Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer.

Little is known regarding the subclone evolution process in advanced bladder cancer, particularly with respect to the genomic alterations that lead to the development of metastatic lesions. In this project, we identify gene expression signatures associated with metastatic bladder cancer through mRNA expression profiling of RNA isolated from 33 primary bladder cancer and corresponding lymph node (LN) metastasis samples. Gene expression profiling (GEP) was performed on RNA isolated using the Illumina DASL platform. We identified the developmental transcription factor TCF21 as being significantly higher in primary bladder cancer compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were conducted in bladder cancer cell lines with high and low expression of TCF21, respectively. We also performed GEP in bladder cancer cell lines following TCF21 overexpression. We identified 2,390 genes differentially expressed in primary bladder cancer and corresponding LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of 1. Among those significantly altered, expression of TCF21 was higher in the primary tumor compared with LN metastasis. We validated this finding with qPCR and IHC on patient samples. Moreover, TCF21 expression was higher in luminal cell lines and knockdown of TCF21 increased invasion, tumor cell dissemination, and metastasis. In contrast, overexpression of TCF21 in highly metastatic basal bladder cancer cell lines decreased their invasive and metastatic potential. IMPLICATIONS: TCF21 is differentially overexpressed in primary bladder cancer compared with matched LN metastasis, with in vitro and in vivo studies demonstrating a metastasis suppressor function of this transcription factor.

Impact of High-risk Features and Effect of Neoadjuvant Chemotherapy in Urothelial Cancer Patients with Invasion into the Lamina Propria on Transurethral Resection in the Absence of Deep Muscle Invasion.

BACKGROUND: High-risk non-muscle-invasive bladder cancer (NMIBC) that invades into the lamina propria is frequently understaged and is associated with a risk of lymph node metastasis and death. OBJECTIVE: To identify high-risk features (HRFs) for NMIBC that may identify patients with poorer prognosis who may benefit from neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC). DESIGN, SETTING, AND PARTICIPANTS: We performed a single-center retrospective review of patients who underwent RC for NMIBC with invasion into the lamina propria between 1995 and 2013. HRFs included hydronephrosis, abnormal examination under anesthesia, lymphovascular invasion, or variant histology. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Pathology at RC, and overall (OS) and disease-specific (DSS) survival were evaluated and analyzed by Fisher's exact test, Student t test, Cox proportional hazards regression analysis, and the Kaplan-Meier method. RESULTS AND LIMITATIONS: We identified 336 patients with a median follow-up of 130 mo. Of these, 159 (47%) had no HRF, 140 (41.5%) had one HRF, and 37 (11%) had ≥2 HRFs. At RC, patients with ≥2 HRFs had a significantly higher rate of pathologic T stage upstaging and lymph node metastasis (p<0.05). Median OS was 139 mo for those with no HRF, 127 mo for those with one HRF, and 56 mo for those with ≥2 HRF (p=0.0057). HRFs are also associated with a decreased DSS (p=0.0009). Patients with ≥2 HRFs (11/37) who received NAC showed improved OS (21% vs 55% 5-yr OS, p=0.0353) and trended toward an improvement in DSS (25% vs 56% 5-yr OS, p=0.0716) compared with RC alone. CONCLUSIONS: The presence of ≥2 HRFs in NMIBC invading the lamina propria is associated with worse pathology at RC and a significant decrease in OS and DSS. NAC appears to provide benefit for these patients. Limitations include retrospective design and limited sample size. PATIENT SUMMARY: The presence of high-risk features in urothelial cancer with invasion into the lamina propria has a worse prognosis that may be mitigated by neoadjuvant chemotherapy.

Generation of patient-derived xenografts from fine needle aspirates or core needle biopsy.

BACKGROUND: Patient-derived xenografts have recently become a powerful tool for cancer research and may be used to guide personalized therapy. Thus far, patient-derived xenografts have been grown from tumor tissue obtained after operative resection; however, many cancer patients never undergo operative intervention for a variety of reasons. We hypothesized that xenograft tumors could be grown from smaller volumes of patient tissue, such as those obtained during diagnostic biopsies. METHODS: Surgical specimens were obtained after resection of primary or metastatic lesions of the following cancers: pancreatic carcinoma, non-small cell lung cancer, bladder (urothelial) carcinoma, and melanoma. At least 10 cases of each cancer were included in this study. To mimic clinical biopsies, small fragments of the surgical specimens were biopsied with a 22-gauge needle, and the needle contents were injected subcutaneously in immunocompromised mice. The tumor fragment from which the biopsy was taken was also implanted subcutaneously in the contralateral side of the same mouse as a control. RESULTS: Success rates of the traditional method of xenograft implantation ranged from 27.3%-70%. Success rates of the fine needle aspirate technique ranged from 0%-36.4%. An attempt to engraft a percutaneous core needle liver biopsy of a metastatic pancreatic adenocarcinoma also was successful. CONCLUSION: We have found that it is possible to engraft fine needle aspirates and core biopsies of solid tumors in order to generate patient-derived xenografts. This may open up xenografting to a wider cancer patient population than previously possible.

Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy.

Muscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered three molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPARγ and estrogen receptor transcription and were enriched with activating FGFR3 mutations and potential FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.

Refining patient selection for neoadjuvant chemotherapy before radical cystectomy.

PURPOSE: We evaluated the survival of patients with muscle invasive bladder cancer undergoing radical cystectomy without neoadjuvant chemotherapy to confirm the utility of existing clinical tools to identify low risk patients who could be treated with radical cystectomy alone and a high risk group most likely to benefit from neoadjuvant chemotherapy. MATERIALS AND METHODS: We identified patients with muscle invasive bladder cancer who underwent radical cystectomy without neoadjuvant chemotherapy at our institution between 2000 and 2010. Patients were considered high risk based on the clinical presence of hydroureteronephrosis, cT3b-T4a disease, and/or histological evidence of lymphovascular invasion, micropapillary or neuroendocrine features on transurethral resection. We evaluated survival (disease specific, progression-free and overall) and rate of pathological up staging. An independent cohort of patients from another institution was used to confirm our findings. RESULTS: We identified 98 high risk and 199 low risk patients eligible for analysis. High risk patients exhibited decreased 5-year overall survival (47.0% vs 64.8%) and decreased disease specific (64.3% vs 83.5%) and progression-free (62.0% vs 84.1%) survival probabilities compared to low risk patients (p <0.001). Survival outcomes were confirmed in the validation subset. On final pathology 49.2% of low risk patients had disease up staged. CONCLUSIONS: The 5-year disease specific survival of low risk patients was greater than 80%, supporting the distinction of high risk and low risk muscle invasive bladder cancer. The presence of high risk features identifies patients with a poor prognosis who are most likely to benefit from neoadjuvant chemotherapy, while many of those with low risk disease can undergo surgery up front with good expectations and avoid chemotherapy associated toxicity.

Use of fluorescence in situ hybridization to predict response to bacillus Calmette-Guérin therapy for bladder cancer: results of a prospective trial.

PURPOSE: No reliable methods currently exist to predict patient response to intravesical immunotherapy with bacillus Calmette-Guérin given after transurethral resection for high risk nonmuscle invasive bladder cancer. We initiated a prospective clinical trial to determine whether fluorescence in situ hybridization results during bacillus Calmette-Guérin immunotherapy can predict therapy failure. MATERIALS AND METHODS: Candidates for standard of care bacillus Calmette-Guérin were offered participation in a clinical trial. Fluorescence in situ hybridization was performed before bacillus Calmette-Guérin, and at 6 weeks, 3 months and 6 months during bacillus Calmette-Guérin therapy with maintenance. Cox proportional hazards regression was used to assess the relationship between fluorescence in situ hybridization results and tumor recurrence or progression. The Kaplan-Meier product limit method was used to estimate recurrence-free and progression-free survival. RESULTS: A total of 126 patients participated in the study. At a median followup of 24 months 31% of patients had recurrent tumors and 14% experienced disease progression. Patients who had positive fluorescence in situ hybridization results during bacillus Calmette-Guérin therapy were 3 to 5 times more likely than those who had negative fluorescence in situ hybridization results to experience recurrent tumors and 5 to 13 times more likely to have disease progression (p <0.01). The timing of positive fluorescence in situ hybridization results also affected outcomes. For example, patients with a negative fluorescence in situ hybridization result at baseline, 6 weeks and 3 months demonstrated an 8.3% recurrence rate compared to 48.1% for those with a positive result at all 3 points. CONCLUSIONS: Fluorescence in situ hybridization results can identify patients at risk for tumor recurrence and progression during bacillus Calmette-Guérin immunotherapy. This information may be used to counsel patients about alternative treatment strategies.