong, Hui Wen Chua, Lily Chiu & Evelyn Siew-Chuan Koay

  2. CellSievo Pte Ltd, Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore

    Wai Chye Cheong & Mo-Huang Li

  3. Tucker Medical Pte Ltd, Novena Specialist Center, Singapore, Singapore

    Steven Tucker

  4. Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, 119074, Singapore, Singapore

    Hui Wen Chua & Evelyn Siew-Chuan Koay

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  1. Meggie Mo Chao Huang
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Correspondence to Evelyn Siew-Chuan Koay.

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Huang, M.M.C., Leong, S.M., Chua, H.W. et al. 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. Mol Diagn Ther 18, 459–468 (2014). https://doi.org/10.1007/s40291-014-0098-z

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