TERT Promoter Mutations as Simple and Non-Invasive Urinary Biomarkers for the Detection of Urothelial Bladder Cancer in a High-Risk Region.

Bladder cancer (BC) is the 10th most common cancer in the world. While there are FDA-approved urinary assays to detect BC, none have demonstrated sufficient sensitivity and specificity to be integrated into clinical practice. Telomerase Reverse Transcriptase (TERT) gene mutations have been identified as the most common BC mutations that could potentially be used as non-invasive urinary biomarkers to detect BC. This study aims to evaluate the validity of these tests to detect BC in the Kerman province of Iran, where BC is the most common cancer in men. Urine samples of 31 patients with primary (n = 11) or recurrent (n = 20) bladder tumor and 50 controls were prospectively collected. Total urinary DNA was screened for the TERT promoter mutations (uTERTpm) by Droplet Digital PCR (ddPCR) assays. The performance characteristics of uTERTpm and the influence by disease stage and grade were compared to urine cytology results. The uTERTpm was 100% sensitive and 88% specific to detect primary BC, while it was 50% sensitive and 88% specific in detecting recurrent BC. The overall sensitivity and specificity of uTERTpm to detect bladder cancer were 67.7% and 88.0%, respectively, which were consistent across different tumor stages and grades. The most frequent uTERTpm mutations among BC cases were C228T (18/31), C250T (4/31), and C158A (1/31) with mutant allelic frequency (MAF) ranging from 0.2% to 63.3%. Urine cytology demonstrated a similar sensitivity (67.7%), but lower specificity (62.0%) than uTERTpm in detecting BC. Combined uTERTpm and urine cytology increased the sensitivity to 83.8%, but decreased the specificity to 52.0%. Our study demonstrated promising diagnostic accuracy for the uTERTpm as a non-invasive urinary biomarker to detect, in particular, primary BC in this population.

Activating Telomerase TERT Promoter Mutations and Their Application for the Detection of Bladder Cancer.

This review summarizes state-of-the-art knowledge in early-generation and novel urine biomarkers targeting the telomerase pathway for the detection and follow-up of bladder cancer (BC). The limitations of the assays detecting telomerase reactivation are discussed and the potential of transcription-activating mutations in the promoter of the TERT gene detected in the urine as promising simple non-invasive BC biomarkers is highlighted. Studies have shown good sensitivity and specificity of the urinary TERT promoter mutations in case-control studies and, more recently, in a pilot prospective cohort study, where the marker was detected up to 10 years prior to clinical diagnosis. However, large prospective cohort studies and intervention studies are required to fully validate their robustness and assess their clinical utility. Furthermore, it may be interesting to evaluate whether the clinical performance of urinary TERT promoter mutations could increase when combined with other simple urinary biomarkers. Finally, different approaches for assessment of TERT promoter mutations in urine samples are presented together with technical challenges, thus highlighting the need of careful technological validation and standardization of laboratory methods prior to translation into clinical practice.

Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil.

In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.

Risk Prediction for Renal Cell Carcinoma: Results from the European Prospective Investigation into Cancer and Nutrition (EPIC) Prospective Cohort Study.

BACKGROUND: Early detection of renal cell carcinoma (RCC) has the potential to improve disease outcomes. No screening program for sporadic RCC is in place. Given relatively low incidence, screening would need to focus on people at high risk of clinically meaningful disease so as to limit overdiagnosis and screen-detected false positives. METHODS: Among 192,172 participants from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (including 588 incident RCC cases), we evaluated a published RCC risk prediction model (including age, sex, BMI, and smoking status) in terms of discrimination (C-statistic) and calibration (observed probability as a function of predicted probability). We used a flexible parametric survival model to develop an expanded model including age, sex, BMI, and smoking status, with the addition of self-reported history of hypertension and measured blood pressure. RESULTS: The previously published model yielded well-calibrated probabilities and good discrimination (C-statistic [95% CI]: 0.699 [0.679-0.721]). Our model had slightly improved discrimination (0.714 [0.694-0.735], bootstrap optimism-corrected C-statistic: 0.709). Despite this good performance, predicted risk was low for the vast majority of participants, with 70% of participants having 10-year risk less than 0.0025. CONCLUSIONS: Although the models performed well for the prediction of incident RCC, they are currently insufficiently powerful to identify individuals at substantial risk of RCC in a general population. IMPACT: Despite the promising performance of the EPIC RCC risk prediction model, further development of the model, possibly including biomarkers of risk, is required to enable risk stratification of RCC.

Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer.

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.

Urinary TERT promoter mutations are detectable up to 10 years prior to clinical diagnosis of bladder cancer: Evidence from the Golestan Cohort Study.

BACKGROUND: Detecting pre-clinical bladder cancer (BC) using urinary biomarkers may provide a valuable opportunity for screening and management. Telomerase reverse transcriptase (TERT) promoter mutations detectable in urine have emerged as promising BC biomarkers. METHODS: We performed a nested case-control study within the population-based prospective Golestan Cohort Study (50,045 participants, followed up to 14 years) and assessed TERT promoter mutations in baseline urine samples from 38 asymptomatic individuals who subsequently developed primary BC and 152 matched controls using a Next-Generation Sequencing-based single-plex assay (UroMuTERT) and droplet digital PCR assays. FINDINGS: Results were obtained for 30 cases and 101 controls. TERT promoter mutations were detected in 14 pre-clinical cases (sensitivity 46·67%) and none of the controls (specificity 100·00%). At an estimated BC cumulative incidence of 0·09% in the cohort, the positive and negative predictive values were 100·00% and 99·95% respectively. The mutant allelic fractions decreased with the time interval from urine collection until BC diagnosis (p = 0·033) but the mutations were detectable up to 10 years prior to clinical diagnosis. INTERPRETATION: Our results provide the first evidence from a population-based prospective cohort study of the potential of urinary TERT promoter mutations as promising non-invasive biomarkers for early detection of BC. Further studies should validate this finding and assess their clinical utility in other longitudinal cohorts. FUNDING: French Cancer League, World Cancer Research Fund International, Cancer Research UK, Tehran University of Medical Sciences, the International Agency for Research on Cancer, and the U.S. National Cancer Institute.

Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.

Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case-control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.

Circulating tumor DNA detection in head and neck cancer: evaluation of two different detection approaches.

The use of non-invasive biomarkers such as circulating tumor DNA (ctDNA) in head and neck tumors may be of relevance in early diagnosis and eventually improved outcome. We evaluated two different approaches from two case series in Europe and South America including (i) targeted screening of ctDNA mutations, and (ii) detection of TP53 mutations in plasma and oral rinses without previous knowledge of mutational status in tumor samples. Targeted sequencing in 5 genes identified ctDNA mutations in plasma among 42% of HNSCC cases, 67% of who were early stage cases. No association was found between ctDNA mutation detection and overall survival. Sequencing of the entire coding region of the TP53 gene resulted in identification of TP53 mutations in 76% of tumor cases. However, concordance of mutation detection was low between tumor, oral rinses (11%) and plasma (2,7%) samples. Identification of 5 pathogenic TP53 mutations in oral rinses from 3 non-cancer controls gives additional evidence of mutation occurrence in individuals without a diagnosed cancer and presents an additional challenge for the development of ctDNA diagnostic assays.