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Comparative Study
. 2016 Aug 2:16:585.
doi: 10.1186/s12885-016-2589-2.

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing

Elisabeth Mack  1 , Kathleen Stabla  1 , Jorge Riera-Knorrenschild  1 , Roland Moll  2 , Andreas Neubauer  1 , Cornelia Brendel  3
Affiliations
Comparative Study

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing

Elisabeth Mack et al. BMC Cancer. .

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 mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS 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 the KRAS gene.

Methods: We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS 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 for KRAS 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 for KRAS mutation testing in colorectal cancer in a clinical setting.

Keywords: Colorectal cancer; High resolution melting analysis; KRAS mutation; Pyrosequencing.

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Figures

Fig. 1
Fig. 1
Sensitivity of HRM. Dilutions of genomic DNA from the KRAS mutant cell line PL45 (codon 12 GGT → GAT, heterozygous mutation) in WT genomic DNA was analysed by HRM. Normalised fluorescence and difference graphs are indicated. Clear discrimination of WT and mutant amplicons is possible using either graph if the fraction of PL45-DNA in the sample exceeds 25 %, corresponding to a mutant allele frequency of 12.5 %
Fig. 2
Fig. 2
Sensitivity of pyrosequencing. Dilutions of genomic DNA from the KRAS mutant cell line PL45 (codon 12 GGT → GAT, heterozygous mutation) in WT genomic DNA was analysed by pyrosequencing on the Pyromark Q24 platform. The WT sequence and the sequence to analyse including wobble bases at the potentially mutant positions in the region of interest (codons 12 and 13) are indicated in the top panel. Lower panels: KRAS codon 12/13 pyrograms for different dilutions of PL45-DNA in WT DNA. Clear mutation calls are obtained for samples containing ≥25 % of PL45-DNA, corresponding to 12.5 % mutant alleles. For lower concentrations, yielding signals below the LOD of the mutation (2.2 %) + 3 %, presence of a potential low level mutation is suggested, which requires technical and/or biological replication of the analysis
Fig. 3
Fig. 3
Outline of the two-step procedure for KRAS codon 12/13 mutation analysis. Genomic DNA from microdissected colorectal cancer cells from FFPE samples is subjected to HRM of a PCR amplicon spanning the mutation-bearing region of interest. For samples that are clearly identified as KRAS mutant or, respectively, WT, the HRM result is incorporated in the final diagnostic report. Samples for which HRM analysis fails technically or which yield ambiguous HRM curves are further evaluated by a second round of HRM and, if results are still invalid, to pyrosequencing. Note that samples for which a WT result is obtained by the diagnostic procedure outlined here require further examination for additional KRAS and NRAS mutations
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References

    1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi: 10.3322/caac.20107. - DOI - PubMed
    1. Stein A, Bokemeyer C. How to select the optimal treatment for first line metastatic colorectal cancer. World J Gastroenterol. 2014;20(4):899–907. doi: 10.3748/wjg.v20.i4.899. - DOI - PMC - PubMed
    1. De Roock W, Jonker DJ, Di Nicolantonio F, Sartore-Bianchi A, Tu D, Siena S, Lamba S, Arena S, Frattini M, Piessevaux H, et al. Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA. 2010;304(16):1812–1820. doi: 10.1001/jama.2010.1535. - DOI - PubMed
    1. De Roock W, Piessevaux H, De Schutter J, Janssens M, De Hertogh G, Personeni N, Biesmans B, Van Laethem JL, Peeters M, Humblet Y, et al. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol. 2008;19(3):508–515. doi: 10.1093/annonc/mdm496. - DOI - PubMed
    1. Di Fiore F, Blanchard F, Charbonnier F, Le Pessot F, Lamy A, Galais MP, Bastit L, Killian A, Sesboue R, Tuech JJ, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer. 2007;96(8):1166–1169. doi: 10.1038/sj.bjc.6603685. - DOI - PMC - PubMed

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