Simplex Droplet Digital PCR Assays for the Detection of TERT Promoter Mutations in Urine Samples for the Non-invasive Diagnosis of Urothelial Cancer.

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter region are highly frequent in urothelial cancer (UC), and their detection in urine (cell-free DNA from the urine supernatant or DNA from exfoliated cells in the urine pellet) has demonstrated promising evidence as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumour-derived mutations in urine requires highly sensitive methods, capable of measuring low-allelic fraction mutations. We developed sensitive droplet digital PCR (ddPCR) assays for detecting urinary TERT promoter mutations (uTERTpm), targeting the two most common mutations (C228T and C250T), as well as the rare A161C, C228A, and CC242-243TT mutations. Here, we described the step-by-step protocol uTERTpm mutation screening using simplex ddPCR assays and give some recommendations for isolation of DNA from urine samples. We also provide limits of detection for the two most frequent mutations and discuss advantages of the method for clinical implementation of the assays for the detection and monitoring of UC.

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.

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.

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.

Urinary TERT promoter mutations as non-invasive biomarkers for the comprehensive detection of urothelial cancer.

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.

[Traumatic dissection of the renal pedicle. Modalities of management in adults and children]. / Dissection traumatique du pédicule rénal. Modalités de prise en charge chez l'adulte et l'enfant.

OBJECTIVE: To evaluate the medium-term results of treatment for traumatic dissection of the renal arteries in a series of 12 cases and to propose emergency management based on recent endovascular revascularization techniques. MATERIAL AND METHODS: Between January 1999 and July 2003, 12 patients were admitted for closed trauma of the renal artery. There were 11 dissections with thrombosis and 1 intimal flap without distal thrombosis. Six patients were revascularized surgically (3 reno-renal bypass grafts, 3 auto-transplantations), 4 patients were treated by an endovascular procedure and 2 patients were treated conservatively with simple surveillance. Renal function, renal artery patency and blood pressure were evaluated immediately and at 3 months by clinical examination, Doppler ultrasound of the renal artery or CT angiography, and renal scintigraphy. RESULTS: In the group of 6 patients undergoing surgical revascularization (mean warm ischaemia time: 8 hours 30 minutes), 2 nephrectomies were performed (1 failure of revascularization, 1 sepsis in a non-functioning kidney). The other 4 patients presented negligible renal function on scintigraphy at 3 months despite patent renal arteries. Among the 4 patients undergoing endovascular revascularization (mean warm ischaemia time: 8 hours 50 minutes), 2 died from associated lesions and 2 had a non-functioning kidney (1 stent thrombosis, 1 silent kidney despite a patent renal artery). No cases of hypertension were observed regardless of the type of management. CONCLUSION: Renal revascularization after thrombosis due to traumatic dissection of the renal artery must not be performed systematically after a warm ischaemia time of more than 4 hours in view of the poor recovery of renal function and the absence of morbidity associated with simple surveillance. When a procedure is performed, evaluation of the results must be based on morphological as well as functional parameters (scintigraphy).

[Management of blunt trauma of the kidney]. / Prise en charge des traumatismes fermés du rein.

The current management of blunt trauma of the kidney is based on the 5-grade classification of lesions established by the ASST (American Society of the Surgery of Trauma). The indications for imaging are now clearly defined and spiral CT represents the reference examination. Over the last decade, the debate concerning the management of severe trauma has divided the supporters of surgical treatment from those who recommended conservative management. The contribution of interventional radiology and endourological treatments and the efficacy of intensive care now limit the complications related to trauma and reduce the need for surgery. However, the morbidity related to trauma is considerable in the presence of fragments of devascularized renal parenchyma, urine extravasation and associated lesions. These complications can be anticipated by a better definition of the traumatic lesions. The American classification presents certain limitations in relation to these combinations of poor prognostic factors. This review was designed to define the most recent biomechanical considerations, the place of imaging and finally the indications and results of management of blunt trauma of the kidney, in the light of the data of the literature.

[Use of MEOPA (nitrogen monoxide-oxygen mixture) as analgesic for prostate biopsies]. / Utilisation du MEOPA (mélange protoxyde d'azote-oxygène) comme méthode analgésique des biopsies de prostate.

OBJECTIVE: To determine the tolerance of prostate biopsies in the case of co-administration of an analgesic gas composed of a ready for use mixture of nitrogen monoxide and oxygen (MEOPA) (marketed under the trade name of Kalinox), and intrarectal instillation of xylocaine gel by comparing the results with those obtained in a control group only receiving intrarectal xylocaine instillation. MATERIAL AND METHODS: Non-randomized, prospective study conducted on 100 cases, with a control group composed of the first 22 patients (before availability of MEOPA in the department), followed by 78 patients treated with MEOPA. A questionnaire was completed by the patient and by the outpatient nurse caring for the patient. RESULTS: A very significant reduction (p=0.003) of the VAS pain score (from 3.86 to 2.38) and a highly significant improvement (p<0.001) of global tolerance of the procedure based on the nurse's estimation of pain (VAS scale) were observed. The MEOPA group also presented a significant improvement of pain in patients who had previously undergone a series of prostate biopsies (p=0.043). The satisfaction rate was higher in patients receiving MEOPA, although 28.2% of patients experienced adverse effects, all minor and bothersome in only 3.81% of cases. CONCLUSION: The MEOPA analgesic method as a complement to intrarectal xylocaine instillation considerably improved tolerance of the procedure with no major adverse effects and without prolonging the procedure or the surveillance. It is simple to use, safe and effective.

[Rigid ureteroscopy in the case of suspected tumor of the upper urinary tract: report of 63 cases]. / L'urétéroscopie rigide en cas de suspicion de tumeur de la voie excrétrice supérieure: à propos de 63 cas.

OBJECTIVE: This retrospective study was designed to determine the place of rigid ureteroscopy in the diagnosis of upper urinary tract tumours. MATERIAL AND METHODS: 63 patients (45 males and 18 females) with a mean age of 60 years, were investigated by rigid ureteroscopy for suspected upper urinary tract tumour. The Wolf 8-9.8 F ureteroscope was used in the case of suspected upper urinary tract tumour in a context of ureteric obstruction on the IVU in 29 cases, haematuria in 23 cases, a defect on retrograde ureteropyelography in 5 cases, suspicious CT scan in 5 cases and positive cytology with normal cystoscopy in 2 cases. RESULTS: The ureteroscope was able to be advanced as far as the suspicious zone in 89% of cases. The mean operating time was 48 minutes and the mean hospital stay was 3.2 days. Diagnostic rigid ureteroscopy has a Sensitivity of 58% (95% confidence interval CI95: +/- 28), a Specificity of 100%, a Positive Predictive Value of 100%, a Negative Predictive Value of 91% (+/- 7) and a low morbidity (3%). CONCLUSION: Rigid ureteroscopy does not appear to be the examination of choice for the diagnosis of upper urinary tract tumours, as it cannot investigate the renal pelvis or calices and the ureteroscope cannot always be advanced to the suspicious zone. The flexible ureteroscope now appears to be a more reliable diagnostic tool.