Naotaka Nishiyama

Genome-wide DNA methylation profiles in urothelial carcinomas and urothelia at the precancerous stage

To clarify genome‐wide DNA methylation profiles during multistage urothelial carcinogenesis, bacterial artificial chromosome (BAC) array‐based methylated CpG island amplification (BAMCA) was performed in 18 normal urothelia obtained from patients without urothelial carcinomas (UCs) (C), 17 noncancerous urothelia obtained from patients with UCs (N), and 40 UCs. DNA hypo‐ and hypermethylation on multiple BAC clones was observed even in N compared to C. Principal component analysis revealed progressive DNA methylation alterations from C to N, and to UCs. DNA methylation profiles in N obtained from patients with invasive UCs were inherited by the invasive UCs themselves, that is DNA methylation alterations in N were correlated with the development of more malignant UCs. The combination of DNA methylation status on 83 BAC clones selected by Wilcoxon test was able to completely discriminate N from C, and diagnose N as having a high risk of carcinogenesis, with 100% sensitivity and specificity. The combination of DNA methylation status on 20 BAC clones selected by Wilcoxon test was able to completely discriminate patients who suffered from recurrence after surgery from patients who did not. The combination of DNA methylation status for 11 BAC clones selected by Wilcoxon test was able to completely discriminate patients with UCs of the renal pelvis or ureter who suffered from intravesical metachronous UC development from patients who did not. Genome‐wide alterations of DNA methylation may participate in urothelial carcinogenesis from the precancerous stage to UC, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication. (Cancer Sci 2009)

Clinicopathological Analysis of Patients with Non-muscle-invasive Bladder Cancer: Prognostic Value and Clinical Reliability of the 2004 WHO Classification System

OBJECTIVE The aim of this study was to clarify the prognostic value and clinical reliability of the 2004 World Health Organization classification system of non-muscle-invasive bladder cancer. METHODS Between January 1995 and November 2010, 153 patients were diagnosed with non-muscle-invasive bladder cancer. We used a substage system that discerns T1-microinvasive (T1m, 42 patients) and T1-extensive-invasive (T1e, 37 patients) cancers. RESULTS There were 2 (1.3%), 89 (58.2%) and 62 (40.5%) cases of Grade 1-3 urothelial carcinoma, respectively, on the basis of the 1973 World Health Organization classification system. Of these, 37 (24.2%) and 116 (75.8%) were graded as low and high on the basis of the 2004 World Health Organization classification system. All of the cases with progression (15 patients) were diagnosed as high grade at the time of primary transurethral resection of the bladder tumor. Based on the Kaplan-Meier analysis, the 2004 World Health Organization classification system accurately predicted tumor recurrence (P = 0.029) and progression (P = 0.031). The 5-year recurrence-free survival rates in patients with low-grade and high-grade tumors were 68.7 and 47.1%, and the 5-year progression-free survival rates were 100 and 89.0%, respectively. In the high-grade T1 cases, the substage (T1m or T1e) was a significant predictor of tumor recurrence (P = 0.001) and progression (P = 0.020). CONCLUSIONS The 2004 World Health Organization classification system accurately predicts the risk of recurrence in primary non-muscle-invasive bladder cancer cases and has the same accuracy when predicting the risk of progression as the 1973 World Health Organization classification. Furthermore, the substaging system for high-grade T1 tumors is useful in predicting both recurrence and progression.

Construction of Predictive Models for Cancer-specific Survival of Patients with Non-muscle-invasive Bladder Cancer Treated with Bacillus Calmette-Guerin: Results from a Multicenter Retrospective Study

OBJECTIVE The aims of this study were to clarify the prognostic factors and to validate the bacillus Calmette-Guérin failure classification advocated by Nieder et al. in patients with non-muscle-invasive bladder cancer who had intravesical recurrence after bacillus Calmette-Guérin therapy. METHODS Data from 402 patients who received intravesical bacillus Calmette-Guérin therapy between January 1990 and November 2011 were collected from 10 institutes. Among these patients, 187 with bacillus Calmette-Guérin failure were analyzed for this study. RESULTS Twenty-nine patients (15.5%) were diagnosed with progression at the first recurrence after bacillus Calmette-Guérin therapy. Eighteen (62.1%) of them died of bladder cancer. A total of 158 patients were diagnosed with non-muscle-invasive bladder cancer at the first recurrence after bacillus Calmette-Guérin therapy. Of them, 23 (14.6%) underwent radical cystectomy. No patients who underwent radical cystectomy died of bladder cancer during the follow-up. On multivariate analysis of the 135 patients with bladder preservation, the independent prognostic factors for cancer-specific survival were age (≥70 [P = 0.002]), tumor size (≥3 cm [P = 0.015]) and the Nieder classification (bacillus Calmette-Guérin refractory [P < 0.001]). In a subgroup analysis, the estimated 5-year cancer-specific survival rates in the groups with no positive, one positive and two to three positive factors were 100, 93.4 and 56.8%, respectively (P < 0.001). CONCLUSIONS Patients with stage progression at the first recurrence after bacillus Calmette-Guérin therapy had poor prognoses. Three prognostic factors for predicting survival were identified and used to categorize patients with non-muscle-invasive bladder cancer treated with bacillus Calmette-Guérin into three risk groups based on the number of prognostic factors in each one.

Copy number alterations in urothelial carcinomas: Their clinicopathological significance and correlation with DNA methylation alterations

The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Array comparative genomic hybridization analysis using a 244K oligonucleotide array was performed on 49 samples of UC tissue. Losses of 2q33.3–q37.3, 4p15.2–q13.1 and 5q13.3–q35.3 and gains of 7p11.2–q11.23 and 20q13.12–q13.2 were correlated with higher histological grade, and gain of 7p21.2–p21.12 was correlated with deeper invasion. Losses of 6q14.1–q27 and 17p13.3–q11.1 and gains of 19q13.12–q13.2 and 20q13.12–q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2–p12.1 and gain of 3q26.32–q29 were correlated with vascular involvement. Losses of 5q14.1–q23.1, 6q14.1–q27, 8p22–p21.3, 11q13.5–q14.1 and 15q11.2–q22.2 and gains of 7p11.2–q11.22 and 19q13.12–q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p32.2–p31.3, 10q11.23–q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands and genome-wide DNA hypo- and hypermethylation were accumulated in clusters A, B1 and B2, respectively. Tumor-related genes that may encode therapeutic targets and/or indicators useful for the diagnosis and prognostication of UCs should be explored in the above regions. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity of UCs.

Simultaneous laparoscopic descending colectomy and nephroureterectomy for descending colon carcinoma and left ureteral carcinoma: Report of a case

To our knowledge, there is no case report of the synchronous resection of colon and ureteral carcinomas by laparoscopy, because of the rareness of this combination and the technical difficulties involved. We report a case of simultaneous descending colon and left ureteral carcinomas, both of which were judged to be relatively early stage carcinoma, which we resected successfully laparoscopically. The patient, a 65-year-old man, recovered uneventfully and was discharged on postoperative day 8. For simultaneous abdominal primary malignancies, laparoscopic surgery should be considered proactively if the procedure is technically feasible and judged to be curative.

Comparison of laparoscopic, hand-assisted, and open surgical nephroureterectomy.

Background and Objectives: The aim of this study was to compare oncologic outcomes after laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, and open nephroureterectomy for upper urinary tract urothelial cancer. Methods: Between April 1995 and August 2010, 189 patients underwent laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, or open nephroureterectomy for upper urinary tract urothelial cancer. Of these patients, 110 with no previous or concurrent bladder cancer or any metastatic disease were included in this study. Cancer-specific survival, recurrence-free survival, and intravesical recurrence-free survival rates were analyzed by the Kaplan-Meier method and compared with the log-rank test. The median follow-up period for the cohort was 70 months (range, 6–192 months). Results: The 3 groups were well matched for tumor stage, grade, and the presence of lymphovascular invasion and concomitant carcinoma in situ. The estimated 5-year cancer-specific survival rates were 81.1%, 65.6%, and 65.2% for laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, and open nephroureterectomy, respectively (P = .4179). The estimated 5-year recurrence-free survival rates were 33.8%, 10.0%, and 41.2% for laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, and open nephroureterectomy, respectively (P = .0245). The estimated 5-year intravesical recurrence-free survival rates were 64.8%, 10.0%, and 76.2% for laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, and open nephroureterectomy, respectively (P < .0001). Conclusion: Although there was no significant difference in cancer-specific survival rate among the laparoscopic nephroureterectomy, hand-assisted laparoscopic nephroureterectomy, and open nephroureterectomy groups, hand-assisted laparoscopic nephroureterectomy may be inferior to laparoscopic nephroureterectomy or open nephroureterectomy with regard to recurrence-free survival and intravesical recurrence-free survival rates.

Epigenetic silencing of miR-200b is associated with cisplatin resistance in bladder cancer

In this study, we identified microRNAs (miRNAs) involved in cisplatin (CDDP) resistance in bladder cancer (BCa). After establishing CDDP-resistant BCa cell lines (T24RC and EJ138RC), TaqMan arrays revealed that members of the miR-200 family (miR-200b, miR-200a and miR-429) were downregulated in T24RC as compared to parental T24 cells. miR-200b was associated with CDDP sensitivity in BCa cells, and its downregulation was associated with CpG island hypermethylation. Pharmacological demethylation using 5-aza-2’-deoxycytidine restored miR-200b expression, and the combination of 5-aza-2’-deoxycytidine + CDDP strongly inhibited T24RC cell proliferation. Microarray analysis revealed that miR-200b + CDDP induced genes involved in CDDP sensitivity or cytotoxicity, including IGFBP3, ICAM1 and TNFSF10, in the resistant cells. Expression and DNA methylation of miR-200b were inversely associated in primary BCa, and low expression/high methylation was associated with poor overall survival. These results suggest downregulation of miR-200b is associated with CDDP resistance in BCa. Epigenetic silencing of miR-200b may be a marker of CDDP resistance and a useful therapeutic target for overcoming CDDP resistance in BCa.

MP98-02 A POTENTIAL ROLE OF ABERRANT DNA METHYLATION IN THE CHEMORESISTANCE IN BLADDER CANCER CELLS. DNA METHYLATION INHIBITORS COULD RE-SENSITIZE DRUG-RESISTANCE BLADDER CANCER CELLS.

INTRODUCTION AND OBJECTIVES: Aberrant DNA methylation is one of the well-known epigenetic changes in cancer, although its involvement in the chemoresistance remains to be elucidated. In this study, we aimed to unravel the roles played by DNA methylation in chemoresistance in bladder cancer (BCa). METHODS: We established gemcitabine (GEM)-resistant (T24RG and UMUC3-RG) or cisplatin (CDDP)-resistant (T24-RC and UNUC3-RC) BCa cell lines by continuously treating their parental cells. Genome-wide DNA methylation was assessed by Infinium HumanMethylation450 BeadChip (HM450). To assess the chromatin accessibilities, cells were treated with a CpG methyltransferase M.SssI, after which DNA methylation was analyzed by HM450. To evaluate whether treatment with epigenetic drugs could overcome the chemoresistance in BCa, cells were treated with a DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR) and/or a histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), after which they were treated with or without GEM or CDDP. RESULTS: HM450 assays revealed increased levels of methylation at a number of CpG sites in the resistant cells as compared the parental cells (730 sites in T24-RC and 3856 sites in UMUC3-RC, respectively), however, a number of CpG sites remained unmethylated but loss chromatin accessibility in the resistant cells (1391 sites in T24RG and 1322 sites in UMUC3-RG, respectively. In CDDP-resistant BCa cells, a combination treatment with 5-Aza-CdR and CDDP synergistically suppressed cell proliferation, suggesting that 5-Aza-CdR restored CDDP-sensitivity (Figure A, B). In contrast, 5-Aza-CdR didn’t show any growth inhibitory effects in GEM-resistant cells (Figure C, D). Moreover, a treatment with SAHA with or without 5-Aza-CdR also failed to restore GEM-resistance in BCa cells (Figure E, F). CONCLUSIONS: Our results suggest that epigenetic alteration may be one of key factors for drug resistance, drug resistance cells could be desensitized by DNA methylation inhibitors especially in the CDDP-resistance in BCa cells.

Upper tract urothelial carcinoma following intravesical bacillus Calmette-Guérin therapy for nonmuscle-invasive bladder cancer: Results from a multi-institutional retrospective study

OBJECTIVES The aim of this study was to clarify the prognostic indicators for upper tract urothelial carcinoma (UTUC) following intravesical bacillus Calmette-Guérin (BCG) therapy for nonmuscle-invasive bladder cancer (NMIBC). METHODS Data from 402 patients who received intravesical BCG therapy between January 1990 and November 2011 were collected from 10 institutes. The median follow-up interval from transurethral resection of the bladder tumor (TURBT) followed by BCG treatment was 50.0 months (IQR: 31.8-77.0). Of these patients, 186 (46.3%) had intravesical recurrence during the follow-up period after BCG therapy. RESULTS Thirty patients (7.5%) were diagnosed with UTUC after BCG therapy. The 10-year recurrence-free survival rates for UTUC (RFS-UTUC) was 87.5%. In univariate and multivariate analyses, the independent predicting factors for UTUC were intravesical recurrence (P = 0.016) and tumor morphology at TURBT before BCG (P = 0.045). The 10-year RFS-UTUC of patients with intravesical recurrence and others, were 80.6% and 95.0%, respectively. The 10-year RFS-UTUC of patients with papillary pedunculated tumors and nonpapillary or nonpedunculated were 96.1% and 84.6%, respectively. CONCLUSIONS The frequency of UTUC in patients with NMIBC after BCG therapy is not negligible. Two independent predicting factors (intravesical recurrence and nonpapillary nonpedunculated at TURBT before BCG) were identified for UTUC. These results might be useful to predict UTUC after BCG therapy for NMIBC.

Bacillus Calmette-Guérin may have clinical benefit for glandular or squamous differentiation in non-muscle invasive bladder cancer patients: retrospective multicenter study

Objectives To clarify the efficacy of intravesical Bacillus Calmette-Guérin (BCG) instillation for non-muscle invasive bladder (NMIBC) cancer with variant histology, especially glandular differentiation or squamous differentiation. Materials and methods From May 1991 through June 2016, 53 patients were diagnosed retrospectively as having NMIBC with variant histology. Among these patients, 47 NMIBC patients with squamous differentiation or glandular differentiation were analyzed for this study. The median follow-up interval from diagnosis of NMIBC with variant histology was 28.9 months (1.5-168.8). Results Of these patients, 38 (80.9%) and 9 (19.1%) were diagnosed as having glandular differentiation and squamous differentiation, respectively. Radical cystectomy was conducted for six (12.8%) immediately after the diagnosis of NMIBC with variant histology. Of the 41 patients with bladder preservation, 20 (48.8%), 3 (7.3%), 3 (7.3%) and 15 (36.6%) underwent BCG, THP, MMC and no additional treatment, respectively. There were significant differences between BCG and other treatments or no additional treatment for recurrence (P = 0.034), progression (P = 0.004) and cancer-specific survival (P = 0.014). Conclusion Overall, our results show that intravesical BCG instillation for variant histology in NMIBC leads to a better prognosis with regard to progression and cause-specific survival than other intravesical treatments or no additional treatment. BCG treatment may also have a clinical benefit for variant histology in non-muscle invasive bladder cancer patients.