Tumor invasion in vivo was studied by light and electron microscopy as well as by immunofluorescence microscopy. Special regard was paid to the grade of tumor differentiation. Dimethylhydrazine-induced murine colonic carcinomas comprising a differentiated and an undifferentiated tumor type with low and high invasiveness respectively, were used. At the invasion front of both tumor types a striking dissociation of the organized tumor cell complexes into isolated tumor cells was found together with a loss of most of the cytological features of differentiation. It is supposed that this process mobilizes the tumor cells from the main tumor bulk enabling them to invade the host tissue by active locomotion. This view is strongly supported by the demonstration of morphological equivalents of active cell movement such as pseudopodia-like cytoplasmic extrusions, adaptive changes of the cell shape and microfilament bundles. Although the proposed mechanism of tumor invasion is essentially the same in both tumor types, the grade of differentiation is nevertheless critical, as in the undifferentiated carcinomas only subtle dedifferentiation steps (loss of basement membrane and cell junctions) are necessary to acquire an invasive status. This fact may explain the comparatively high invasiveness and poor prognosis of undifferentiated carcinomas.

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 b2-microglobulin (b2m) gene. To examine the implications of b2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of b2m mutations in MSI-H colorectal adenomas (n 5 38) and carcinomas (n 5 104) 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 b2m 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 b2m 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. b2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of b2m expression may promote local progression of colorectal MSI-H tumors. However, no b2m mutations were observed in metastasized CRCs (UICC stage IV, p 5 0.04). These results suggest that functional b2m may be necessary for distant metastasis formation in CRC patients. ' 2007 Wiley-Liss, Inc.

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 T 25 mononucleotide marker in the 3Vuntranslated 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 falsepositive 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. (Cancer Res 2005; 65(18): 8072-8)

Colorectal cancers (CRC) 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 the present 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 still remained 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 (IFN-γ) -inducible HLA class II antigen expression in the RFX5-mutant cell line HDC108. We conclude that somatic mutations of the RFX5 gene represent a

An X-ray method to determine the transformation zone size in ceramics containing tetragonal ZrO, is described. Results obtained on plane fracture surfaces of various composites were correlated to mechanical property data and TEM observations and were found to be in good agreement, This indicates that the method presented is a useful tool for estimating stress-induced transformation zone sizes and for checking the effectiveness of tetragonal particles introduced in a ceramic matrix.H E tetragonal-monoclinic transforma-