Due to its low frequency, high grade colorectal carcinomas (HG-CRCs) are underrepresented in molecular series. We intended to further characterize the pathological and molecular features of these tumors. In addition, morphologically different areas when present, were analyzed separately to study tumor heterogeneity. We found that most (72.5%) of HG-CRCs showed mismatch repair (MMR) deficiency. MMR status conditioned the frequency and the clonality of the molecular alterations found. Thus, whereas BRAF mutations and gene fusions were observed only in MMR deficient (MMRd) tumors, TP53, KRAS, and gene amplifications predominated in MMR proficient (MMRp) tumors. In MMRp tumors, gene amplification was a mechanism of progression, whereas the accumulation of mutations in genes of different pathways such as NOTCH, MMR or PIK3CA was involved in the clonal diversity of MMRd HG-CRC. In summary, intertumor and intratumor molecular heterogeneity in HG-CRCs is mainly due to MMR status.

High grade colorectal carcinomas (HG-CRCs), which comprise 15% of colorectal carcinomas, are underrepresented in reported molecular studies. Clinicopathological, immunohistochemical, and molecular features of 40 HG-CRCs are described. Moreover, glandular and solid areas of 25 tumors were separately analyzed. The expression of MLH1, PMS2, MSH2, MSH6, p53, E-cadherin, CDX2, CK20, CD8, PDL1, PAN-TRK, c-MET, SMARCB1, ARID1A, SMARCA2, and SMARCA4 was analyzed by immunohistochemistry. Promoter MLH1 methylation was analyzed in tumors with MLH1/PMS2 loss. Next-generation sequencing was used to screen 161 genes for hotspot mutations, copy number variations and gene fusions. In this series, 72.5% of HG-CRCs showed mismatch repair deficiency (MMRd). MMR deficient tumor and MMR proficient (MMRp) tumors showed striking molecular differences. Thus, whereas BRAF mutations were only observed in MMRd tumors, mutations in KRAS and TP53 were more frequent in MMR proficient tumors. Moreover, gene fusions (NTRK1 and MET) were detected only in MMRd tumors, whereas gene amplification (MYC, CCND1 and EGFR) predominated in MMRp/TP53-mutated tumors. Loss of expression of proteins involved in chromatin remodeling, such as ARID1A, was observed only in MMRd HG-CRCs, which also showed more frequently PD-L1 expression and a higher number of tumor infiltrating lymphocytes. The separate analysis of glandular and solid areas indicated that the clonal or subclonal nature of the molecular alterations also depended on MMR status. Mutations in genes such as TP53 and KRAS were always clonal in MMRp-CRCs but occurred as subclonal events in MMRd-CRCs. Gene amplification was implicated in the progression of MMRp tumors, but not in MMRd tumors, in which clonal diversity was due to accumulation of mutations in genes of different pathways such as NOTCH, MMR, or PIK3CA. In summary, intertumor and intratumor molecular heterogeneity in HG-CRCs is mainly due to MMR status.