PURPOSE: Although several molecular markers predicting resistance to cetuximab- or panitumumab-based therapy of metastatic colorectal cancer were described, mutations in RAS proto-oncogenes remain the only predictors being used in daily clinical practice. However, 35%-45% of wild-type RAS patients still do not respond to this anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody-based therapy, and therefore the definition of other predictors forms an important clinical need. The aim of the present retrospective single-institutional study was to evaluate potential genes responsible for resistance to anti-EGFR therapy in relation to mutational analysis of primary versus metastatic lesions. PATIENTS AND METHODS: Twenty-four paired primary and corresponding metastatic tissue samples from eight nonresponding and four responding metastatic colorectal cancer patients treated with cetuximab-based therapy were sequenced using a next-generation sequencing panel of 26 genes involved in EGFR signaling pathway and colorectal carcinogenesis. RESULTS: Mutational status of primary tumors and metastatic lesions was highly concordant in TP53, APC, CTNNB1, KRAS, PIK3CA, PTEN, and FBXW7 genes. Metastatic samples harbor significantly more mutations than primary tumors. Potentially negative predictive value of FBXW7 mutations in relationship to anti-EGFR treatment outcomes was confirmed. Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. CONCLUSION: The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily clinical practice, as it brings a significant added value in terms of validity of the diagnostic procedure.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Masaryk University Brno Czech Republic

Department of Comprehensive Cancer Care Masaryk Memorial Cancer Institute Masaryk University Brno Czech Republic; Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Oncological and Experimental Pathology Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Radiation Oncology Masaryk Memorial Cancer Institute Masaryk University Brno Czech Republic

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Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–E386. PubMed

Van Cutsem E, Cervantes A, Nordlinger B, Arnold D, ESMO Guidelines Working Group Metastatic colorectal cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2014;25(suppl 3):iii1–iii9. PubMed

Tay RY, Wong R, Hawkes EA. Treatment of metastatic colorectal cancer: focus on panitumumab. Cancer Manag Res. 2015;7:189–198. PubMed PMC

Khattak MA, Martin H, Davidson A, Phillips M. Role of first-line anti-epidermal growth factor receptor therapy compared with anti-vascular endothelial growth factor therapy in advanced colorectal cancer: a meta-analysis of randomized clinical trials. Clin Colorectal Cancer. 2015;14(2):81–90. PubMed

Misale S, Di Nicolantonio F, Sartore-Bianchi A, Siena S, Bardelli A. Resistance to anti-EGFR therapy in colorectal cancer: from heterogeneity to convergent evolution. Cancer Discov. 2014;4(11):1269–1280. PubMed

Mirone G, Shukla A, Marfe G. Signaling mechanisms of resistance to EGFR- and anti-angiogenic inhibitors cancer. Crit Rev Oncol Hematol. 2016;97:85–95. PubMed

Mlcochova J, Faltejskova P, Nemecek R, Svoboda M, Slaby O. MicroRNAs targeting EGFR signalling pathway in colorectal cancer. J Cancer Res Clin Oncol. 2013;139(10):1615–1624. PubMed PMC

Mlcochova J, Faltejskova-Vychytilova P, Ferracin M, et al. MicroRNA expression profiling identifies miR-31-5p/3p as associated with time to progression in wild-type RAS metastatic colorectal cancer treated with cetuximab. Oncotarget. 2015;6(36):38695–38704. PubMed PMC

Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013;369(11):1023–1034. PubMed

Lee MS, Kopetz S. Current and future approaches to target the epidermal growth factor receptor and its downstream signaling in metastatic colorectal cancer. Clin Colorectal Cancer. 2015;14(4):203–218. PubMed

Laurent-Puig P, Cayre A, Manceau G, et al. Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. J Clin Oncol. 2009;27(35):5924–5930. PubMed

Martin V, Landi L, Molinari F, et al. HER2 gene copy number status may influence clinical efficacy to anti-EGFR monoclonal antibodies in metastatic colorectal cancer patients. Br J Cancer. 2013;108(3):668–675. PubMed PMC

Jacobs B, De Roock W, Piessevaux H, et al. Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab. J Clin Oncol. 2009;27(30):5068–5074. PubMed

Sartore-Bianchi A, Di Nicolantonio F, Nichelatti M, et al. Multi-determinants analysis of molecular alterations for predicting clinical benefit to EGFR-targeted monoclonal antibodies in colorectal cancer. PLoS One. 2009;4(10):e7287. PubMed PMC

The Cancer Gebnome Atlas Network Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330–337. PubMed PMC

Wong SQ, Li J, Tan AY, et al. CANCER 2015 Cohort Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genomics. 2014;7:23. PubMed PMC

Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248–249. PubMed PMC

Maughan TS, Adams RA, Smith CG, et al. MRC COIN Trial Investigators Addition of cetuximab to oxaliplatin-based first-line combination chemotherapy for treatment of advanced colorectal cancer: results of the randomised phase 3 MRC COIN trial. Lancet. 2011;377(9783):2103–2114. PubMed PMC

Modzelewska K, Elgort MG, Huang J, et al. An activating mutation in sos-1 identifies its Dbl domain as a critical inhibitor of the epidermal growth factor receptor pathway during Caenorhabditis elegans vulval development. Mol Cell Biol. 2007;27(10):3695–3707. PubMed PMC

Oliner K, Douillard JY, Sienna S, et al. Analysis of KRAS/NRAS and BRAF mutations in the phase III PRIME study of panitumumab (pmab) plus FOLFOX versus FOLFOX as first-line treatment for metastatic colorectal cancer (mCRC) J Clin Oncol. 2013;31(15_suppl):3511.

De Roock W, Claes B, Bernasconi D, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 2010;11(8):753–762. PubMed

Richman SD, Seymour MT, Chambers P, et al. KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol. 2009;27(35):5931–5937. PubMed

Eklöf V, Wikberg ML, Edin S, et al. The prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer. Br J Cancer. 2013;108(10):2153–2163. PubMed PMC

Sartore-Bianchi A, Martini M, Molinari F, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res. 2009;69(5):1851–1857. PubMed

Loupakis F, Pollina L, Stasi I, et al. PTEN expression and KRAS mutations on primary tumors and metastases in the prediction of benefit from cetuximab plus irinotecan for patients with metastatic colorectal cancer. J Clin Oncol. 2009;27(16):2622–2629. PubMed

Lupini L, Bassi C, Mlcochova J, et al. Prediction of response to anti-EGFR antibody-based therapies by multigene sequencing in colorectal cancer patients. BMC Cancer. 2015;15:808. PubMed PMC

Liang J, Wu YL, Chen BJ, Zhang W, Tanaka Y, Sugiyama H. The C-kit receptor-mediated signal transduction and tumor-related diseases. Int J Biol Sci. 2013;9(5):435–443. PubMed PMC

Launonen V. Mutations in the human LKB1/STK 11 gene. Hum Mutat. 2005;26(4):291–297. PubMed

Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature. 2007;448(7152):439–444. PubMed

Yoshida K, Sanada M, Ogawa S. Deep sequencing in cancer research. Jpn J Clin Oncol. 2013;43(2):110–115. PubMed

Lièvre A, Bachet JB, Le Corre D, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006;66(8):3992–3995. PubMed

Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008;359(17):1757–1765. PubMed

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. 2008;26(10):1626–1634. PubMed

Vaughn CP, Zobell SD, Furtado LV, Baker CL, Samowitz WS. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer. 2011;50(5):307–312. PubMed

Schwartzberg LS, Rivera F, Karthaus M, et al. PEAK: a randomized, multicenter phase II study of panitumumab plus modified fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) or bevacizumab plus mFOLFOX6 in patients with previously untreated, unresectable, wild-type KRAS exon 2 metastatic colorectal cancer. J Clin Oncol. 2014;32(21):2240–2247. PubMed

Heinemann V, von Weikersthal LF, Decker T, et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol. 2014;15(10):1065–1075. PubMed

Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol. 2008;26(35):5705–5712. PubMed

Souglakos J, Philips J, Wang R, et al. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. Br J Cancer. 2009;101(3):465–472. PubMed PMC

Rowland A, Dias MM, Wiese MD, et al. Meta-analysis of BRAF mutation as a predictive biomarker of benefit from anti-EGFR monoclonal antibody therapy for RAS wild-type metastatic colorectal cancer. Br J Cancer. 2015;112(12):1888–1894. PubMed PMC

Vakiani E, Janakiraman M, Shen R, et al. Comparative genomic analysis of primary versus metastatic colorectal carcinomas. J Clin Oncol. 2012;30(24):2956–2962. PubMed PMC

Wertz IE, Kusam S, Lam C, et al. Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and 658 FBW7. Nature. 2011;471(7336):110–114. PubMed