Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both p = 0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (p = 0.003 and p = 0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).

Impact of EWS-ETS fusion type on disease progression in Ewing's sarcoma/peripheral primitive neuroectodermal tumor: prospective results from the cooperative Euro-E.W.I.N.G. 99 trial.

PURPOSE EWS-ETS fusion genes are the driving force in Ewing's sarcoma pathogenesis. Because of the variable breakpoint locations in the involved genes, there is heterogeneity in fusion RNA and protein architecture. Since previous retrospective studies suggested prognostic differences among patients expressing different EWS-FLI1 fusion types, the impact of fusion RNA architecture on disease progression and relapse was studied prospectively within the Euro-E.W.I.N.G. 99 clinical trial. PATIENTS AND METHODS Among 1,957 patients who registered before January 1, 2007, 703 primary tumors were accessible for the molecular biology study. Fusion type was assessed by polymerase chain reaction on frozen (n = 578) or paraffin-embedded materials (n = 125). The primary end point was the time to disease progression or relapse. Results After exclusion of noninformative patients, 565 patients were entered into the prognostic factor analysis comparing type 1 (n = 296), type 2 (n = 133), nontype 1/nontype 2 EWS-FLI1 (n = 91) and EWS-ERG fusions (n = 45). Median follow-up time was 4.5 years. The distribution of sex, age, tumor volume, tumor site, disease extension, or histologic response did not differ between the four fusion type groups. We did not observe any significant prognostic value of the fusion type on the risk of progression or relapse. The only slight difference was that the risk of progression or relapse associated with nontype 1/nontype 2 EWS-FLI1 fusions was 1.38 (95% CI, 0.96 to 2.0) times higher than risk associated with other fusion types, but it was not significant (P = .10). CONCLUSION In contrast to retrospective studies, the prospective evaluation did not confirm a prognostic benefit for type 1 EWS-FLI1 fusions.

The promise of telomere length, telomerase activity and its regulation in the translocation-dependent cancer ESFT; clinical challenges and utility.

The Ewing's sarcoma family of tumours (ESFT) are diagnosed by EWS-ETS gene translocations. The resulting fusion proteins play a role in both the initiation and maintenance of these solid aggressive malignant tumours, suppressing cellular senescence and increasing cell proliferation and survival. EWS-ETS fusion proteins have altered transcriptional activity, inducing expression of a number of different target genes including telomerase. Up-regulation of hTERT is most likely responsible for the high levels of telomerase activity in primary ESFT, although telomerase activity and expression of hTERT are not predictive of outcome. However levels of telomerase activity in peripheral blood may be useful to monitor response to some therapeutics. Despite high levels of telomerase activity, telomeres in ESFT are frequently shorter than those of matched normal cells. Uncertainty about the role that telomerase and regulators of its activity play in the maintenance of telomere length in normal and cancer cells, and lack of studies examining the relationship between telomerase activity, regulators of its activity and their clinical significance in patient samples have limited their introduction into clinical practice. Studies in clinical samples using standardised assays are critical to establish how telomerase and regulators of its activity might best be exploited for patient benefit.

The use of real-time reverse transcription-PCR for prostate-specific antigen mRNA to discriminate between blood samples from healthy volunteers and from patients with metastatic prostate cancer.

PURPOSE: A clinical role for nonquantitative reverse transcription-PCR (RT-PCR) using prostate-specific antigen in blood samples from patients with prostate cancer remains undefined. Assay variation and detection of prostate-specific antigen mRNA illegitimate transcription may explain inconsistent results between studies. Defining levels of prostate-specific antigen mRNA expression in blood samples from healthy volunteers and patients with prostate cancer would allow cutoffs to be established to distinguish the two groups. EXPERIMENTAL DESIGN: Quantitative real-time RT-PCR for prostate-specific antigen mRNA was established and levels of prostate-specific antigen mRNA measured in bloods samples from healthy volunteers (n=21) and patients with localized (n=27) and metastatic (n=40) prostate cancer. RESULTS: Levels of prostate-specific antigen mRNA were significantly higher in blood samples from patients with metastatic prostate cancer than in blood samples from patients with localized prostate cancer (P <0.001) or in blood samples from healthy volunteers (P <0.01); levels between patients with localized prostate cancer and healthy volunteers were no different. Assay sensitivity to detect patients with metastatic prostate cancer was 68% with specificity of 95%. In patients with newly diagnosed metastatic prostate cancer, monitoring response to hormonal therapy was possible with this assay. No correlation between levels of prostate-specific antigen mRNA and serum prostate-specific antigen protein levels was found, suggesting that prostate-specific antigen mRNA and serum prostate-specific antigen protein levels reflect different features of prostate cancer, i.e., circulating tumor cells and total tumor bulk, respectively. CONCLUSIONS: Quantitative RT-PCR discriminates patients with metastatic prostate cancer from healthy volunteers and patients with localized prostate cancer but cannot discriminate patients with localized prostate cancer from healthy volunteers. A role for quantitative RT-PCR has been identified in the assessment and monitoring of patients with metastatic prostate cancer.