br KRAS and NRAS mutations are also expected to
KRAS and NRAS mutations are also expected to be mutu-ally exclusive, although preclinical data have shown divergent oncogenic properties of the KRAS p.G12D and NRAS p.G12D mutations . Coexisting KRAS and NRAS mutations were not seen in 2 studies with approximately 500 to 700 CRC pa-tients [6,38] but were reported in 11 (0.9%) of 1294 European CRC patients  and in 1 (0.2%) of 621 and 8 (0.7%) of 1110 Chinese CRC patients [28,29]. In this study, the coexistence of activating KRAS and NRAS mutation was initially reported in 3 of 744 CRCs. Retrospective quality assessment confirmed false detection of coexistence in one of these specimens. Mul-tiregional analysis revealed presence of KRAS and NRAS in separate invasive adenocarcinoma components submitted within the same tissue blocks (CRC0068) and presence of both KRAS and NRAS mutations in an adenoma component (CRC0202).
Dual KRAS mutations have also been reported in 4 (0.5%) of 747 European CRC patients and 8 (0.7%) of 1110 Chinese CRC patients [6,29]. Whether the presence of 2 KRAS muta-tions was seen in the same or different tumor populations is not reported. In this study, NGS detected 3 (0.4%) of 744 CRCs with coexistence of 2 activating KRAS mutations. Mul-tiregional analysis confirmed different KRAS mutations within the invasive adenocarcinoma component and the adjacent ad-enoma component in CRC0256 and CRC0449. Coexistence of 2 activating KRAS mutations within an individual adenocar-cinoma component was not seen in 744 CRCs of this study. Analysis of the entire NGS panel revealed an APC mutation (p.R1450* in CRC0265 and p.P1443fs in CRC0449, data not shown) in the adenoma but not the adenocarcinoma, indicating that CRC0256 and CRC0449 each contained a col-lisional adenoma and adenocarcinoma originating from differ-ent ancestral clones. The adenocarcinoma may have expanded laterally and upward to invade a synchronous adenoma. In the absence of molecular profiling, the histomorphology could have been simply interpreted as colonic adenocarcinoma aris-ing from an adenoma.
Invasion or abutting of a colonic adenocarcinoma into an adenoma of different clonal origin may have clinical implica-tions. In the current guidelines for standard of care of metasta-tic CRC published by the College of American Pathologists and the Association of Molecular Pathology, KRAS, NRAS, and BRAF are the only 3 Ethylmalonyl Coenzyme A recommended for mutational profiling . In the multistep model for colorectal tumorigen-esis [4,5], KRAS, NRAS, and BRAF mutations, which are all part of the MAPK pathway, represent trunk (initiating) drivers to promote progression from small adenoma to large adenoma, a step before the formation of the founder cell of
Coexisting mutations in colorectal cancers 19
adenocarcinomas. These trunk drivers should be present in the invasive adenocarcinomas and their adjacent precursor adeno-mas. Therefore, inclusion of the precursor adenoma for muta-tional profiling of trunk drivers, such as the KRAS mutational status, is expected to be concordant with those from the inva-sive adenocarcinoma component. However, 2 or more colonic polyps may develop in close proximity, which may lead an ad-enocarcinomas to invade upward and collide with a neighbor-ing unrelated adenomas. Endoscopic biopsy of a superficial lesion may therefore contain a minor invasive adenocarcinoma component insufficient for molecular profiling and a dominant adenoma component originating from a different ancestral clone carrying different trunk driver mutations.
We propose an operating procedure for clinical validation of coexisting activating mutations of the BRAF, KRAS, and NRAS genes within the MAPK pathway, which are expected to be mutually exclusive in colorectal tumorigenesis. Multire-gional analysis according to this operating procedure con-firmed a rare incidence of coexisting activating RAS mutations, determined their presence in the same or different tumor populations, and identified invasion of a synchronous adenoma by a collisional adenocarcinoma of different clonal original. Further studies are warranted to elucidate the biolog-ical significance and clinical implications of coexisting activat-ing mutations within the MAPK pathway.
 Sepulveda AR, Hamilton SR, Allegra CJ, et al. Biomarkers for the eval-uation of colorectal cancer: guideline summary from the American Soci-ety for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J Clin Oncol 2017;35:1453-86.
 Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clin-ical Oncology provisional clinical opinion: testing for KRAS gene muta-tions in patients with metastatic colorectal carcinoma to predict response to anti–epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009;27:2091-6.