No differences in miRNA expression in the stroma of ERG+ and ERG- prostate cancers.

Prostate cancer (PC) is one of the most common cancers in males. A significant proportion of PCs bear TMPRSS2-ETS translocation and overexpress ERG transcription factor, allowing classification into ERG+ and ERG- groups, which differ in several features including the tumor microenvironment. The aim of the study was to verify whether they differ in expression of the miRNA in the microenvironment. The material consisted of 150 radical prostatectomies. Immunohistochemistry (IHC) for ERG was done using a routine method. FISH for TMPRSS2-ETS translocation was done with a ZytoLight SPEC ERG/TMPRSS2 TriCheck Probe. From each case, a representative section was selected, and tumor and non-tumor were microdissected with the LMD7000 device. RNA was isolated using the RNeasy Mini Kit system (Qiagen) and miRNA libraries were prepared with the NEBNext Multiplex Small RNA Library Prep Set for Illumina and their sequencing was performed on the NexSeq 500. Statistical analysis was done with Statistica and R software. When analyzing the expression of miRNAs some differences could be seen, but after correction for multiple comparisons was applied, these were found to be non- significant.

Patterns of gene expression characterize T1 and T3 clear cell renal cell carcinoma subtypes.

Renal carcinoma is the 20th most common cancer worldwide. Clear cell renal cell carcinoma is the most frequent type of renal cancer. Even in patients diagnosed at an early stage, characteristics of disease progression remain heterogeneous. Up-to-date molecular classifications stratify the ccRCC samples into two clusters. We analyzed gene expression in 23 T1 or T3 ccRCC samples. Unsupervised clustering divided this group into three clusters, two of them contained pure T1 or T3 samples while one contained a mixed group. We defined a group of 36 genes that discriminate the mixed cluster. This gene set could be associated with tumor classification into a higher stage and it contained significant number of genes coding for molecular transporters, channel and transmembrane proteins. External data from TCGA used to test our findings confirmed that the expression levels of those 36 genes varied significantly between T1 and T3 tumors. In conclusion, we found a clustering pattern of gene expression, informative for heterogeneity among T1 and T3 tumors of clear cell renal cell carcinoma.

BRAF mutations in sporadic colorectal carcinoma from polish patients.

BRAF mutations are second to KRAS mutations in activation of the MAPK pathway in colorectal carcinoma cells. In addition to mutated KRAS, BRAF V600E mutation is associated with resistance to EGFR-targeted therapy in colorectal cancer; thus mutated BRAF might serve as a predictive factor. In this study, 163 routinely resected adenocarcinomas were screened for mutations in exons 11 and 15 of the BRAF gene. Only 6 (3.7%) tumours had a missense point mutation (G469A, D594G, G596R, K601N and twice V600E). The tumours were locally advanced with multiple metastases to lymph nodes. Mutations were associated with microsatellite instability (2 cases MSI-H, 2 cases MSI-L) and mutually exclusive with a mutated KRAS gene. In this sample set, mutations in the BRAF gene were more diverse and less frequent than usually reported.

Immunophenotype and cytogenetics of mucinous tubular and spindle cell carcinoma of the kidney.

Mucinous tubular and spindle cell carcinoma (MTSCC) of the kidney is a rare, recently described entity. The authors present three new cases. The histological picture was that of classic MTSCC, with alternating small tubules located in a mucin-containing stroma, and spindle cell areas composed of bland, monomorphic cells. On immunohistochemistry, the tumors were positive for epithelial markers, including CK7 and CK18, vimentin, CD15, AMACR, and neuroendocrine markers, such as NSE and CD57. On FISH analysis we found losses on chromosomes 1 and 8, and gains of chromosomes 7 and 17. This is the first report of this rare entity in Polish medical literature.

Tissue microarray FISH applied to colorectal carcinomas with various microsatellite status.

Colorectal carcinoma is etiopathologically heterogenic. It may develop through a sequence of mutations leading to chromosome instability or be a result of defects in DNA repair mechanisms manifested by microsatellite instability of varying degrees. Colorectal carcinoma can thus be classified into microsatellite-stable (MSS), highly microsatellite unstable (MSI-H) and intermediate low-level microsatellite unstable (MSI-L) groups. Fluorescent hybridization in situ (FISH) is a method of detecting specific sequences of nucleic acids that is based on specific bonding of a fluorescent marker-associated probe and specific DNA fragment. The material consisted of 146 non-selected cases of colorectal carcinoma patients operated on at First Chair of General Surgery, Collegium Medicum, Jagiellonian University in Cracow, Poland. Following a standard histopathological evaluation, tissue microarrays were prepared using a Tissue MicroArray Builder, and FISH was performed employing probes specific for chromosomes 1, 8, 17 and 18. Microsatellite instability was evaluated in frozen material using the PCR reaction with gel and capillary electrophoresis. The mean number of signals obtained for chromosome 1 in the entire material was 2.06, while the corresponding mean values in the MSS group equaled 2.07, in the MSI-L group - 2.07, and in the MSI-H group - 2.01. The mean number of signals for chromosome 17 in the entire material was 2.1, in the MSS group - 2.11, in the MSI-L group - 2.13, and in the MSI-H group - 2.01. The number of signals for chromosome 18 in the entire material was 2, in the MSS group - 2, in the MSI-L group - 2, and in the MSI-H group - 2. The means number of signals for chromosome 8 in the entire material was 2.07, in the MSS group - 2.08, in the MSI-L group - 2.01, and in the MSI-H group - 2. These differences are not sufficient for distinguishing colorectal carcinoma molecular forms.

Correlation of microsatellite status, proliferation, apoptotic and selected immunohistochemical markers in colorectal carcinoma studied with tissue microarray.

Colorectal carcinoma is a frequent malignant tumor, characterized by varying clinical course and response to treatment. At the molecular level, colorectal carcinomas are divided into tumors with chromosomal instability (microsatellite-stable, MSS), microsatellite instability (MSI-H) and low microsatellite instability (MSI-L). The method of tissue microarrays allows for combining materials originating from multiple patients into a single slide, what makes possible to simultaneously investigate large material for the presence of numerous, diversified markers. The study material consisted of 208 cases of colorectal carcinoma. Microsatellite instability was evaluated in frozen material employing the PCR reaction with gel and capillary electrophoresis. Following a standard histopathological assessment, tissue microarrays were prepared using a MTA-1 microarrayer (Beecher) and standard immunohistochemical reactions were performed to detect the presence of bcl-2, CDX-2, Ki67, MLH1, MSH2, MSH6, p16, p53 and survivin. Apoptotic cells were detected using the TUNEL method. The correlations between the reactions were investigated and differences in the expression of the investigated proteins noted in carcinomas with various degrees of microsatellite instability. The agglomeration analysis showed differences in patterns of expression between MSS, MSI-L and MSI-H carcinomas. The discriminant function analysis demonstrated that the MSI-H carcinomas were best differentiated by MLH1, survivin and Ki67 expression, while the MSI-L tumors differed from the remaining colorectal carcinomas by their apoptotic index, local tumor stage (pT), the presence of angioinvasion and mucin production.

The relationship between MSI status and vessel density in colorectal carcinoma.

The development of new blood vessels is a prerequisite for progression of malignant neoplasms. Factors that induce neoangiogenesis include VEGF, VEGF-C, VEGF-D, PD-ECG, ANG-2, TSP-1, HIF-1 and HIF-2. From the etiopathogenetic viewpoint, colorectal carcinoma is heterogenic. It may develop via a sequence of mutations leading to chromosome instability or else result from DNA repair defects, which are manifested as microsatellite instability. The objective of the present investigations was the comparison of neoangiogenesis in microsatellite-stable colorectal carcinomas, as well as in tumors with low and high instability levels. The material included 71 surgical cases of colorectal carcinoma. Vessel density was assessed by immunohistochemical reactions to CD34 and vWf, calculating the number of vessel sections within the invasion margin, in visual fields selected at random, and within hot spots. Microsatellite instability was evaluated in frozen materials employing the PCR reaction with gel and capillary electrophoresis. In all the cases, the authors detected CD34+ and less numerous vWf+ vessels within the tumor and in its vicinity. In 45 cases, no microsatellite instability was found, in 13 cases, low level instability (MSI-L) was observed, and in another 13 - high microsatellite instability (MSI-H). Some differences in vessel density were noted between the above groups, yet they were not statistically significant. On the other hand, the authors observed more numerous CD34+ vessels in cases with metastases to the regional lymph nodes. In conclusion, it is suggested that neoangiogenesis in sporadic colorectal carcinoma is directly related to metastatic potential, but not to MSI status.

High thymidylate synthase expression is typical for sporadic MSI-H colorectal carcinoma.

Colorectal carcinoma is etiopathologically heterogenic. It may develop through a sequence of mutations leading to chromosome instability or be a result of defects in DNA repair mechanisms manifested by microsatellite instability. Carcinomas of this type are supposed to be characterized by a better prognosis and a different response to chemotherapy. The main target of 5-fluorouracil (5-FU) treatment is thymidylate synthase (TS). High TS expression has been identified as promoting resistance to 5-FU. The objective of the present investigation was to determine whether microsatellite instability is associated with thymidylate synthase expression. Ninety-eight cases of colorectal carcinoma were studied. Microsatellite instability was evaluated in frozen material employing the PCR reaction with gel and capillary electrophoresis. TS expression levels were assessed in preparations stained immunohistochemically using a semiquantitative method on a scale with scores from 0 to 3. The MSI-H phenotype was detected in ten cases, MSI-L in 16, and MSS in 72. The mean TS expression score was 1.79. In the MSS group, the mean TS expression score was 1.69, in the MSI-L group the mean TS expression score was 1.73, and in the MSI-H group the mean TS expression score was 2.67. The differences between MSI-H and MSS/MSI-L were statistically significant (p<0.0002 and p<0.004, respectively). The results may explain the different response of MSI-H carcinomas to 5-FU treatment.

CDH1 gene promoter hypermethylation in gastric cancer: relationship to Goseki grading, microsatellite instability status, and EBV invasion.

Hypermethylation of the CDH1 promoter region seems to be the most common epigenetic mechanism in this gene silencing in gastric cancer. In this study, CDH1 promoter hypermethylation was observed in 54.8% (46/84) of the analyzed sporadic gastric carcinomas. We introduce a new relation: clustering of Goseki grading into 3 grade was determined by CDH1 promoter hypermethylation. The percentage of methylation in Goseki III cancers was significantly higher (83%) when compared with other grades; the lowest proportion was detected in IV (36%) and II (38%) groups, whereas grade I demonstrated typical percentage of promoter hypermethylation. A novel polymorphism R732R in exon 14 of the CDH1 gene was detected by mutational analysis. Additionally, all cases with the MSI-high phenotype revealed CDH1 promoter hypermethylation. In MSI-low and MSS gastric cancers, this percentage was lower, reaching 71% and 41%, respectively. Moreover, the methylation status was correlated with the LOH phenotype. We detected CDH1 promoter hypermethylation in all EBV-positive gastric cancers (5/5), whereas methylation in the EBV-negative group occurred in 58% of cases. We also report that "methylated" tumors were slightly larger than "nonmethylated," whereas the second group revealed a higher probability of longer patient survival, though these relationships were not statistically significant. These results suggest that downregulation of E-cadherin, caused by promoter hypermethylation, in sporadic gastric carcinomas may be associated with a worse prognosis and specific tumor phenotype.