Evaluation of immunoexpression and MDR1 promoter methylation levels in prostatic tissue samples

Os objectivos deste estudo foram: (1) Determinar os níveis de metilação do promotor do MDR1 em tecido prostático com adenocarcinoma (CaP), neoplasia intraepitelial prostática de alto grau (HGPIN), hiperplasia benigna (BPH) e tecido morfologicamente normal (MNP). (2) Correlacionar os níveis de metilação com a imunoexpressão da gp-P. Os nossos resultados demonstram que a hipermetilação do MDR1 constitui um mecanismo eficaz de regulação da sua expressão. Estudos futuros permitirão avaliar o impacto destes resultados na terapêutica do cancro da próstata. Our aims were: (1) To determine the MDR1 methylation levels in tissue of Prostate cancer (PCa), high grade prostatic intraepithelial neoplasia (HGPIN), benign prostatic hyperplasia (BPH) and morphologically normal prostate tissue (MNP); (2) to correlate the methylation levels of the MDR1 promoter with the immunoexpression of P-gp. Our results demonstrate that MDR1 hypermethylation constitutes an effective mechanism of P-gp expression regulation. Future studies will be able to evaluate the impact of these results in the treatment of PCa patients

Evaluation of immunoexpression and MDR1 promoter methylation levels in prostatic tissue samples

Os objectivos deste estudo foram: (1) Determinar os níveis de metilação do promotor do MDR1 em tecido prostático com adenocarcinoma (CaP), neoplasia intraepitelial prostática de alto grau (HGPIN), hiperplasia benigna (BPH) e tecido morfologicamente normal (MNP). (2) Correlacionar os níveis de metilação com a imunoexpressão da gp-P. Os nossos resultados demonstram que a hipermetilação do MDR1 constitui um mecanismo eficaz de regulação da sua expressão. Estudos futuros permitirão avaliar o impacto destes resultados na terapêutica do cancro da próstata. Our aims were: (1) To determine the MDR1 methylation levels in tissue of Prostate cancer (PCa), high grade prostatic intraepithelial neoplasia (HGPIN), benign prostatic hyperplasia (BPH) and morphologically normal prostate tissue (MNP); (2) to correlate the methylation levels of the MDR1 promoter with the immunoexpression of P-gp. Our results demonstrate that MDR1 hypermethylation constitutes an effective mechanism of P-gp expression regulation. Future studies will be able to evaluate the impact of these results in the treatment of PCa patients

A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis

<p>Abstract</p> <p>Background</p> <p>The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings.</p> <p>Results</p> <p>We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as <it>TCF3:PBX1</it>, <it>ETV6:RUNX1</it>, and <it>TMPRSS2:ERG</it>) from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies.</p> <p>Conclusion</p> <p>This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.</p

Cysteine-Rich Secretory Protein-3 (CRISP3) Is Strongly Up-Regulated in Prostate Carcinomas with the TMPRSS2-ERG Fusion Gene

A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (rs = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

<p>Abstract</p> <p>Background</p> <p>Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors.</p> <p>Methods</p> <p>A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters.</p> <p>Results</p> <p>Significant differences in methylation levels among the four subtypes of renal tumors were found for <it>CDH1 </it>(<it>p </it>= 0.0007), <it>PTGS2 </it>(<it>p </it>= 0.002), and <it>RASSF1A </it>(<it>p </it>= 0.0001). <it>CDH1 </it>hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (<it>p </it>= 0.00016 and <it>p </it>= 0.0034, respectively), whereas <it>PTGS2 </it>methylation levels were significantly higher in ccRCC compared to pRCC (<it>p </it>= 0.004). <it>RASSF1A </it>methylation levels were significantly higher in pRCC than in normal tissue (<it>p </it>= 0.035). In pRCC, <it>CDH1 </it>and <it>RASSF1A </it>methylation levels were inversely correlated with tumor stage (<it>p </it>= 0.031) and nuclear grade (<it>p </it>= 0.022), respectively.</p> <p>Conclusion</p> <p>The major subtypes of renal epithelial neoplasms display differential aberrant <it>CDH1</it>, <it>PTGS2</it>, and <it>RASSF1A </it>promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.</p

Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

<p>Abstract</p> <p>Background</p> <p>A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in <it>MLL</it>-related leukemia. Recently, we have established the <it>MLL-SEPT2 </it>gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified <it>MLL </it>and <it>SEPT2 </it>gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of <it>MLL-SEPT2</it>-associated myeloid neoplasms so far described in the literature.</p> <p>Methods</p> <p>Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: <it>CBFB-MYH11 </it>(n = 13), <it>PML-RARA </it>(n = 12); <it>RUNX1-RUNX1T1 </it>(n = 12), normal karyotype (n = 11), and <it>MLL </it>gene fusions other than <it>MLL-SEPT2 </it>(n = 10). We also studied all three <it>MLL-SEPT2 </it>myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.</p> <p>Results</p> <p>When compared with normal controls, we found a 12.8-fold reduction of wild-type <it>SEPT2 </it>and <it>MLL-SEPT2 </it>combined expression in cases with the <it>MLL-SEPT2 </it>gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type <it>MLL </it>and <it>MLL-SEPT2 </it>combined expression (p = 0.028). The down-regulation of <it>SEPT2 </it>in <it>MLL-SEPT2 </it>myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other <it>MLL </it>gene fusions). In addition, <it>MLL </it>expression was also down-regulated in the group of <it>MLL </it>fusions other than <it>MLL-SEPT2</it>, when compared with the normal control group (p = 0.023)</p> <p>Conclusion</p> <p>We found a significant down-regulation of both <it>SEPT2 </it>and <it>MLL </it>in <it>MLL-SEPT2 </it>myeloid neoplasias. In addition, we also found that <it>MLL </it>is under-expressed in AML patients with <it>MLL </it>fusions other than <it>MLL-SEPT2</it>.</p

TMPRSS2-ERG Gene Fusion Causing ERG Overexpression Precedes Chromosome Copy Number Changes in Prostate Carcinomas and Paired HGPIN Lesions

TMPRSS2-ETS gene fusions have been found recurrently in prostate carcinomas, but not in the presumed precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN). However, HGPIN lesions may share chromosomal changes with prostate cancer. To determine the relative order of genetic events in prostate carcinogenesis, we have analyzed 34 prostate carcinomas, 19 paired HGPIN lesions, 14 benign prostate hyperplasias, and 11 morphologically normal prostatic tissues for TMPRSS2-ERG and TMPRSS2-ETV1 rearrangements and genomic imbalances. TMPRSS2 exon 1 was fused in-frame with ERG exon 4 in 17 of 34 (50%) prostate carcinomas and in 4 of 19 (21%) HGPIN lesions, but in none of controls. The findings were further validated by sequencing analysis and by the real-time polymerase chain reaction quantification of TMPRSS2-ERG fusion transcript and the ERG exons 5/6:exons 1/2 expression ratio. Chromosome copy number changes were detected by comparative genomic hybridization in 42% of clinically confined carcinomas and in none of the 16 HGPIN lesions analyzed. We demonstrate for the first time that the TMPRSS2-ERG fusion gene can be detected in a proportion of HGPIN lesions and that this molecular rearrangement is an early event that may precede chromosome-level alterations in prostate carcinogenesis