Depth of invasion to the bladder wall as a prognostic factor and its association with circulating cell-free DNA levels in patients with muscle-invasive bladder cancer.

Background: Radical cystectomy (RC) is the standard surgical treatment for patients with muscle-invasive bladder cancer, but the prognosis is not favorable, and new prognostic factors need to be discovered. We investigated the potential of depth of invasion (DOI) as a prognostic factor in patients with muscle-invasive bladder cancer who underwent RC. Furthermore, we examined the association between preoperative levels of circulating cell-free DNA and DOI. Materials and methods: We retrospectively reviewed patients who underwent RC between January 2007 and December 2017; those who received neoadjuvant chemotherapy were excluded. Depth of invasion was measured using hematoxylin-eosin-stained RC specimens. Results: Of the 121 patients selected, 41 (33.9%) were eligible for analysis. The median follow-up period was 14 months and mean DOI was 17 mm (range, 2-75 mm). Long DOI (>17 mm) was significantly associated with shorter progression-free survival (hazard ratio, 14.5; 95% confidence interval, 3.9-53.97, p < 0.0001) and cancer-specific survival (hazard ratio, 18.97; 95% confidence interval, 4.04-88.99, p = 0.0002) compared with short DOI. Multivariate analysis revealed that DOI was an independent risk factor for cancer-specific survival. The levels of circulating cell-free DNA were significantly higher in patients with a longer DOI than in those with short DOI (65 vs. 20 ng/mL, respectively; p = 0.028). Conclusions: Depth of invasion predicted with levels of circulating cell-free DNA and thus could be a useful prognostic factor.

Diagnostic and Prognostic Roles of Urine Nectin-2 and Nectin-4 in Human Bladder Cancer.

The clinical utility of urine nectins in bladder cancer (BCa) is unclear. We investigated the potential diagnostic and prognostic values of urine Nectin-2 and Nectin-4. Levels of urine Nectin-2, Nectin-4, and NMP-22 were quantified using an enzyme-linked immunosorbent assay in 122 patients with BCa, consisting of 78 with non-muscle-invasive BCa (NMIBC) and 44 with muscle-invasive BCa (MIBC), and ten healthy controls. Tumor nectin expression in MIBC was evaluated with immunohistochemical staining of transurethral resection specimens. The level of urine Nectin-4 (mean: 18.3 ng/mL) was much higher than that of urine Nectin-2 (mean: 0.40 ng/mL). The sensitivities of Nectin-2, Nectin-4, NMP-22, and cytology assays were 84%, 98%, 52%, and 47%, respectively; their specificities were 40%, 80%, 100%, and 100%, respectively. Both urine Nectin-2 and Nectin-4, though not NMP-22, were found to be significantly more sensitive than cytology. A four-titer grouping based on levels of urine Nectin-2/Nectin-4 (low/high, high/high, low/low, and high/low) showed a high capability for discriminating between NMIBC and MIBC. Neither urine Nectin-2 nor Nectin-4 levels had a significant prognostic value in NMIBC or MIBC. Urine levels correlated with tumor expression and serum levels in the Nectin-4 analysis, but not in the Nectin-2 analysis. Urine nectins are potential diagnostic biomarkers for BCa.

Probiotics enhances anti-tumor immune response induced by gemcitabine plus cisplatin chemotherapy for urothelial cancer.

Chemotherapy drugs, such as gemcitabine and cisplatin (GC), are frequently administered to patients with advanced urothelial carcinoma, however the influence of the gut microbiota on their action is unclear. Thus, we investigated the effects of GC on the gut microbiome and determined whether oral supplementation with a probiotics mixture of Lactobacillus casei Shirota and Bifidobacterium breve enhanced the anti-tumor immune response. After subcutaneous inoculation with MBT2 murine bladder cancer cells, syngenic C3H mice were randomly allocated into eight groups. The gut microbiome cluster pattern was altered in both the GC and oral probiotics groups (p = 0.025). Both tumor-bearing conditions (no treatment) and GC chemotherapy influenced Pseudoclostridium, Robinsoniella, Merdimonas, and Phocea in the gut. Furthermore, comparison of the GC-treated and GC + probiotics groups revealed an association of four methyltransferase family enzymes and two short-change fatty acid-related enzymes with oral probiotics use. A significant difference in tumor volume was observed between the GC and GC + probiotics groups at week 2 of treatment. Additionally, decreased recruitment of cancer-associated fibroblasts and regulatory T cells, and activation of CD8+ T cells and dendritic cells were observed in the tumor microenvironment. Our findings reveal the positive effects of a probiotics mixture of Lactobacillus and Bifidobacterium in enhancing anti-tumor effects through the gut-tumor immune response axis. Future clinical trials are needed to evaluate the full benefits of this novel supplement with oral probiotics in patients with advanced urothelial carcinoma.

Tumor expression of Nectin-1-4 and its clinical implication in muscle invasive bladder cancer: An intra-patient variability of Nectin-4 expression.

There is limited evidence regarding the tumor expression of nectins and their clinical implications in muscle invasive bladder cancer. Herein, we evaluated expression of Nectins 1-4 in 64 patients with muscle invasive bladder cancer who underwent radical cystectomy using a histochemical scoring method (H-score; immunohistochemical staining intensity multiplied by the percentage of positive-staining cells). The cutoff values were defined based on the median H-scores. Of the 64 patients, 45 (70%) had residual tumors in radical cystectomy specimens, while 13 (20%) had lymph node metastasis. The median (interquartile range) H-scores of Nectin-1, - 2, - 3, and - 4 expression were 0 (0-10), 80 (30-180), 5 (0-30), and 100 (33-160), respectively. The Nectin-4 H-score of the neuroendocrine variant was significantly lower than that of pure urothelial carcinoma (P = 0.015). Post-neoadjuvant chemotherapy pathological response (

Association between urine 6-sulfatoxy-melatonin level and intravesical Bacillus Calmette-Guerin treatment-induced sleep quality deterioration in patients with non-muscle invasive bladder cancer.

PURPOSE: The level of 6-sulfatoxy-melatonin (SaMT), a metabolite of melatonin, in first-void morning urine reflects blood melatonin levels from the previous night. We investigated the association between urine SaMT and sleep quality deterioration in patients with non-muscle invasive bladder cancer (NMIBC) treated with intravesical Bacillus Calmette-Guerin induction therapy (iBCG). METHODS: We enrolled 51 patients who received iBCG once weekly for 6 or 8 weeks. Patient-reported outcomes were assessed with questionnaires including the International Prostate Symptom Score (IPSS) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (QLQC30). Questionnaires were completed before (baseline), during, at completion, and 1 and 3 months after iBCG. Melatonin and SaMT levels at baseline were measured in serum and first-void morning urine samples, respectively. RESULTS: Based on changes in the QLQC30 insomnia subscale, 28 (55%) patients experienced sleep quality deterioration (deterioration group). Urine SaMT values in the deterioration group were lower than those in the non-deterioration group (P = 0.0015; 7.5 vs 15.4 ng/mg creatinine, respectively). Nocturia scores in the non-deterioration group decreased over time, while those of the deterioration group remained high after completion of iBCG. A binary logistic regression analysis revealed that low urine SaMT levels (≤ 9.6 ng/mg creatinine), high IPSS nocturia scores at baseline, and high IPSS storage subscores at baseline were associated with BCG-induced sleep quality deterioration. CONCLUSIONS: This study confirmed the association among urine SaMT levels, nocturia, and sleep disturbance in patients with NMIBC who receive iBCG. We should be aware of treatment-induced impairments to aid in appropriate decision-making.

Hexylaminolevulinate-mediated fluorescent urine cytology with a novel automated detection technology for screening and surveillance of bladder cancer.

OBJECTIVES: To evaluate the diagnostic performance of fluorescent voided urine cytology (FVUC) using a novel automated detection technology to screen for primary bladder cancer and for surveillance of recurrent bladder tumour. PATIENTS AND METHODS: We created a rapid, objective, automated, and high-throughput detection device for hexylaminolevulinate-mediated FVUC, named the cellular fluorescence analysis unit-II (CFAU-II). Two different cohorts were used in this study: (i) screening test for primary bladder cancer (165 patients with bladder cancer and 52 controls), and (ii) surveillance test for detecting intravesical recurrent tumour (192 patients with treated non-muscle-invasive bladder cancer and 15 with post-nephroureterectomy upper urinary tract cancer). Voided urine samples were subjected to urine analysis, conventional VUC (cVUC), and FVUC. Diagnostic performance was compared between cVUC, FVUC, and a combination of the two. RESULTS: A total of 614 urine samples were successfully collected, processed, and analysed. Comparative analysis of the screening test cohort demonstrated that the overall sensitivity of FVUC (63%, P < 0.001) and combination testing (72%, P < 0.001) was significantly higher than that of cVUC (29%). FVUC was found to be superior in most of the subgroups, especially in low-grade, Ta, and small tumours. Analysis of the surveillance test cohort showed that combination testing achieved a sensitivity of 82% and a negative predictive value of 98%, whereas those of cVUC were 39% and 96%, respectively. According to the pathological finding of recurrent tumours presenting false-negative result in the FVUC, the majority of the overlooked recurrent diseases were Ta low-grade tumours. Logistic regression analysis suggested an association between the risk of false-positive results and high density of urine white blood cells and alkaluria. CONCLUSION: The present findings clearly demonstrate that FVUC using the newly developed automation technology has superior sensitivity to cVUC for both screening for primary bladder cancer and recurrent tumour detection. It is essential to confirm the clinical usefulness of this method via further large-scale studies, in addition to ensuring its affordability and availability.

Integrative assessment of clinicopathological parameters and the expression of PD-L1, PD-L2 and PD-1 in tumor cells of retroperitoneal sarcoma.

Evidence is limited regarding the immunologic profile and immune microenvironment of soft tissue sarcoma subtypes. The aim of the present study was to describe the clinical significance and prognostic implications of PD-L1, PD-L2, and PD-1 in patients with retroperitoneal sarcoma (RSar). In this retrospective, multicenter, collaborative study, medical charts were reviewed and the immunohistochemical staining results of resected tissue specimens from 51 patients with RSar were examined. Immunohistochemical staining was performed with primary antibodies against PD-L1, PD-L2, PD-1, and Ki-67. The correlations between the baseline clinical parameters and expression levels of the four molecules in sarcoma cells were evaluated, and their prognostic values after tumor resection were assessed. Dedifferentiated liposarcoma (41%), leiomyosarcoma (20%), and undifferentiated pleomorphic sarcoma (16%) were the three major types identified. Dedifferentiated liposarcoma and leiomyosarcoma showed higher levels of PD-L1 expression than did other sarcomas. The Spearman correlation analysis revealed that baseline serum lactate dehydrogenase levels were moderately and positively correlated with PD-L1 (P=0.02, r=0.41) and PD-L2 (P=0.006, r=0.47) expression. The median recurrence-free and disease-specific survival was 58 and 16 months, respectively, during the 29-month median follow-up after surgery. On univariate analysis, a higher expression level of PD-1 was associated with a higher risk of recurrence, whereas multivariate analyses revealed that independent predictors of recurrence-free and disease-specific survival indicated a high expression of Ki-67 (P=0.03; hazard ratio, 2.29 vs. low expression) and prognostic stage IIIB (P=0.04; hazard ratio, 5.11 vs. stage I-II), respectively. Findings of the current study provide novel insights about the prognostic value of PD-L1, PD-L2, and PD-1 expression in RSar. Serum lactate dehydrogenase levels constitute a potential predictor of PD-L1 and PD-L2 expression levels in RSar. Further investigations are needed to determine the immunologic landscape of RSar and provide a foundation for therapeutic intervention using immune checkpoint inhibitors.

Supplementary Oral Anamorelin Mitigates Anorexia and Skeletal Muscle Atrophy Induced by Gemcitabine Plus Cisplatin Systemic Chemotherapy in a Mouse Model.

Chemotherapy-induced adverse effects can reduce the relative dose intensity and quality of life. In this study, we investigated the potential benefit of supplementary anamorelin and 5-aminolevulinic acid (5-ALA) as preventive interventions against a gemcitabine and cisplatin (GC) combination chemotherapy-induced adverse effects in a mouse model. Non-cancer-bearing C3H mice were randomly allocated as follows and treated for 2 weeks-(1) non-treated control, (2) oral anamorelin alone, (3) oral 5-ALA alone, (4) gemcitabine and cisplatin (GC) chemotherapy, (5) GC plus anamorelin, and (6) GC plus 5-ALA. GC chemotherapy significantly decreased body weight, food intake, skeletal muscle mass and induced severe gastric mucositis, which resulted in decreased ghrelin production and blood ghrelin level. The supplementation of oral anamorelin to GC chemotherapy successfully mitigated decrease of food intake during the treatment period and body weight loss at day 8. In addition, analysis of the resected muscles and stomach revealed that anamorelin suppressed chemotherapy-induced skeletal muscle atrophy by mediating the downregulation of forkhead box protein O-1 (FOXO1)/atrogin-1 signaling and gastric damage. Our findings suggest the preventive effect of anamorelin against GC combination chemotherapy, which was selected for patients with some types of advanced malignancies in clinical practice.

Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder.

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.

A Potential Application of Dynamic Contrast-Enhanced Magnetic Resonance Imaging Combined with Photodynamic Diagnosis for the Detection of Bladder Carcinoma in Situ: Toward the Future 'MRI-PDD Fusion TURBT'.

The detection of carcinoma in situ (CIS) is essential for the management of high-risk non-muscle invasive bladder cancers. Here, we focused on dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) combined with photodynamic diagnosis (PDD) for the detection of CIS. A total of 45 patients undergoing pre-surgical DCE-MRI and PDD-assisted endoscopic surgery accompanied by biopsies of the eight segmentations were analyzed. Immunohistochemical analysis of the biopsies revealed hypervascularity of CIS lesions, a cause of strong submucosal contrast-enhancement. It was found that 56 (16.2%) of 344 biopsies had pathologically proven CIS. In the DCE-MRI, the overall sensitivity and specificity for detecting CIS were 48.2% and 81.9%, respectively. We set out two different combinations of PDD and DCE-MRI for detecting CIS. Combination 1 was positive when either the PDD or DCE-MRI were test-positive. Combination 2 was positive only when both PDD and DCE-MRI were test-positive. The overall sensitivity of combinations 1 and 2 were 75.0% and 37.5%, respectively (McNemar test, vs PDD alone; p = 0.041 and p < 0.001, respectively). However, the specificity was 74.0% and 91.7%, respectively (vs PDD alone; both p < 0.001). Our future goal is to establish 'MRI-PDD fusion transurethral resction of the bladder tumor (TURBT), which could be an effective therapeutic and diagnostic approach in the clinical management of high-risk disease.

Supplementary granulocyte macrophage colony-stimulating factor to chemotherapy and programmed death-ligand 1 blockade decreases local recurrence after surgery in bladder cancer.

Despite advances and refinements in surgery and perioperative chemotherapy, there are still unmet medical needs with respect to radical cystectomy for muscle-invasive bladder cancer (MIBC). We investigated the potential benefit of supplementary granulocyte macrophage colony-stimulating factor (GM-CSF) to chemoimmunotherapy with programmed cell death protein-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis blockade and standard neoadjuvant chemotherapy in bladder cancer. We inoculated 2 × 105 MBT2 cells s.c. in C3H mice to create a syngeneic animal model of local recurrence (LR). When the tumor diameter reached 12 mm, the mice were allocated randomly as follows: (i) non-treated control (vehicle only); (ii) anti-mPD-L1 monotherapy; (iii) mGM-CSF monotherapy; (iv) anti-mPD-L1 plus mGM-CSF; (v) gemcitabine and cisplatin (GC); (vi) GC plus anti-mPD-L1; (vii) GC plus mGM-CSF; and (viii) GC plus anti-mPD-L1 plus mGM-CSF. After completing 2-week neoadjuvant therapy, tumors were resected for resection margin evaluation and immunohistochemical staining and blood was collected for flow cytometry and ELISA. Operative wounds were sutured, and the operative site was monitored to detect LR. Addition of anti-mPD-L1 and mGM-CSF to neoadjuvant GC chemotherapy enhanced the antitumor effect and reduced positive resection margins (50% vs 12.5%). Combination of GC, anti-mPD-L1, and mGM-CSF resulted in longer LR-free survival and cancer-specific survival compared to those in other groups. These effects involved an immunotherapy-related decrease in oncological properties such as tumor invasion capacity and epithelial-mesenchymal transition. mGM-CSF significantly decreased the accumulation of myeloid-derived suppressor cells in both the blood and tumor microenvironment and blood interleukin-6 levels. Supplementary GM-CSF to neoadjuvant GC plus PD-L1 blockade could decrease LR after radical surgery by immune modulation in the blood and tumor microenvironment.

Clinical Impact of the Increase in Immunosuppressive Cell-Related Gene Expression in Urine Sediment during Intravesical Bacillus Calmette-Guérin.

BACKGROUND: The aim of this study is to evaluate the clinical impact of intravesical Bacillus Calmette-Guérin (BCG)-induced changes in blood/urinary immune markers. METHODS: Time-course changes in blood/urinary clinical parameters and mRNA expression of 13 genes in urine sediment taken eight times during the treatment course of intravesical BCG (before, every 2 weeks for 8 weeks, and after) in 24 patients with non-muscle invasive bladder cancer. The genes examined include cellular markers of four immune checkpoint proteins (PD-L1, PD-L2, PD-1, and CTLA-4), immunosuppressive cells (regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells), pan-T lymphocytes, B lymphocytes, and neutrophils. RESULTS: Significant transient increase in gene expression was observed for PD-L1, PD-1, FOXP3, and CD204 at 6-8 doses of BCG. The patients were stratified into two groups depending on the number of genes with increased mRNA expression. Fourteen (58%) had 0-1 genes upregulated, while 10 (42%) had 2-4 genes with increased expression. No patient in the 0-1 group experienced recurrence, while 70% of patients in the 2-4 group experienced recurrence (p value = 0.037, hazard ratio = 5.93). CONCLUSIONS: Our findings suggested that increases in more than one of PD-L1, PD-1, FOXP3, and CD204, expression in the urine sediments was associated with resistance to BCG treatment.

Mycoplasma genitalium Infection and Chronic Inflammation in Human Prostate Cancer: Detection Using Prostatectomy and Needle Biopsy Specimens.

The evidence of association between sexually transmitted infection and prostatic inflammation in human prostate cancer (PCa) is limited. Here, we sought to examine the potential association of prostatic infection with the inflammatory environment and prostate carcinogenesis. We screened surgical and biopsy specimens from 45 patients with PCa against a panel of sexually transmitted infection-related organisms using polymerase chain reaction and examined the severity of intraprostatic inflammation by pathologic examination. Among tested organisms, the rate of Mycoplasma genitalium (Mg) infection was significantly different between the prostate cancer cohort and benign prostate hyperplasia (BPH) cohort (P = 0.03). Mg infection in the surgical specimens was associated with younger patients. The rate of extensive disease (pT2c⁻3b) was higher in Mg-positive patients than in Mg-negative patients (P = 0.027). No significant correlation was observed between Mg infection status and the grade of intraprostatic inflammation. The detection sensitivity of biopsy specimens was 61% for Mg and 60% for human papillomavirus (HPV)18, indicating possible clinical application of this material. A comprehensive understanding of the correlation between the urogenital microbiome and inflammation would facilitate the development of strategies for PCa prevention. Further studies are required to explore its clinical utility in recommendations of early re-biopsy, close follow-up, and treatment by antibiotics.

Dual benefit of supplementary oral 5-aminolevulinic acid to pelvic radiotherapy in a syngenic prostate cancer model.

BACKGROUND: Normal tissue damage caused by radiotherapy remains the largest dose-limiting factor in radiotherapy for cancer. Therefore, the aim of this study was to investigate the supplementary oral 5-aminolevulinic acid (ALA) to standard radiation therapy as a novel radioprotective approach that would not compromise the antitumor effect of radiation in normal rectal and bladder mucosa in a syngenic prostate cancer (PCa) model. METHODS: To evaluate the radiosensitizing effect of ALA in vitro, clonogenic survival assays were performed in DU145, PC3, and MyC-CaP cell lines. To evaluate the effect of ALA in vivo a single dose (25 Gy) of radiation with or without ALA was given to healthy mice. Next, a syngenic PCa model of MyC-CaP cells in FVB mice was created, and multiple doses (12 Gy total) of radiation were administered to the mouse pelvic area with or without ALA administration. Resected tumors, recta, and urinary bladders were immunostained with antibodies against Ki-67, γ-H2AX, CD204, and uroplakin-III. Total RNA levels in recta and urinary bladders were analyzed via RT2 Profiler polymerase chain reaction (PCR) arrays related to "Stress & Toxicity PathwayFinder," "Mitochondria," and "Inflammasomes." RESULTS: The addition of in vitro single or in vivo repeated administration of exogenous ALA acted as a radiosensitizer for PCa cells. Rectal toxicity was characterized by histological changes including loss of surface epithelium, fibrosis, severe DNA damage, and the aggregation of M2 macrophages. Urinary bladder toxicity was characterized by bladder wall thickening and urothelium denuding. The higher dose (300 mg/kg/day) of ALA exerted a better radioprotective profile than the lower dose (30 mg/kg/day) in normal recta and urinary bladders. Out of the 252 genes tested, 35 (13.4%) were detected as relevant genes which may be involved in the radioprotective role of ALA administration. These included interleukin-1a (IL-1a), IL-1b, IL-12, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL3, and NLRP3. CONCLUSIONS: Our study provides novel and comprehensive insights into the dual benefits including radiosensitizing PCa tumor tissues and radioprotection of normal pelvic organs from radiation therapy. Knowledge of the underlying mechanism will facilitate the search for optimal treatment parameters for supplemental oral ALA during radiotherapy for PCa.

Regulatory T Cells and Tumor-Associated Macrophages in the Tumor Microenvironment in Non-Muscle Invasive Bladder Cancer Treated with Intravesical Bacille Calmette-Guérin: A Long-Term Follow-Up Study of a Japanese Cohort.

The clinical significance of regulatory T cells (Treg) and tumor-associated macrophages (TAM) in the tumor microenvironment of human bladder cancer remains unclear. The aim of this study is to explore their relevance to oncological features in non-muscle invasive bladder cancer (NMIBC). We carried out immunohistochemical analysis of forkhead box P3 (FOXP3, Treg maker), CD204 (TAM marker), and interleukin-6 (IL6) using surgical specimens obtained from 154 NMIBC patients. The Treg and TAM counts surrounding the cancer lesion and IL6-positive cancer cell counts were evaluated against clinicopathological variables. We focused on the ability of the Treg and TAM counts around the cancer lesion to predict outcomes after adjuvant intravesical Bacille Calmette-Guérin (BCG) treatment. High Treg counts were associated with female patients, older age, T1 category, and high tumor grade. TAM count was significantly correlated with Treg count and with IL6-positive cancer cell count. In our analysis of 71 patients treated with BCG, high counts of Treg and TAM were associated with shorter recurrence-free survival, and the former was an independent predictor of recurrence. Poor response to intravesical BCG was associated with Treg and TAM in the tumor microenvironment. Disrupting the immune network can be a supplementary therapeutic approach for NMIBC patients receiving intravesical BCG.

Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder.

Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder.

A multiplex urinary immunoassay for bladder cancer detection: analysis of a Japanese cohort.

BACKGROUND: Bladder cancer (BCa) is among the most commonly diagnosed malignancies worldwide, and due the high rate of post-operative disease recurrence, it is one of the most prevalent in many countries. The development of non-invasive molecular assays that can accurately detect and monitor BCa would be a major advance, benefiting both patients and healthcare systems. We have previously identified a urinary protein biomarker panel that is being developed for application in at-risk patient cohorts. Here, we investigated the potential utility of the multiplex assay in a Japanese cohort. METHODS: The Japanese study cohort collected from urology clinics at two institutions was comprised of a total of 288 subjects. The protein biomarker panel (IL8, MMP9, MMP10, ANG, APOE, SDC1, A1AT, PAI1, CA9, VEGFA) was monitored in voided urine samples collected prior to cystoscopy using a custom multiplex ELISA assay. The diagnostic performance of the biomarker panel was assessed using receiver operator curves, predictive modeling and descriptive statistics. RESULTS: Urinary biomarker concentrations were significantly elevated in cases versus controls, and in cases with high-grade and muscle-invasive tumors. The AUC for the 10-biomarker assay was 0.892 (95 % confidence interval 0.850-0.934), with an overall diagnostic sensitivity specificity of 0.85 and 0.81, respectively. A predictive model trained on the larger institutional cohort correctly identified 99 % of the cases from the second institution. CONCLUSIONS: Urinary levels of a 10-biomarker panel enabled discrimination of patients with BCa. The multiplex urinary diagnostic assay has the potential to be developed for the non-invasive detection of BCa in at-risk Japanese patients.

5-fluorouracil enhances the antitumor effect of sorafenib and sunitinib in a xenograft model of human renal cell carcinoma.

Sorafenib and sunitinib are multi-kinase inhibitors with antitumor activity in patients with advanced renal cell carcinoma (RCC). Several studies have evaluated the effect of sorafenib/sunitinib in combination with chemotherapeutic agents in different types of tumor. However, few studies have addressed the activity of fluorinated pyrimidine in combination with sorafenib/sunitinib. In this study, we examined the potential of combination therapy with 5FU and sorafenib/sunitinib in human RCC cell lines. Three human RCC cell lines, ACHN, Caki-1 and Caki-2, were used to assess sensitivity to 5-fluorouracil (5FU), sorafenib and sunitinib alone or in combination using an in vitro cell survival assay. Caki-2 cells demonstrated significantly higher resistance to 5FU and sorafenib as compared to ACHN and Caki-1. Additive antitumor effects of the combination therapy were observed in the in vitro study. There was a tendency for a positive correlation between the sensitivity to sunitinib and platelet-derived growth factor ß (PDGFR-ß) expression levels, which were examined by reverse transcription polymerase chain reaction. Caki-1 xenograft models were prepared by inoculating cells subcutaneously into nude mice, which were divided randomly into six groups: control, 5FU (8 mg/kg/day, intraperitoneally), sorafenib (15 mg/kg/day, orally), sunitinib (20 mg/kg/day, orally), and 5FU with sorafenib or sunitinib. The treatments were administered on 5 days each week, and tumor growth was monitored for 42 days following inoculation of cells. Synergistic antitumor effects of the combination therapy were observed in an in vivo study. The resected tumors were evaluated using the Ki-67 labeling index and microvessel density. Both the Ki-67 labeling index and microvessel density were decreased in tumors treated with the combination therapy compared to those treated with sorafenib/sunitinib alone. These findings suggest that the combination therapy of 5FU with sorafenib/sunitinib may be an effective therapeutic modality for advanced RCC patients.