Oncogenic gene fusions represent attractive targets for therapy of cancer. However, the frequency of actionable genomic rearrangements in colorectal cancer (CRC) is very low, and universal screening for these alterations seems to be impractical and costly. To address this problem, several large scale studies retrospectivelly showed that CRC with gene fusions are highly enriched in groups of tumors defined by MLH1 DNA mismatch repair protein deficiency (MLH1d), and hypermethylation of MLH1 promoter (MLH1ph), and/or the presence of microsatellite instability, and BRAF/KRAS wild-type status (BRAFwt/KRASwt). In this study, we used targeted next generation sequencing (NGS) to explore the occurence of potentially therapeutically targetable gene fusions in an unselected series of BRAFwt/KRASwt CRC cases that displayed MLH1d/MLH1ph. From the initially identified group of 173 MLH1d CRC cases, 141 cases (81.5%) displayed MLH1ph. BRAFwt/RASwt genotype was confirmed in 23 of 141 (~16%) of MLH1d/MLH1ph cases. Targeted NGS of these 23 cases identified oncogenic gene fusions in nine patients (39.1%; CI95: 20.5%-61.2%). Detected fusions involved NTRK (four cases), ALK (two cases), and BRAF genes (three cases). As a secondary outcome of NGS testing, we identified PIK3K-AKT-mTOR pathway alterations in two CRC cases, which displayed PIK3CA mutation. Altogether, 11 of 23 (~48%) MLH1d/MLH1ph/BRAFwt/RASwt tumors showed genetic alterations that could induce resistance to anti-EGFR therapy. Our study confirms that targeted NGS of MLH1d/MLH1ph and BRAFwt/RASwt CRCs could be a cost-effective strategy in detecting patients with potentially druggable oncogenic kinase fusions.

2nd Faculty of Medicine Charles University Prague Czech Republic

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Bioptická Laboratoř s r o Pilsen Czech Republic

Cytopathos s r o Bratislava Slovakia

School of Biological Sciences Georgia Institute of Technology Atlanta Georgia USA

Šikl's Department of Pathology The Faculty of Medicine and Faculty Hospital in Pilsen Charles University Pilsen Czech Republic

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International Agency for Research on Cancer. Global cancer obsrvatory. https://gco.iarc.fr/. Accessed February 12, 2020.

Piawah S, Venook AP. Targeted therapy for colorectal cancer metastases: a review of current methods of molecularly targeted therapy and the use of tumor biomarkers in the treatment of metastatic colorectal cancer. Cancer. 2019;125(23):4139-4147.

Khan K, Valeri N, Dearman C, et al. Targeting EGFR pathway in metastatic colorectal cancer- tumour heterogeniety and convergent evolution. Crit Rev Oncol Hematol. 2019;143:153-163.

Bensen AB, Venook AP, Al-Hawary MM, et al. NCCN Clinical Practice Guidelines in oncology: colon cancer. Version 1.2020. NCCN.org. Accessed February 12, 2020.

Therkildsen C, Bergmann TK, Henrichsen-Schnack T, Ladelund S, Nilbert M. The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer: a systematic review and meta-analysis. Acta Oncol. 2014;53(7):852-864.

Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular biomarkers for the evaluation of colorectal cancer: guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology. J Clin Oncol. 2017;35:1453-1486.

Costigan DC, Dong F. The extended spectrum of RAS-MAPK pathway mutations in colorectal cancer. Genes Chromosomes Cancer. 2020;59(3):152-159.

Pagani F, Randon G, Guarini V, et al. The landscape of actionable gene fusions in colorectal cancer. Int J Mol Sci. 2019;20(21):5319.

Kloosterman WP, van den Braak RRJ C, Pieterse M, et al. A systematic analysis of oncogenic gene fusions in primary colon cancer. Cancer Res. 2017;77(14):3814-3822.

Gatalica Z, Xiu J, Swensen J, Vranic S. Molecular characterization of cancers with NTRK gene fusions. Mod Pathol. 2019;32(1):147-153.

Lasota J, Chłopek M, Lamoureux J, et al. Colonic adenocarcinomas harboring NTRK fusion genes: a clinicopathologic and molecular genetic study of 16 cases and review of the literature. Am J Surg Pathol. 2020;44(2):162-173.

Solomon JP, Linkov I, Rosado A, et al. NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls. Mod Pathol. 2020;33(1):38-46.

Aisner DL, Nguyen TT, Paskulin DD, et al. ROS1 and ALK fusions in colorectal cancer, with evidence of intratumoral heterogeneity for molecular drivers. Mol Cancer Res. 2014;12(1):111-118.

Pietrantonio F, Di Nicolantonio F, Schrock AB, et al. ALK, ROS1, and NTRK rearrangements in metastatic colorectal cancer. J Natl Cancer Inst. 2017;109(12):10.

Pietrantonio F, Di Nicolantonio F, Schrock AB, et al. RET fusions in a small subset of advanced colorectal cancers at risk of being neglected. Ann Oncol. 2018;29(6):1394-1401.

Li AY, McCusker MG, Russo A, et al. RET fusions in solid tumors. Cancer Treat Rev. 2019;81:101911.

Ross JS, Wang K, Chmielecki J, et al. The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int J Cancer. 2016;138(4):881-890.

Chou A, Fraser T, Ahadi M, et al. NTRK gene rearrangements are highly enriched in MLH1/PMS2 deficient, BRAF wild-type colorectal carcinomas-a study of 4569 cases. Mod Pathol. 2019;33(5):924-932.

Cocco E, Benhamida J, Middha S, et al. Colorectal carcinomas containing hypermethylated MLH1 promoter and wild-type BRAF/KRAS are enriched for targetable kinase fusions. Cancer Res. 2019;79(6):1047-1053.

Wang J, Yi Y, Xiao Y, et al. Prevalence of recurrent oncogenic fusion in mismatch repair-deficient colorectal carcinoma with hypermethylated MLH1 and wild-type BRAF and KRAS. Mod Pathol. 2019;32(7):1053-1064.

Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821-9826.

Skalova A, Vanecek T, Martinek P, et al. Molecular profiling of mammary analog secretory carcinoma revealed a subset of tumors harboring a novel ETV6-RET translocation: report of 10 cases. Am J Surg Pathol. 2018;42(2):234-246.

Newcombe RG. Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat Med. 1998;17(8):857-872.

Knudsen S. Promoter2.0: for the recognition of PolII promoter sequences. Bioinformatics. 1999;15(5):356-361.

Pedersen AG, Nielsen H. Neural network prediction of translation initiation sites in eukaryotes: perspectives for EST and genome analysis. Proc Int Conf Intell Syst Mol Biol. 1997;5:226-233.

Tate JG, Bamford S, Jubb HC, et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res. 2019;47(D1):D941-D947.

Gao C, Wang Y, Broaddus R, Sun L, Xue F, Zhang W. Exon 3 mutations of CTNNB1 drive tumorigenesis: a review. Oncotarget. 2017;9(4):5492-5508.

Ikenoue T, Ijichi H, Kato N, et al. Analysis of the beta-catenin/T cell factor signaling pathway in 36 gastrointestinal and liver cancer cells. Jpn J Cancer Res. 2002;93(11):1213-1220.

Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275(5307):1787-1790.

Jour G, Oultache A, Sadowska J, et al. GNAS mutations in fibrous dysplasia: a comparative study of standard sequencing and locked nucleic acid PCR sequencing on decalcified and nondecalcified formalin-fixed paraffin-embedded tissues. Appl Immunohistochem Mol Morphol. 2016;24(9):660-667.

Madsen RR, Vanhaesebroeck B, Semple RK. Cancer-associated PIK3CA mutations in overgrowth disorders. Trends Mol Med. 2018;24(10):856-870.

Johnson V, Volikos E, Halford SE, et al. Exon 3 beta-catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome. Gut. 2005;54(2):264-267.

Shihab HA, Gough J, Cooper DN, Day IN, Gaunt TR. Predicting the functional consequences of cancer-associated amino acid substitutions. Bioinformatics. 2013;29(12):1504-1510.

Bray SM, Lee J, Kim ST, et al. Genomic characterization of intrinsic and acquired resistance to cetuximab in colorectal cancer patients. Sci Rep. 2019;9(1):15365.

Puccini A, Berger MD, Naseem M, et al. Colorectal cancer: epigenetic alterations and their clinical implications. Biochim Biophys Acta Rev Cancer. 2017;1868(2):439-448.

Fang M, Ou J, Hutchinson L, Green MR. The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG Island Methylator phenotype. Mol Cell. 2014;55(6):904-915.

Bond CE, Whitehall VLJ. How the BRAF V600E mutation defines a distinct subgroup of colorectal cancer: molecular and clinical implications. Gastroenterol Res Pract. 2018;2018:9250757.

Farchoukh L, Kuan SF, Dudley B, Brand R, Nikiforova M, Pai RK. MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation harbor KRAS mutations and arise from conventional adenomas. Am J Surg Pathol. 2016;40(10):1390-1399.

Sato K, Kawazu M, Yamamoto Y, et al. Fusion kinases identified by genomic analyses of sporadic microsatellite instability-high colorectal cancers. Clin Cancer Res. 2019;25(1):378-389.

Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23(3):609-618.

Petrelli F, Ghidini M, Cabiddu M, et al. Microsatellite instability and survival in stage II colorectal cancer: a systematic review and meta-analysis. Anticancer Res. 2019;39(12):6431-6441.

Copija A, Waniczek D, Witkoś A, Walkiewicz K, Nowakowska-Zajdel E. Clinical significance and prognostic relevance of microsatellite instability in sporadic colorectal cancer patients. Int J Mol Sci. 2017;18(1):107.

Wang B, Li F, Zhou X, Ma Y, Fu W. Is microsatellite instability-high really a favorable prognostic factor for advanced colorectal cancer? A meta-analysis. World J Surg Oncol. 2019;17(1):169.

Sargent DJ, Marsoni S, Monges G, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28(20):3219-3226.

Sinicrope FA, Foster NR, Thibodeau SN, et al. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst. 2011;103(11):863-875.

Guastadisegni C, Colafranceschi M, Ottini L, Dogliotti E. Microsatellite instability as a marker of prognosis and response to therapy: a meta-analysis of colorectal cancer survival data. Eur J Cancer. 2010;46(15):2788-2798.

Webber EM, Kauffman TL, O'Connor E, Goddard KA. Systematic review of the predictive effect of MSI status in colorectal cancer patients undergoing 5FU-based chemotherapy. BMC Cancer. 2015;15:156.

Des Guetz G, Schischmanoff O, Nicolas P, Perret GY, Morere JF, Uzzan B. Does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer? A systematic review with meta-analysis. Eur J Cancer. 2009;45(10):1890-1896.

Des Guetz G, Uzzan B, Nicolas P, Schischmanoff O, Perret GY, Morere JF. Microsatellite instability does not predict the efficacy of chemotherapy in metastatic colorectal cancer. A systematic review and meta-analysis. Anticancer Res. 2009;29(5):1615-1620.

Thomas J, Leal A, Overman MJ. Clinical development of immunotherapy for deficient mismatch repair colorectal cancer. Clin Colorectal Cancer. 2020; In Press.

Juric D, Rodon J, Tabernero J, et al. Phosphatidylinositol 3-kinase α-selective inhibition with Alpelisib (BYL719) in PIK3CA-altered solid tumors: results from the first-in-human study. J Clin Oncol. 2018;36(13):1291-1299.

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