Colonic carcinogenesis along different genetic routes: glycophenotyping of tumor cases separated by microsatellite instability/stability.

Different genetic routes account for colonic carcinogenesis. However, when analyzing colon cancer specimens, separation into different groups based on genetic alterations is commonly not performed. Thus, we here initiate the comparative phenotyping considering microsatellite instability/stability for clinical specimens. The focus is given to glycan epitopes, expression of which is known to be modulated by signal-transducing proteins that act as key regulators of normal colon epithelial growth and differentiation. In addition to six plant lectins used as sensors, the presence of two adhesion/growth-regulatory galectins is studied. Overall, a considerable level of intra- and interindividual heterogeneity is revealed. Alterations in the proportion of stained cells between tumor-adjacent and malignant epithelia concerned plant lectins, which bind substituted N-glycan cores, α2,6-sialylated branch ends, core 1 O-glycans and N-acetylgalactosamine. A tendency for changes was noted between microsatellite-unstable and microsatellite-stable cases for core substitution (bisected N-glycan, presence of ß1,6-branching) and status of α2,6-sialylation. Statistical significance was reached for presence of galectin-3, found to be elevated in microsatellite-stable compared to microsatellite-unstable tumors. These results emphasize the potential of distinct signaling pathways to regulate certain aspects of the glycophenotype in vivo and thus delineate a perspective to discern functionally relevant deviations in expression of endogenous lectins and their counter-receptors.

Lack of HLA class II antigen expression in microsatellite unstable colorectal carcinomas is caused by mutations in HLA class II regulatory genes.

Colorectal cancers (CRCs) develop on the basis of a deficient DNA mismatch repair (MMR) system in about 15% of cases. MMR-deficient CRC lesions show high-level microsatellite instability (MSI-H) and accumulate numerous mutations located at coding microsatellite loci that lead to the generation of immunogenic neopeptides. Consequently, the host's antitumoral immune response is of high importance for the course of the disease in MSI-H CRC patients. Accordingly, immune evasion mediated by impairment of HLA class I antigen presentation is frequently observed in these cancers. In this study, we aimed at a systematic analysis of alterations affecting HLA class II antigen expression in MSI-H CRC. HLA class II antigens are expressed by only two-thirds of MSI-H CRCs. The mechanisms underlying the lack of HLA class II antigens in a subset of MSI-H CRCs remain unknown. We here screened HLA class II regulatory genes for the presence of coding microsatellites and identified mutations of the essential regulator genes RFX5 in 9 (26.9%) out of 34 and CIITA in 1 (2.9%) out of 34 MSI-H CRCs. RFX5 mutations were related to lack of or faint HLA class II antigen expression (p = 0.006, Fisher's exact test). Transfection with wild-type RFX5 was sufficient to restore interferon gamma-inducible HLA class II antigen expression in the RFX5-mutant cell line HDC108. We conclude that somatic mutations of the RFX5 gene represent a novel mechanism of loss of HLA class II antigen expression in tumor cells, potentially contributing to immune evasion in MSI-H CRCs.

T25 repeat in the 3' untranslated region of the CASP2 gene: a sensitive and specific marker for microsatellite instability in colorectal cancer.

DNA mismatch repair deficiency is observed in about 10% to 15% of all colorectal carcinomas and in up to 90% of hereditary nonpolyposis colorectal cancer (HNPCC) patients. Tumors with mismatch repair defects acquire mutations in short repetitive DNA sequences, a phenomenon termed high-level microsatellite instability (MSI-H). The diagnosis of MSI-H in colon cancer is of increasing relevance, because MSI-H is an independent prognostic factor in colorectal cancer, seems to influence the efficacy of adjuvant chemotherapy, and is the most important molecular screening tool to identify HNPCC patients. To make MSI typing feasible for the routine pathology laboratory, highly reproducible and cost effective laboratory tests are required. Here, we describe a novel T25 mononucleotide marker in the 3'untranslated region of the CASP2 gene (CAT25) that displayed a quasimonomorphic repeat pattern in normal tissue of 200 unrelated individuals of Caucasian origin. In addition, CAT25 was monomorphic also in all tested donors of African and Asian origin (n = 102 and n = 79, respectively) and thus differs from the most commonly used markers BAT25 and BAT26. Without the analysis of corresponding normal tissue, CAT25 correctly detected 56 of 57 colorectal cancer specimens classified as MSI-H by using the standard National Cancer Institute/International Collaborative Group-HNPCC marker panel. Combined with the standard markers BAT25 and BAT26 in a multiplex PCR, all MSI-H colorectal cancer samples were typed correctly. No false-positive results were obtained in 60 non-MSI-H control colorectal cancer specimens. These data suggest that CAT25 should be included into novel marker panels for microsatellite testing thus allowing for a significant reduction of the complexity and costs of MSI typing. Moreover, CAT25 represents a highly promising marker for early detection of colorectal cancer in HNPCC germ line mutation carriers.

Identification of differentially expressed genes in colorectal adenoma compared to normal tissue by suppression subtractive hybridization.

Altered gene expression that results in unrestricted growth and loss of differentiation is a hallmark of neoplastic cells. The goal of the present study was to determine early changes in gene expression profiles during colorectal carcinogenesis with emphasis on those genes whose expression is upregulated in adenoma tissue compared to adjacent normal colon. Using the suppression subtractive hybridization (SSH) approach on enriched colonic crypts we identified 14 candidate genes showing increased expression levels in the original adenoma sample. Subsequent dot blot, Northern blot, standard and real-time RT-PCR analysis confirmed increased transcript levels of ten genes in various subsets of colorectal adenoma and cancer samples. SERPINB5 and a yet uncharacterized HERV-H sequence showed the highest expression in adenoma and tumor samples without significant expression in the corresponding normal tissue. These genes might represent promising new candidates for colorectal cancer early detection.

Beta2-microglobulin mutations in microsatellite unstable colorectal tumors.

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the beta2-microglobulin (beta2m) gene. To examine the implications of beta2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of beta2m mutations in MSI-H colorectal adenomas (n=38) and carcinomas (n=104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of beta2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of beta2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. beta2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of beta2m expression may promote local progression of colorectal MSI-H tumors. However, no beta2m mutations were observed in metastasized CRCs (UICC stage IV, p=0.04). These results suggest that functional beta2m may be necessary for distant metastasis formation in CRC patients.