Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach
Abstract
Aims: Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-EGF receptor monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Materials & methods: We developed a cost-effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available real-time PCR-based Therascreen® kit (DxS Ltd). Results & conclusion: The PCR/sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527 out of 540 (97.6%) formalin-fixed paraffin-embedded tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases, in which low-intensity peaks suggestive of potential mutations were identified. The DxS assay, which showed a sensitivity of 1%, identified mutations in 11 out of 13 inconclusive cases. Interestingly, five of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with more than 30% tumor cells. However, in 24 samples with less than 30% tumor cells, DxS showed an higher sensitivity. In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have more than 30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells.
Papers of special note have been highlighted as: ▪ of interest ▪▪ of considerable interest
Bibliography
- 1 Malumbres M, Barbacid M: Ras oncogenes: the first 30 years. Nat. Rev. Cancer3(6),459–465 (2003).
- 2 Schubbert S, Shannon K, Bollag G: Hyperactive Ras in developmental disorders and cancer. Nat. Rev. Cancer7(4),295–308 (2007).
- 3 Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell61(5),759–767 (1990).▪▪ Seminal review summarizing the key events that lead to colon tumorigenesis.
- 4 Normanno N, Tejpar S, Morgillo F, De Luca A, van Cutsem E, Ciardiello F: Implications for kras status and EGFR-targeted therapies in metastatic CRC. Nat. Rev. Clin. Oncol.6(9),519–527 (2009).▪ Summarizes the rationale for KRAS testing in colorectal cancer patients and discusses the available methods for this analysis.
- 5 van Krieken JH, Jung A, Kirchner T et al.: Kras mutation testing for predicting response to anti-egfr therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch.454(2),233–235 (2009).▪▪ Indicates guidelines for KRAS testing in colorectal cancer patients.
- 6 Amado RG, Wolf M, Peeters M et al.: Wild-type kras is required for panitumumab efficacy in patients with metastatic colorectal cancer. J. Clin. Oncol.26(10),1626–1634 (2008).
- 7 Karapetis CS, Khambata-Ford S, Jonker DJ et al.: K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N. Engl. J. Med.359(17),1757–1765 (2008).
- 8 Bokemeyer C, Bondarenko I, Makhson A et al.: Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J. Clin. Oncol.27(5),663–671 (2009).
- 9 van Cutsem E, Kohne C-H, Hitre E et al.: Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N. Engl. J. Med.360(14),1408–1417 (2009).
- 10 Allegra CJ, Jessup JM, Somerfield MR et al.: American society of clinical oncology provisional clinical opinion: testing for kras gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J. Clin. Oncol.27(12),2091–2096 (2009).
- 11 Jimeno A, Messersmith WA, Hirsch FR, Franklin WA, Eckhardt SG: Kras mutations and sensitivity to epidermal growth factor receptor inhibitors in colorectal cancer: practical application of patient selection. J. Clin. Oncol.27(7),1130–1136 (2009).
- 12 Tol J, Dijkstra JR, Vink-Borger ME et al.: High sensitivity of both sequencing and real-time PCR analysis of kras mutations in colorectal cancer tissue. J. Cell Mol. Med. (2009) (Epub ahead of print).▪ Compares PCR/sequencing and the real-time PCR-based kit by DxS Ltd (Manchester, UK) in a large cohort of colorectal cancer patients.
- 13 Zavodna K, Konecny M, Krivulcik T et al.: Genetic analysis of kras mutation status in metastatic colorectal cancer patients. Neoplasma56(3),275–278 (2009).
- 14 Whitehall V, Tran K, Umapathy A et al.: A multicenter blinded study to evaluate kras mutation testing methodologies in the clinical setting. J. Mol. Diagn.11(6),543–552 (2009).
- 15 Juan T, Suggs S, Wolf M et al.: A comparability study of 4 commercial kras tests. Presented at: 99th AACR Annual Meeting. CA, USA, 12–16 April (2008) (Abstract 1811).
- 16 Franklin WA: Not all tests for EGFR and k-ras are equivalent. Presented at: Education Session Tumor Biology Track 2009 ASCO Annual Meeting. FL, USA, 29 May–2 June (2009).
- 17 Franklin WA, Haney J, Sugita M, Bemis L, Jimeno A, Messersmith WA: Kras mutation: comparison of testing methods and tissue sampling techniques in colon cancer. J. Mol. Diagn.12(1),43–50 (2010).
- 18 Ren ZP, Sallstrom J, Sundstrom C, Nister M, Olsson Y: Recovering DNA and optimizing PCR conditions from microdissected formalin-fixed and paraffin-embedded materials. Pathobiology68(4–5),215–217 (2000).
- 19 van Beers EH, Joosse SA, Ligtenberg MJ et al.: A multiplex PCR predictor for ACGH success of FFPE samples. Br. J. Cancer94(2),333–337 (2006).
- 20 Jonsson M, Ekstrand A, Edekling T et al.: Experiences from treatment-predictive kras testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor. BMC Clin. Pathol.9,8(2009).
- 21 Bos JL: Ras oncogenes in human cancer: a review. Cancer Res.49(17),4682–4689 (1989).
- 22 Weichert W, Schewe C, Lehmann A et al.: Kras genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology. J. Mol. Diagn.12(1),35–42 (2009).
- 23 Ogino S, Kawasaki T, Brahmandam M et al.: Sensitive sequencing method for kras mutation detection by pyrosequencing. J. Mol. Diagn.7(3),413–421 (2005).
- 24 Poehlmann A, Kuester D, Meyer F, Lippert H, Roessner A, Schneider-Stock R: K-ras mutation detection in colorectal cancer using the pyrosequencing technique. Pathol. Res. Pract.203(7),489–497 (2007).
- 25 Zuo Z, Chen SS, Chandra PK et al.: Application of cold-PCR for improved detection of kras mutations in clinical samples. Mod. Pathol.22(8),1023–1031 (2009).
- 26 Jung A, Baretton G, Dietel M: The German quality assurance system for the molecular-pathological detection of kras-mutations in colorectal cancer. J. Clin. Oncol.27,15S (2009) (Suppl. Abstr. 4018).
- 27 Angulo B, Garcia-Garcia E, Martinez R et al.: A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect kras mutations: a morphology-based approach in colorectal carcinoma. J. Mol. Diagn.12(3),292–299 (2010).
- 28 Linardou H, Dahabreh IJ, Kanaloupiti D et al.: Assessment of somatic k-ras mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol.9(10),962–972 (2008).▪ Contributes to demonstrate that KRAS mutations are associated with resistance to anti-EGF receptor therapies in colon and lung cancer patients
- 29 Marchetti A, Gasparini G, Albitar M et al.: K-ras mutations and cetuximab in colorectal cancer. N. Engl. J. Med.360(8),833–836 (2009).
