N. Peter Wiklund

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism

Significance This study shows that the telomerase reverse transcriptase (TERT) promoter mutations, which create de novo E-twenty six/ternary complex factors (Ets/TCF) transcription binding sites, besides being the most common somatic genetic lesions, influence both survival and disease recurrence in bladder cancer patients. The effect of the TERT promoter mutations on both survival and recurrence is modified by a common polymorphism within the preexisting Ets binding site in the TERT promoter. The data were supported by the results from reporter assays carried out in two urothelial carcinoma cell lines. The findings of the study suggest that the TERT promoter mutations in conjunction with the common polymorphism have potential of being used as clinical biomarkers in bladder cancer. The telomerase reverse transcriptase (TERT) promoter, an important element of telomerase expression, has emerged as a target of cancer-specific mutations. Originally described in melanoma, the mutations in TERT promoter have been shown to be common in certain other tumor types that include glioblastoma, hepatocellular carcinoma, and bladder cancer. To fully define the occurrence and effect of the TERT promoter mutations, we investigated tumors from a well-characterized series of 327 patients with urothelial cell carcinoma of bladder. The somatic mutations, mainly at positions −124 and −146 bp from ATG start site that create binding motifs for E-twenty six/ternary complex factors (Ets/TCF), affected 65.4% of the tumors, with even distribution across different stages and grades. Our data showed that a common polymorphism rs2853669, within a preexisting Ets2 binding site in the TERT promoter, acts as a modifier of the effect of the mutations on survival and tumor recurrence. The patients with the mutations showed poor survival in the absence [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.02–4.70] but not in the presence (HR 0.42, 95% CI 0.18–1.01) of the variant allele of the polymorphism. The mutations in the absence of the variant allele were highly associated with the disease recurrence in patients with Tis, Ta, and T1 tumors (HR 1.85, 95% CI 1.11–3.08). The TERT promoter mutations are the most common somatic lesions in bladder cancer with clinical implications. The association of the mutations with patient survival and disease recurrence, subject to modification by a common polymorphism, can be a unique putative marker with individualized prognostic potential.

Robot-Assisted Radical Cystectomy with Intracorporeal Urinary Diversion in Patients with Transitional Cell Carcinoma of the Bladder

BACKGROUND Robot-assisted radical cystectomy (RARC) may reduce morbidity after cystectomy. Descriptions of the surgical techniques of RARC with intracorporeal orthotopic neobladder or ileal conduit are sparse and oncologic and functional outcome data have not been reported. OBJECTIVE We present our technique with RARC and intracorporeal urinary diversion (neobladder or ileal conduit) and present oncologic and functional outcomes, as well as complication rates. DESIGN, SETTING, AND PARTICIPANTS Single-hospital institution case-series from 2004 to 2009 including 45 selected patients (38 male, 7 female) with high-grade and/or muscle-invasive urothelial cancer of the bladder. SURGICAL PROCEDURE We performed RARC; pelvic lymph node dissection using three different templates; and a totally intracorporeal urinary diversion, either orthotopic neobladder (n=36) or ileal conduit (n=9). MEASUREMENTS Perioperative variables, pathology data, early and late complication rates, urinary continence, potency, and cancer-specific survival were evaluated as outcome measures. RESULTS AND LIMITATIONS Median patient age, operative time, estimated blood loss, and lymph node yield were 62 yr (range: 37-79), 477 min (range: 325-760), 550 ml (range: 200-2200), and 19 (range: 10-52), respectively. Nine patients were diagnosed with positive lymph nodes. Surgical margins were clear in all but one patient. Early complications occurred in 18 patients (40%). Median postoperative stay was 9 d (range: 4-78), and median postoperative follow-up time was 25 mo. Four patients died due to metastatic disease. The study is limited by a relative small sample size and no comparative group. CONCLUSIONS RARC with totally intracorporeal urinary diversion is technically feasible with good intermediate-term oncologic results. This is a nonrandomised study including a limited number of patients with a restricted follow-up time, however, and so precautions must be considered when interpreting the outcomes.

Transurethral Resection of Non–Muscle-Invasive Bladder Transitional Cell Cancers With or Without 5-Aminolevulinic Acid Under Visible and Fluorescent Light: Results of a Prospective, Randomised, Multicentre Study

BACKGROUND Fluorescent light (FL)-guided cystoscopy induced by 5-aminolevulinic acid (5-ALA) has been reported to detect more tumours compared with standard white-light (WL) cystoscopy. Most reports are from single centres with relatively few patients. OBJECTIVE To evaluate whether 5-ALA-induced FL and WL cystoscopy at transurethral resection (TUR) is superior compared with standard procedures under WL only with respect to tumour recurrence and progression in patients with non-muscle-invasive bladder cancer. DESIGN, SETTING, AND PARTICIPANTS This randomised, multicentre, observer- and pathologist-blinded, prospective phase 3 clinical trial enrolled 300 patients, and of those patients, 153 were randomised to FL cystoscopy and 147 were randomised to standard WL cystoscopy. INTERVENTION All patients were first inspected under WL and all lesions were recorded. Patients randomised to FL underwent a second inspection. TUR was carried out in both groups. MEASUREMENTS Control cystoscopy under WL was performed in all patients every 3 mo during the first year after randomisation and biannually thereafter. RESULTS AND LIMITATIONS At the first TUR, the mean number of resection specimens per patient was 2.5 (FL: 2.5; WL: 2.4; p=0.37) and the resulting mean number of resected tumours was 1.7 with FL and 1.8 with WL (p=0.85). More patients were diagnosed with carcinoma in situ (CIS) in the WL group (13%) than in the FL group (4.2%). Within-patient comparison of FL patients only showed that FL detected more lesions than WL. Tumour lesions solely detected by FL cystoscopy that would not otherwise be detected by WL cystoscopy included 52% dysplasia, 33% CIS, 18% papillary neoplasms, 13% pT1, and 7% pTa. Outcome at 12 mo did not show any difference between groups with regard to recurrence-free and progression-free survival rates. CONCLUSIONS In this prospective, randomised, multi-institutional study, we found no clinical advantage of FL cystoscopy compared with WL cystoscopy and TUR.

Modulation of cholinergic and substance P‐like neurotransmission by nitric oxide in the guinea‐pig ileum

1 The role of endogenous nitric oxide (NO) as a modulator of enteric neurotransmission was investigated in longitudinal muscle myenteric plexus (LMMP) preparations of guinea‐pig isolated ileum. 2 In tissues previously incubated with [3H]‐choline, exogenous NO inhibited electrically‐evoked [3H]‐choline overflow as well as responses to exogenous agonists, indicating that NO has the potential of neuromodulation both pre‐ and postjunctionally. 3 A series of NO synthase inhibitors enhanced contractile responses to nerve stimulation indicating inhibitory neuromodulation by endogenous NO. 4 The potency order of the NO synthase inhibitors and their consistent effects after dexamethasone, on responses to nerve stimulation, indicate action on a constitutive NO synthase. 5 Responses enhanced by NO synthase inhibitors were inhibited by the substance P receptor antagonist, spantide, suggesting a neuromodulatory influence on substance P‐like neurotransmission by the endogenous NO. 6 NO synthase inhibition did not modify contractile responses to application of acetylcholine or substance P, or [3H]‐choline overflow, indicating that endogenous NO mainly has a prejunctional inhibitory action on substance P‐like neurotransmission. Nor did it modify responses to direct electrical muscle stimulation in the presence of tetrodotoxin. This suggests a prejunctional enhancing effect by NO synthesis inhibition. 7 Evidence for endogenous NO modulation of acetylcholine release was obtained when NO synthase inhibition modified atropine‐sensitive, nerve‐mediated contractile responses. However, [3H]‐choline overflow was unaltered by NO synthase inhibition. 8 NO synthase inhibition did not modify responses to inhibitory neurotransmission. 9 The findings suggest that endogenous NO inhibits substance P‐like motor neurotransmission, probably via prejunctional mechanisms. Cholinergic transmission may also be reduced by endogenous NO, acting prejunctionally.

Nitric oxide metabolite determinations reveal continuous inflammation in multiple sclerosis.

Nitric oxide (NO) is formed as a consequence of induction of the iNOS enzyme during inflammatory disorders. To investigate NO production in multiple sclerosis (MS), we determined the concentrations of its oxidation products (NOx) in the cerebrospinal fluid (CSF) and plasma of 61 MS patients. The patients were divided into three groups on the basis of their clinical disease activity. The total levels of NOx in CSF were significantly increased in all MS groups as compared to healthy controls and tension headache patients. CSF nitrite correlated with clinical disease activity. At exacerbation, the CSF nitrite levels exceed the plasma level. This suggests that clinical disease activity is due to a CNS inflammatory response, which is more intense and qualitatively different from that during clinical stable phases. This study supports NO involvement in the pathogenesis of MS and determination of nitrite levels may be useful a surrogate marker for disease activity.

Oncologic, Functional, and Complications Outcomes of Robot-assisted Radical Cystectomy with Totally Intracorporeal Neobladder Diversion

BACKGROUND Robot-assisted radical cystectomy (RARC) with totally intracorporeal neobladder diversion is a complex procedure that has been reported with good outcomes in small series. OBJECTIVE To present complications and oncologic and functional outcomes of this procedure. DESIGN, SETTING, AND PARTICIPANTS Between 2003 and 2012 in a tertiary referral center, 70 patients were operated on by two experienced robotic surgeons. Data were collected prospectively and reviewed retrospectively. INTERVENTION RARC with totally intracorporeal modified Studer ileal neobladder formation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The overall outcome of RARC with a totally intracorporeal neobladder was presented by assessing (1) surgical margins, (2) recurrence or cancer-specific death at 24 mo, (3) 30-d and 90-d complications graded according to the modified Clavien-Dindo system, (4) daytime and nighttime continence (no or one pad per day) at 6 and 12 mo, and (5) satisfactory sexual activity or potency at 6 mo and 12 mo. Survival rates were estimated by Kaplan-Meier plots. RESULTS AND LIMITATIONS Median follow-up of the cohort was 30.3 mo (interquartile range: 12.7-35.6). We recorded negative margins in 69 of 70 patients (98.6%). Clavien 3-5 complications occurred in 22 of 70 patients (31.4%) at 30 d and 13 of 70 (18.6%) at >30 d. At 90 d, the overall complication rate was 58.5%. Clavien <3 and Clavien ≥3 complications were recorded in 15 of 70 patients (21.4%) and 26 of 70 (37.1%), respectively. Kaplan-Meier estimates for recurrence-free, cancer-specific, and overall survival at 24 mo were 80.7%, 88.9%, and 88.9%, respectively. Daytime continence and satisfactory sexual function or potency at 12 mo ranged between 70% and 90% in both men and women. Limitations of this study include its retrospective design, selection bias due to the learning curve phase, and missing data. CONCLUSIONS In this expert center for RARC, outcomes after RARC with totally intracorporeal neobladder diversion appear satisfactory and in line with contemporary open series.

Cholinergic neuromodulation by endothelin in guinea pig ileum

The effect of endothelin on cholinergic neuroeffector transmission in guinea pig ileum was investigated. Endothelin was shown to inhibit the nerve-induced contractions and concomitantly to increase the basal muscle tone. Furthermore, endothelin inhibited the nerve-induced release of [3H]acetylcholine whereas the contractile response to exogenous acetylcholine was enhanced. In conclusion, our findings suggest that endothelin is a modulator of cholinergic neuroeffector transmission in guinea pig ileum with possible action via both inhibitory prejunctional and stimulatory postjunctional mechanisms.

Adrenergic neuromodulation by endothelin in guinea pig pulmonary artery.

Endothelin was tested for possible interactions with the noradrenergic neuroeffector transmission in guinea pig pulmonary artery. Endothelin concentration-dependently enhanced nerve-induced contractile responses and concomitantly increased basal tone. Furthermore, endothelin inhibited the stimulation-evoked release of [3H]noradrenaline and enhanced the contractile response to exogenously applied noradrenaline. These results suggest that endothelin, in addition to its direct contractile effect, may function as a modulator of adrenergic neuroeffector transmission in the guinea pig pulmonary artery via both stimulatory post- and inhibitory prejunctional mechanisms.

Modulation of neuroeffector transmission by endogenous nitric oxide: a role for acetylcholine receptor-activated nitric oxide formation, as indicated by measurements of nitric oxide/ nitrite release

Nitric oxide (NO) synthase inhibitors enhanced nerve-mediated contractile responses in guinea pig ileum longitudinal muscle, likely via a prejunctional effect on substance P-like neuroeffector transmission. Supporting a modulatory role for NO, application of NO through administration of acid sodium nitrite evoked marked inhibitory effects on responses to transmural nerve stimulation. Substance P-like responses to nerve stimulation were abolished by substance P receptor antagonists and were enhanced by atropine, indicating a cholinergic influence on substance P-like neuroeffector transmission. Since acetylcholine can evoke release of NO from endothelium, the possible role of acetylcholine in NO release in ileum was examined. The release of NO/nitrite, determined by chemiluminescence, was inhibited by NG-monomethyl-L-arginine (L-NMMA), by calcium removal, by tetrodotoxin or by atropine, indicating a nerve-mediated control of NO production. A basis for the NO release is likely to be spontaneous neuronal activity, where release of acetylcholine, with subsequent muscarinic receptor activation, contributes to stimulation of NO formation.

Robot-assisted Radical Cystectomy: Description of an Evolved Approach to Radical Cystectomy

BACKGROUND Although open radical cystectomy (ORC) remains the gold standard of care for muscle-invasive bladder cancer, robot-assisted radical cystectomy (RARC) continues to gain wider acceptance. In this article, we focus on the steps of RARC, describing our approach, which has been developed over the past 10 yr. Totally intracorporeal RARC aims to offer the benefits of a complete minimally invasive approach while replicating the oncologic outcomes of open surgery. OBJECTIVE We report our outcomes of a totally intracorporeal RARC procedure, describing step by step our technique and highlighting the variations on this standard template of nerve-sparing and female organ-preserving approaches in men and women. DESIGN, SETTING, AND PARTICIPANTS Between December 2003 and October 2012, a total of 113 patients (94 male and 19 female) underwent totally intracorporeal RARC. SURGICAL PROCEDURE We performed RARC, extended pelvic lymph node dissection, and a totally intracorporeal urinary diversion (UD) in all patients. In the accompanying video, we focus on the standard template for RARC, also describing nerve-sparing and female organ-preserving approaches. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Complications and oncologic outcomes are reported, including overall survival (OS) and cancer-specific survival (CSS) using Kaplan-Meier analysis. RESULTS AND LIMITATIONS RARC with intracorporeal UD was performed in 113 patients. Mean age was 64 yr (range: 37-84). Forty-three patients underwent intracorporeal ileal conduit, and 70 had intracorporeal neobladder. On surgical pathology, 48% of patients had ≤ pT1 disease, 27% had pT2 disease, 13% had pT3 disease, and 12% had pT4 disease. The mean number of lymph nodes removed was 21 (range: 0-57). Twenty percent of patients had lymph node-positive disease. Positive surgical margins occurred in six cases (5.3%). Median follow-up was 25 mo (range: 3-107). We recorded a total of 70 early complications (0-30 d) in 54 patients (47.8%), with 37 patients (32.7%) having Clavien grade ≥ 3. Thirty-six late complications (>30 d) were recorded in 30 patients (26.5%), with 20 patients (17.7%) having Clavien grade ≥ 3. One patient (0.9%) died within 90 days of operation from pulmonary embolism. Using Kaplan-Meier analysis, CSS was 81% at 3 yr and 67% at 5 yr. CONCLUSIONS Our structured approach to RARC has enabled us to develop this complex service while maintaining patient outcomes and complication rates comparable with ORC series. Our results demonstrate acceptable oncologic outcomes and encouraging long-term CSS rates.