Basement membrane components are key players in specialized extracellular matrices

More than three decades ago, basement membranes (BMs) were described as membrane-like structures capable of isolating a cell from and connecting a cell to its environment. Since this time, it has been revealed that BMs are specialized extracellular matrices (sECMs) with unique components that support important functions including differentiation, proliferation, migration, and chemotaxis of cells during development. The composition of these sECM is as unique as the tissues to which they are localized, opening the possibility that such matrices can fulfill distinct functions. Changes in BM composition play significant roles in facilitating the development of various diseases. Furthermore, tissues have to provide sECM for their stem cells during development and for their adult life. Here, we briefly review the latest research on these unique sECM and their components with a special emphasis on embryonic and adult stem cells and their niches

Immunohistochemical detection of sporadic and hereditary colorectal carcinomas with defective DNA mismatch repair

Background. Identification of colorectal carcinomas (CRCs) with defective DNA mismatch repair (MMR) is of great clinical relevance. Aim of the present study was to precisely determine the role of immunohistochemistry as a screening test for the detection of these tumors. Design. A consecutive series of 330 CRCs was included in the study. Expression of MMR proteins (MLH1, MSH2, MSH6 and PMS2) was evaluated by immunohistochemistry and microsatellite instability (MSI) by a fluorescence PCR method using mononucleotide (BAT26, BAT25 and BAT40) and dinucleotide markers. MLH1 promoter methylation was determined by methylation specific PCR. Results. Deficit of MMR was observed in 51 tumors (MMR-D, 15,5%), whereas the remaining 279 CRCs showed normal protein expression and microsatellite stability and were classified as MMR proficient (MMR-P, 84,5%). 50/51 MMR-D tumors showed high frequency MSI and 50/51 demonstrated loss of protein expression. In detail 39 tumors showed complete loss of MLH1 and PMS2 expression, 3 tumors loss of MSH2 and MSH6 expression, and 6 tumors selective loss of the MSH6 protein. A single carcinoma showed selective loss of PMS2 expression. The large majority (84,3%) of MMR-D tumors were localized in the proximal colon. MLH1 promoter methylation was observed in 34 MLH1/PMS2 negative carcinomas. On the basis of immunohistochemical data and MLH1 promoter methylation status, we can hypothesize that about 70% of MMR-D tumors were sporadic and 30% hereditary. Conclusion. Our results indicate that immunohistochemistry is a rapid and suitable method for the identification of MMR-D CRCs. Pathologic screening of colorectal tumors should include analysis of expression of MLH1, MSH2 and MSH6 proteins, as the observed frequency of cases with selective loss of MSH6 expression was much higher than expected

Analysis of MLH1 promoter methylation in colorectal carcinomas with microsatellite instability

Background Microsatellite analysis and immunohistochemistry for DNA mismatch repair proteins (MMRPs) demonstrated great utility in the identification of Lynch syndrome. However, the majority of MMR-deficient colorectal carcinomas are sporadic and produced by hypermethylation of the MLH1 promoter. The aim of our study was to evaluate the role of MLH1 promoter methylation analysis in the distinction between MLH1- negative sporadic and hereditary carcinomas. Methods The study included 370 colorectal adenocarcinomas. Microsatellite analysis was performed using the five markers of Bethesda plus BAT40 and a fluorescence based PCR method. MMRPs expression (MLH1, MSH2, MSH6, PMS2) was evaluated by immunohistochemistry. MLH1 promoter methylation (C-region, proximal relative to the transcription start of the MLH1 gene) was determined by methylation-specific PCR. Results MLH1 promoter methylation was detected in 198 of 272 (72.8%) MSI-H carcinomas, whereas all the 98 MSS/MSI-L tumors analyzed were unmethylated. Among MSI-H tumors, MLH1 methylation was found in 196/222 (88.3%) MLH1-negative carcinomas and in 2 of 50 (4%) MLH1-positive carcinomas (p<0.001). MLH1-negative tumors of patients aging <56 years were less frequently methylated (8/14, 57.1%) than tumors of patients aging 56–70 years (46/55, 83.6%) and of patients older than 70 years (142/153, 92.8%) (p<0.001). Conclusions Our data confirm that MLH1 promoter methylation is the major mechanism leading to MSI-H in colorectal cancer. Analysis of MLH1 methylation in conjunction with clinical data and MMRPs expression pattern may be relevant in the selection of patients with suspected Lynch syndrome for genetic testing

Analysis of BRAF Gene Mutation and MLH1 Promoter Methylation in MSI-H Colorectal Carcinomas with Loss of MLH1 Protein Expression

Purpose of the study. Hereditary and sporadic colorectal carcinomas (CRCs) with deficit of DNA mismatch repair (MMR-D) should be identified in all patients to ensure accurate treatment and risk assessment for relatives. Almost all MMR-D CRCs can be detected by microsatellite instability (MSI) and immunohistochemical testing. Analysis of MLH1 promoter methylation and evaluation of BRAF gene mutational status can help to differentiate hereditary from sporadic MSI-H MLH1-negative colorectal carcinomas. Methods. The study included a consecutive series of 2248 CRCs surgically resected from January 2004 to March 2012. Immunohistochemical analysis of MLH1, MSH2, MSH6 protein was performed in all tumours, PMS2 protein only in selected cases. MSI status was determined by a fluorescent PCR method using the Bethesda panel markers plus BAT40; tumours were classified as MSI-H, MSI-L and MSS according to the guidelines of Bethesda. In MMR-D tumours, analysis of MLH1 promoter methylation was assessed by methylation specific PCR and evaluation of V600E BRAF mutation was investigated by direct DNA sequencing. Summary of results. Deficit of MMR was observed in 332 tumours (14.7%). Most MMR-D tumours showed loss of MLH1 expression (272/332). V600E BRAF mutation was observed in 118/174 MLH1-negative MMR-D tumours and in only 1/42 MLH1-positive MMR-D cancers. MLH1 methylation was detected in 208/235 MMR-D MLH1-negative carcinomas and in 2/50 MMR-D MLH1-positive carcinomas. BRAF mutation was identified in 115/150 MLH1-negative tumours showing MLH1 methylation and only in 3 MLH1-negative tumours without MLH1 methylation. Conclusions. Our study indicate that MLH1 methylation and BRAF mutation occur frequently in MMR-D CRCs, are closely associated and may be used to identify CRC patients with Lynch syndrome

Prognostic value of flow cytometric DNA ploidy in colorectal cancer

Several studies demonstrated that flow cytometric nuclear DNA content and Mismatch Repair (MMR) status are relevant prognostic factors in colorectal cancer (CRC). It has also been suggested that the prognostic value of DNA ploidy is mainly determined by its relationship with MMR status. Aim of the study was to evaluate the prognostic significance of DNA ploidy in a large series of CRCs, characterized for several clinical and pathologic variables and MMR status. Results demonstrate that DNA ploidy is related to pathologic and molecular features in CRC and suggest that flow cytometric nuclear DNA content analysis provides prognostic information additional to MMR status

Assessment of MLH1 promoter methylation and BRAF gene mutation in colorectal carcinomas with microsatellite instability

Background: Recent studies indicate that analysis of MLH1 promoter methylation and especially evaluation of BRAF gene mutational status can be employed to differentiate hereditary from sporadic MSI-H MLH1-negative colorectal carcinomas. In particular BRAF was demontrated to be frequently mutated in MSI-H sporadic but not in hereditary carcinomas. Design: The study was conducted on a consecutive series of 2162 colorectal adenocarcinomas surgically resected from January 2004 to June 2010. Mismatch repair (MMR) status has been prospectively evaluated by immunohistochemical analysis of MMR protein expression (MLH1, MSH2, MSH6 and, in selected cases, PMS2) and microsatellite instability (MSI) analysis, using a fl uorescent PCR method and the Bethesda panel markers (BAT25, BAT26, D2S123, D5S346, D17S250) plus BAT40. Tumors were classifi ed as MSI-H, MSI-L and MSS according to the guidelines of the International Workshop of Bethesda. In MMR-defi cient (MMR-D) tumors, analysis of MLH1 promoter methylation (C- region) was assessed by methylation specifi c PCR and evaluation of V600E BRAF mutation was investigated by direct DNA sequencing. Results: 316 (14.6%) carcinomas were classifi ed as MMR-D (loss of MMR protein expression and/or MSI-H). Most MMR-D tumors showed loss of MLH1 expression (256, 81%). MLH1 methylation was detected in 196/219 (89%) MLH1-negative carcinomas and in 2/50 (4%) MMR-D MLH1-positive carcinomas. V600E BRAF mutations were observed in 108/158 (68%) MLH1-negative and in only 1/42 (2%) MLH1-positive MMR-D cancers. BRAF mutations were identifi ed only in tumors showing MLH1 promoter methylation (107/142, 75%). All the MLH1-negative carcinomas without MLH1 methylation examined (15 cases) did not demonstrate BRAF mutation. Both MLH1 promoter methylation and BRAF mutation were more frequently observed in older patients. Conclusions: Our results confi rm that MLH1 promoter methylation and BRAF mutation occur in a large fraction of MMR-defi cient MLH1-negative colorectal carcinomas and are closely associated. Furthermore our data indicate that assessment of MLH1 promoter methylation and especially of BRAF mutation might be used in the selection of colorectal cancer patients with presumptive Lynch syndrome

Pathologic and molecular features of BRAF mutated colorectal adenocarcinomas

There is growing evidence of the diagnostic, prognostic and predictive role of BRAF mutation in colorectal cancer. BRAF mutation, together with MSI and CIMP, is also a key feature of large bowel tumors that develop through the serrated pathway. Aim of this study was to examine the clinical and pathological features associated with BRAF mutated colorectal cancers in relation to microsatellite instability status and to compare in the group of Mismatch Repair proficient (MMR-P) carcinomas the characteristics of BRAF-mutated, KRAS-mutated and KRAS/BRAF-wild type tumors

Survival prediction in high-grade gliomas using CT perfusion imaging

Patients with high-grade gliomas usually have heterogeneous response to surgery and chemoirradiation. The objectives of this study were (1) to evaluate serial changes in tumor volume and perfusion imaging parameters and (2) to determine the value of these data in predicting overall survival (OS). Twenty-nine patients with World Health Organization grades III and IV gliomas underwent magnetic resonance (MR) and computed tomography (CT) perfusion examinations before surgery, and 1, 3, 6, 9, and 12 months after radiotherapy. Serial measurements of tumor volumes and perfusion parameters were evaluated by receiver operating characteristic analysis, Cox proportional hazards regression, and Kaplan–Meier survival analysis to determine their values in predicting OS. Higher trends in blood flow (BF), blood volume (BV), and permeability-surface area product in the contrast-enhancing lesions (CEL) and the non-enhancing lesions (NEL) were found in patients with OS 80 % in predicting 24 months OS in patients with grade IV gliomas. Our study indicated that CT perfusion parameters were predictive of survival and could be useful in assessing early response and in selecting adjuvant treatment to prolong survival if verified in a larger cohort of patients