Background Recurrent mutations in the promoter of the telomerase reverse transcriptase ( TERT) gene (C228T and C250T) detected in tumours and cells shed into urine of urothelial cancer (UC) patients are putative biomarkers for UC detection and monitoring. However, the possibility of detecting these mutations in cell-free circulating DNA (cfDNA) in blood and urine, or DNA from urinary exfoliated cells (cellDNA) with a single-gene sensitive assay has never been tested in a case-control setting. Methods We developed a single-plex assay (UroMuTERT) for the detection of low-abundance TERT promoter mutations. We tested 93 primary and recurrent UC cases and 94 controls recruited in France (blood, urine samples and tumours for the cases), and 50 primary UC cases and 50 controls recruited in Portugal (urinary exfoliated cell samples). We compared our assay with urine cytology. Findings In the French series, C228T or C250T were detected in urinary cfDNA or cellDNA in 81 cases (87·1%; 95% CI 78·6–93·2), and five controls (Specificity 94·7%; 95%CI 88·0–98·3), with 98·6% (95% CI 92·5–99·96) concordance in matched tumours. Detection rate in plasma cfDNA among cases was 7·1%. The UroMuTERT sensitivity was (i) highest for urinary cfDNA and cellDNA combined, (ii) consistent across primary and recurrent cases, tumour stages and grades, (iii) higher for low-risk non-muscle invasive UC (86·1%) than urine cytology (23·0%) ( P < 0·0001) and (iv) 93·9% when combined with cytology. In the Portuguese series – the sensitivity and specificity for detection of UC with urinary cellDNA was 68·0% (95% CI 53·3–80·5) and 98·0% (95% CI 89·3–100·0). Interpretation TERT promoter mutations detected by the UroMuTERT assay in urinary DNA (cfDNA or cellDNA) show excellent sensitivity and specificity for the detection of UC, significantly outperforming that of urine cytology notably for detection of low-grade early stages UC. Fund French Cancer League; French Foster Research in Molecular Biology and European Commission FP7 Marie Curie COFUND.
Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.
Sirtuins participate in hormone imbalance, metabolism and aging, which are important processes for endometrial cancer (EC) development. Sirtuins mRNA expression (SIRT1 to 7) was determined in 76 ECs (63 Type I, 12 Type II and one mixed EC), and 30 non-neoplastic endometria (NNE) by quantitative real-time PCR. SIRT1 and SIRT7 protein expression was evaluated by immunohistochemistry using Allred score. Compared to NNE, ECs showed SIRT7 (p < 0.001) mRNA overexpression, whereas SIRT1 (p < 0.001), SIRT2 (p < 0.001), SIRT4 (p < 0.001) and SIRT5 (p < 0.001) were underexpressed. No significant differences were observed for SIRT3 and SIRT6. Type II ECs displayed lower SIRT1 (p = 0.032) and SIRT3 (p = 0.016) transcript levels than Type I ECs. Concerning protein expression, SIRT1 immunostaining median score was higher in ECs compared to NNE epithelium (EC = 5 vs. NNE = 2, p < 0.001), while SIRT7 was lower in ECs (EC = 6 vs. NNE = 7, p < 0.001). No significant associations were found between SIRT1/7 immunoexpression and histological subtype, grade, lymphovascular invasion or stage. Our data shows that sirtuins are deregulated in EC. The diversity of expression patterns observed suggests that sirtuins may have distinctive roles in endometrial cancer similarly to what has been described in other cancer models.
Bladder cancer is one of the most incident neoplasms worldwide, and its treatment remains a significant challenge, since the mechanisms underlying disease progression are still poorly understood. The epithelial to mesenchymal transition (EMT) has been proven to play an important role in the tumorigenic process, particularly in cancer cell invasiveness and metastatic potential. Several studies have reported the importance of epigenetic mechanisms and enzymes, which orchestrate them in several features of cancer cells and, specifically, in EMT. In this paper, we discuss the epigenetic enzymes, protein-coding and non-coding genes, and mechanisms altered in the EMT process occurring in bladder cancer cells, as well as its implications, which allows for improved understanding of bladder cancer biology and for the development of novel targeted therapies.
Background:Urothelial carcinoma (UC) is the most common cancer affecting the urinary system, worldwide. Lack of accurate early detection tools entails delayed diagnosis, precluding more efficient and timely treatment. In a previous study, we found that miR-129-2 and miR-663a were differentially methylated in UC compared with other genitourinary tract malignancies. Here, we evaluated the diagnostic performance of those microRNAs in urine.Methods:Promoter methylation levels of miR-129-2 and miR-663a were assessed, using real-time quantitative methylation-specific PCR, in UC tissue samples (using normal urothelium as control) and, subsequently, in urine samples from UC and other genitourinary malignancies. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.Results:Promoter methylation levels of both microRNAs were significantly higher in UC tissue samples compared with normal urothelium. In urine, the assay was able to distinguish UC from other genitourinary tract carcinomas with 87.7% sensitivity and 84% specificity, resulting in 85.85% overall accuracy.Conclusions:This panel of miRNAs promoter methylation accurately detects UC in urine, comparing well with other promising epigenetic-based biomarkers. This may constitute the basis for a non-invasive assay to detect UC.
Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.
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