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INTRODUCTION AND OBJECTIVE:The standard of care for muscle invasive bladder cancer (MIBC) is cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy. Patients with residual MI...

INTRODUCTION AND OBJECTIVE:Patients with metastatic upper tract urothelial carcinoma (UTUC) have limited treatment options: cisplatin-based adjuvant chemotherapy has been shown to provide a surviva...

INTRODUCTION AND OBJECTIVE:High-grade non-muscle invasive bladder cancer (NMIBC) is treated with intravesical BCG immunotherapy. Unfortunately, 40% of patients develop recurrence with a subset prog...

INTRODUCTION AND OBJECTIVE:Emerging data have shown that patients with locally advanced and metastatic urothelial cancer frequently harbor rare germline variants in cancer predisposition genes, par...

A 16-year-old male was diagnosed with Ewing sarcoma of the ribcage with pulmonary metastases. Six months after completion of scheduled therapy, he was found to have a new intracardiac mass, presumed recurrent Ewing sarcoma. EWSR1 fusion was not detected by droplet digital polymerase chain reaction from blood plasma. After no improvement with salvage chemotherapy, he underwent surgical resection that identified a low-grade spindle cell sarcoma. Despite the near-synchronous presentation of 2 unrelated sarcomas, extensive genomic analyses did not reveal any unifying somatic or germline mutations nor any apparent cancer predisposition. This case also highlights the potential role of utilizing plasma cell-free DNA for diagnosing tumors in locations where biopsy confers high morbidity.

Basal cell carcinosarcoma is a rare biphenotypic malignant skin tumor, in which one tumor component has light microscopic features of basal cell carcinoma, whereas the other has features of sarcoma. Clinical experience with this tumor is limited, and associated molecular genetic alterations are unknown. Herein, we report a unique case of metastatic basal cell carcinosarcoma, in which we analyzed the 2 components of the primary tumor as well as the metastasis by next-generation sequencing. The patient was a 72-year-old man who presented with a 7-year history of a large tumor of the left forearm. The tumor showed mixed features of basal cell carcinoma and undifferentiated sarcoma. The patient underwent a wide local excision and sentinel lymph node biopsy, which revealed microscopic subcapsular deposits of metastatic sarcomatoid tumor. One year later, intra-abdominal metastatic tumor was detected and resected. It had sarcomatoid features by light microscopy and failed to stain for epithelial markers by immunohistochemistry. DNA was extracted separately from the epithelial and sarcomatoid component of the primary tumor, intra-abdominal metastasis, and normal tissue. All exons of 230 cancer-associated genes were sequenced to an average read depth of >500-fold. This revealed multiple identical mutations in epithelial and sarcomatoid tumor compartments. Both compartments harbored 2 identical mutations, a truncating and a missense mutation, in the patched gene (PTCH1). This finding is not only of interest for a shared heritage of different subpopulations in a biphenotypic tumor, but also relevant clinically. It provides a rationale for the clinical use of hedgehog pathway inhibitors for treatment of patients affected by this tumor. Unfortunately, the patient reported herein died of metastatic disease before targeted therapy could be initiated.

(5.4% of all TC cases). In terms of ART 225 men (8.5%) with TC were prescribed testosterone. The OR of receiving a prescription was 4.5 95% CI [4.2,5.5] times higher than in men without TC and disproportionately high in younger men, with an 8.1 fold increase in the third decade of life. CONCLUSIONS: Men with a diagnosis of TC are four times more likely to have a serum testosterone checked, be diagnosed with hypogonadism, and receive ART. Serum testosterone is measured and more frequently found to be low in elderly patients with a history of TC compared to age-matched controls. Additionally, hypogonadism often predates the diagnosis of testis cancer. Finally, despite higher detection in elderly patients, ART is prescribed more often to young men with TC.

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established.We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes.A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P0.001).In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

*Time-dependent covariate analysis of the effect of first on-study SRE (starting 28 days before the event onset) on time to >1⁄4 2-point increase from baseline in worst pain score in patients with a baseline score of

INTRODUCTION AND OBJECTIVES: Genomic characterization of urothelial carcinoma of the bladder (UCB) has begun to reveal significant intertumor heterogeneity when comparing samples from different subjects. As in other malignancies, intratumor heterogeneity, which may allow for tumor evolution and adaption, poses a significant challenge to personalized-medicine strategies. METHODS: To examine UCB tumor evolution and heterogeneity, we performed next-generation targeted sequencing on multiple temporally and spatially separated bladder tumors obtained at time of transurethral resection (TUR) and radical cystectomy (RC). Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes. RESULTS: Phylogenetic reconstruction revealed evidence of branched evolutionary growth. Evaluation of multiple tumors from individual subjects identified both shared and unique potential driver mutations. Evidence of convergent phenotypic evolution was detected through analysis of multiple distinct tumors from several subjects. For example, three separate tumors in one subject (see Figure) shared a common PIK3CA mutation (E453K) and had unique second mutations in PIK3CA (E542V, E545K, and E545Q, respectively). In another subject, distinct inactivatingmutations of EP300were identified in two temporally separated tumor samples. Macrodissection of single tumors into non-invasive and invasive components revealed significant intratumor heterogeneity; one case illustrates howanalysis of amuscleinvasive TURspecimen could result in undersampling and therebymiss the tumor clone that persisted at time of RC. CONCLUSIONS: We demonstrate branched evolution of UCB through genomic analyses of multiple temporally and spatially distinct bladder tumors from individual subjects. Macrodissection of individual tumor samples identified significant intratumor heterogeneity. These concepts may present major challenges to personalized-medicine approaches that rely on sampling of a single tumor at a specific timepoint in the evolution of a patient’s UCB.

The aim of this study was to describe the effect of intensified antihypertensive therapy based on a structured teaching and treatment programme on the prognosis of hypertensive type 1 (insulin-dependent) diabetic patients with kidney disease.The study was a controlled, prospective, parallel, 10-year follow-up trial.A sequential sample of 91 hypertensive type 1 diabetic patients with overt diabetic nephropathy was prospectively followed for 10 years. Forty-five patients (intensified antihypertensive therapy; IT group) participated in an intensified antihypertensive therapy programme and 46 patients received routine antihypertensive treatment as provided by family physicians, consultants and local hospitals (routine antihypertensive therapy; RT group).The main endpoint was death; secondary endpoints were renal replacement therapy, blindness and amputation.Blood pressure was reduced in the IT group and increased in the RT group. During the follow-up period, 29 patients died, seven in the IT group and 22 in the RT group. The survival curves were significantly different (P = 0.0008). The main causes of death were cardiac. In a multiple Cox proportional hazards model, allocation to the IT group reduced the mortality risk [relative risk (RR) = 0.213; 95% confidence interval 0.089-0.509, P = 0.00051, while age (P = 0.0039) and mean blood pressure (P= 0.0113) increased this risk. In multiple Cox or multiple logistic regression models, the risks of dialysis (RR = 0.269, 95% confidence interval 0.110-0.656, P = 0.0039), blindness (odds ratio = 0.158, 95% confidence interval 0.037-0.684, P= 0.0136), and amputation (RR = 0.181, 95% confidence interval 0.047-0.703, P= 0.0135) were significantly lower in the IT group compared with the RT group (log rank P = 0.0008).We conclude that intensified antihypertensive treatment, based on a hypertension teaching and treatment programme, reduces long-term morbidity and mortality in patients with diabetic nephropathy.

INTRODUCTION AND OBJECTIVES: High grade urothelial carcinoma (UC) is an aggressive malignancy with limited therapeutic options in the advanced, metastatic, or recurrent disease setting. Targeted genetic analysis of high grade tumors was performed to determine the prevalence of known cancer genes and to identify potential targets for therapy. METHODS: Frozen high grade bladder tumors and matched germline blood DNA were obtained from 60 patients undergoing transurethral resection (TUR) or cystectomy. Specimens were analyzed using a targeted, deep-sequencing assay designed to identify point mutations, indels, and copy number alterations in 275 cancer-associated genes. RESULTS: The cohort was predominantly male (N 49, 81%) with a median age of 71 (IQR 61, 76). Frozen tumor was obtained from radical cystectomy(RC) in 53 patients (88%), TUR in 6 (10%), and partial cystectomy in 1 (2%). 11 patients (18%) had a history of intravesical therapy. Tumor pathologic stage was Ta in 5 (8%), T1 in 5 (8 %), T2 in 11 (18%), T3 in 30 (50%), and T4 in 9 (15%). Of the 53 patients undergoing RC and lymph node dissection, 17 received neoadjuvant chemotherapy (32%) and 24 (45%) had lymph node metastases. Mean target coverage for all sequenced exons was 512X. The most common genomic event was somatic mutation of TP53, identified in 33 patients (55%). RB mutations were identified in 11 (18%). Alterations in the PI3K/AKT/mTOR signaling pathway were also common; 12 patients had mutations in PIK3CA (20%), and non-overlapping mutations were identified in other genes within the pathway including AKT1, PTEN, and MTOR. Frequent mutations in chromatin remodeling genes were also identified. KDM6A mutations were identified in 21 tumors (35%), MLL2 in 18 (30%), and ARID1A in 17 (28%). Overall, 60% of all analyzed tumors harbored potentially targetable genomic alterations, including those with alterations in ERBB2, BRAF, and FGFR3 (Figure). CONCLUSIONS: Genetic alterations are frequent in high grade bladder UC. Over half of the tumors analyzed harbor alterations in genes that have been targeted for therapeutic benefit in other solid tumors, thus providing a compelling case for clinical trials using novel agents. Analysis for correlations with clinical outcomes is ongoing to assess for the prognostic significance of these genetic events. Source of Funding: Sidney Kimmel Center for Prostate and Urologic Cancers, Michael and Zena Wiener Research and Therapeutics Program in Bladder Cancer

Objective : To determine the prognosis of treated hypertensive type 1 (insulin-dependent) diabetic patients with overt nephropathy. Design : A controlled, prospective, parallel, 5-year follow-up trial. Setting : The tertiary care centre of the Heinrich Heine University Hospital in Dusseldorf, Germany. Patients and interventions : A sequential sample of 91 hypertensive patients with overt diabetic nephropathy participated in a diabetes treatment programme. Thereafter 45 patients received intensified antihypertensive therapy including blood pressure self-monitoring and self-adjustment of antihypertensive drug treatment with the goal of permanent normalization of blood pressure values below 140/90 mmHg. The remaining 46 patients were administered routine antihypertensive therapy and formed the control group. At baseline both groups were comparable in age, sex, metabolic control and renal function. The groups differed at baseline in their duration of diabetes and blood pressure values, which were higher in the intensified antihypertensive therapy group. Outcome measures : Total mortality and the need for renal replacement therapy. Main results : Blood pressure control was significantly improved in patients who were subjected to intensified antihypertensive therapy, whereas it deteriorated in the group of patients who received routine antihypertensive therapy. At follow-up, primary end points of the study occurred in five (11%) patients of the intensified therapy group and in 19 (41%) patients of the routine therapy group. According to life table analysis, intensified antihypertensive therapy was associated with less frequent primary end points (P= 0.0058) and longer survival (P= 0.01). The differences between the groups remained significant after adjustment for covariates in the proportional hazards model. Conclusion : Participation in a treatment programme aimed at intensification of antihypertensive therapy is associated with a reduction of mortality in hypertensive type 1 diabetic patients with overt nephropathy.

rs1512268 on chromosome 8p21 is associated with PC susceptibility, which is located at 14kb downstream of a prostate tumor suppressor gene NKX3.1. In the present study, we aimed to identify functional or causative variants in this region conferring PC susceptibility. METHODS: To identify candidates of functional or causative variants, we performed re-sequencing and fine mapping of this region. The variants in the 3 UTR of NKX3.1 were further screened by RNA stability assay. The variants in the intron, the 5 UTR or the upstream of NKX3.1 were screened by electrophoretic mobility shift assay. The differences in the binding affinity to nuclear proteins between the alleles were confirmed by competition assays, and the nuclear protein that binds to this region was determined by supershift assays. The association between the alleles and transcriptional activities were assessed by reporter assays. NKX3.1 expression in relation to the alleles were examined in vivo by quantative real time PCR and allele specific transcript quantification assays using cDNA from normal prostate tissues. RESULTS: We identified 12 candidates of causative SNPs that were absolutely linked with each otherby re-sequencing and fine mapping of this region. Screening of these variants by RNA stability assay, electrophoretic mobility shift assay, and reporter assay indicated that rs11781886 in the 5 UTR of NKX3.1 displayed different binding affinity to nuclear proteins between the alleles, and that the transcriptional activity of the NKX3.1 promoter was significantly lower in the susceptible allele of this SNP. Sp1 was determined to be the transcription factor that binds to the susceptible G allele, but not to the nonsusceptible A allele. Allele-specific transcript quantification and quantitative PCR analyses showed that the expression of NKX3.1 transcript in the prostate was significantly lower in the subjects with the haplotype carrying the susceptible variant at rs11781886. CONCLUSIONS: These results suggest that the functional variant rs11781886 in the 5 UTR of NKX3.1 can affect its transcription by altering the binding affinity of a transcriptional factor Sp1, and results in PC susceptibility by lowering expression of NKX3.1 transcript in the prostate.