Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences.

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P 2 , but this function appears normal in Synaptojanin RQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P 2 , two lipids found in autophagosomal membranes. Using advanced imaging, we show that Synaptojanin RQ mutants accumulate the PI(3)P/PI(3,5)P 2 -binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in Synaptojanin RQ flies.Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease.

Stichting ParkinsonFonds, Dorpmans-Wigmans Stichting, Erasmus Medical Center, ZonMw-Memorabel programme, EU Joint Programme Neurodegenerative Disease Research (JPND), Parkinson's UK, Avtal om Läkarutbildning och Forskning (ALF) and Parkinsonfonden (Sweden), Lijf and Leven foundation, and cross-border grant of Alzheimer Netherlands-Ligue Européene Contre la Maladie d'Alzheimer (LECMA).

Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non-muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard.Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study. Sensitivity of the CGIs for recurrences detection was investigated on a test set of 101 preTUR urines. Specificity was determined on 70 urines from healthy males aged more than 50 years. A 3-plex assay for the best combination was developed and validated on an independent set of 95 preTUR, recurrence free, and nonmalignant urines (n ¼ 130).Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57%, respectively.Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range as cystoscopy and paves the way for a subsequent study that investigates a modified surveillance protocol consisting of the urine test followed by cystoscopy only when the urine test is positive.

The potential risk of recurrence and progression in patients with non-muscle-invasive bladder cancer necessitates followup by cystoscopy. The risk of progression to muscle-invasive bladder cancer is estimated based on the European Organisation of Research and Treatment of Cancer score, a combination of several clinicopathological variables. However, pathological assessment is not objective and reproducibility is insufficient. The use of molecular markers could contribute to the estimation of tumor aggressiveness. We recently demonstrated that methylation of GATA2, TBX2, TBX3, and ZIC4 genes could predict progression in Ta tumors. In this study, we aimed to validate the markers in a large patient set using DNA from formalin-fixed and paraffin-embedded tissue. PALGA: the Dutch Pathology Registry was used for patient selection. We included 192 patients with pTaG1/2 bladder cancer of whom 77 experienced progression. Methylation analysis was performed and log-rank analysis was used to calculate the predictive value of each methylation marker for developing progression over time. This analysis showed better progression-free survival in patients with low methylation rates compared with the patients with high methylation rates for all markers (Po0.001) during a followup of ten-years. The combined predictive effect of the methylation markers was analyzed with the Coxregression method. In this analysis, TBX2, TBX3, and ZIC4 were independent predictors of progression. On the basis of methylation status of TBX2 and TBX3, patients were divided into three new molecular grade groups. Survival analysis showed that only 8% of patients in the low molecular grade group progressed within 5 years. This was 29 and 63% for the intermediate-and high-molecular grade groups. In conclusion, this new moleculargrade based on the combination of TBX2 and TBX3 methylation is an excellent marker for predicting progression to muscle-invasive bladder cancer in patients with primary pTaG1/2 bladder cancer.

See Uzquiano and Francis (doi: ) for a scientific commentary on this article. Mutations in RTTN , which encodes Rotatin, give rise to various brain malformations. Vandervore et al. reveal mitotic failure, aneuploidy, apoptosis and defective ciliogenesis in patient cells. Rotatin binds to myosin subunits in the leading edge of human neurons, which may explain the proliferation and migration defects observed.

Cancers of the urinary bladder (BC) present as muscle-invasive (MIBC) or non-muscle invasive (NMIBC). Major problems with NMIBC are that 70% of the tumors will recur and 10-20% will eventually progress to MIBC. Therefore the patients are monitored by cystoscopy every 3-6 months after transurethral resection of the tumor in order to spot potential recurrences. DNA methylation has been shown to contribute to the pathogenesis cancer and may serve as useful biomarker. Recent studies showed promising urine methylation biomarkers for BC, but none of these were specifically tested for detection of recurrent BCs which are often smaller and hence more difficult to detect than primary tumors. Therefore we aimed to develop an assay specific for the diagnosis of recurrent bladder tumors in voided urine. From our earlier genome-wide study, we selected 8 candidate CGIs (CpG islands) methylated in BC to screen for the detection of recurrent bladder tumors in voided urine. We first screened these 8 CGIs on an independent set of 50 FFPE bladder tumors and 70 urines from age matched individuals without any history of BC as controls using BS-SNaPshot (Bisulfite specific single nucleotide primer extension assay). Subsequently, the 8 CGIs were investigated in a test set of 100 preTUR (before Trans Urethral Resection) urines associated with a concomitant recurrent tumor. We analyzed the sensitivity, specificity, AUC (area under the curve), PPV and NPV of all the individual markers and five best combinations. We then validated this on a separate cohort of 100 preTUR urines samples. Single marker OTX1 identified recurrent bladder tumors in voided urine with a sensitivity of 71% at a specificity of 90% with an AUC of 0.85 (CI: 0.80-0.91, P<0.0001). A 3 gene methylation panel OTX1, ONECUT2 and OSR1 showed a sensitivity of 73% at a specificity of 90% with an AUC of 0.86 (CI: 0.80-0.91, P< 0.0001). Combining this 3-gene panel methylation assay with FGFR3 (fibroblast growth factor receptor 3) mutation assay achieved a sensitivity of 78% at a specificity of 90% with an AUC of 0.87 (CI: 0.82-0.93, P< 0.0001). This panel of markers showed a sensitivity of 100% in detecting bladder tumor tissue DNA. These markers were also investigated on 40 urines collected from patients who are recurrence free for a period of longer than 6 months. A total of 9/40 (22%) urines were positive in these recurrence free patients. The lower specificity of these samples could be due to the anticipatory effect that has been attributed to urine tests, i.e. urines test sees the tumor earlier than cystocopy. We also observed that multiple tumors from a patient are highly concordant in their methylation percentage, which underlines the usefulness of the markers. In summary the panel biomarkers that are identified will be able to detect the recurrent bladder tumors in voided urine, thereby helping in surveillance of patients with bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4023. doi:1538-7445.AM2012-4023

<p>PDF file, 15K, ROC curve of methylation assay (dotted line) and methylation + FGFR3 assay (thick line) for the validation set.</p>