A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Mack, Elisabeth; Stabla, Kathleen; Riera-Knorrenschild, Jorge; Moll, Roland; Neubauer, Andreas; Brendel, Cornelia
• Is Part Of: BMC cancer. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 12
• Publication Date: 2016-12
• Subjects: KRAS mutation -- Colorectal cancer -- High resolution melting analysis -- Pyrosequencing; Cancer -- Periodicals; 616.994005
• DOI: 10.1186/s12885-016-2589-2
• Languages: English
• ISSNs: 1471-2407

Abstract

Abstract Background KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutantKRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved twoin-vitro diagnostics kits for determination ofKRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of theKRAS gene. Methods We describe a two-step approach toKRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using theTherascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified asKRAS wildtype or mutant by HRM. First, we determinedKRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evaluated practicability and costs of our concept in an independent set of 120 colorectal cancer samples in a routine diagnostic setting. Results HRM and pyrosequencing appeared to be equally sensitive, allowing for clear detection of mutant alleles at a mutant allele frequency ≥12.5 %. Pyrosequencing yielded more exploitable results due to lower input requirements and a lower rate of analysis failures.KRAS codon 12/13 status was called concordantly for 98.2 % (56/57) of all samples that could be successfully analysed by both methods and 100 % (19/19) of samples that were identified mutant by HRM. Reviewing the actual effort and expenses forKRAS mutation testing in our laboratory revealed, that the selective use of pyrosequencing for only those samples that could not be analysed by HRM increased the fraction of valid results from 87.5 % for HRM alone to 99.2 % (119/120) while allowing for a net reduction of operational costs of >75 % compared to pyrosequencing alone. Conclusions Combination of HRM and pyrosequencing in a two-step diagnostic procedure constitutes a reliable and economic analysis platform forKRAS mutation testing in colorectal cancer in a clinical setting.