Diagnostic markers of urothelial cancer based on DNA methylation analysis.

BACKGROUND: Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. METHODS: In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. RESULTS: Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. CONCLUSION: Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.

Salvage I seed implantation for prostate cancer with postradiation local recurrence.

INTRODUCTION: Although radiotherapy has been important in the therapy for localized prostate cancer, prostate-specific antigen failure may occur. This study evaluated the effects and side effects of (125)I low-dose-rate brachytherapy for patients with postradiation local failure. PATIENTS AND METHODS: 15 patients who received salvage brachytherapy were analyzed. A prescribed dose of 144 Gy was selected. Median follow-up calculated from the date of salvage brachytherapy was 33.0 months (range 6-51). RESULTS: 5 patients (33.3%) developed prostate-specific antigen failure. The biochemical relapse-free survival rate was 100% at 1 year, 91.7% at 2 years, and 60.2% at 3 years. All acute genitourinary and gastrointestinal adverse events were in grade 1-2 according to Common Terminology Criteria for Adverse Events version 3. As for late adverse events, 1 patient (6.7%) developed grade 3 hematuria at 17 months postsalvage. CONCLUSIONS: Although careful patient selection is needed, salvage (125)I prostate brachytherapy appears to provide good prostate cancer control with an acceptable rate of complications for patients with local recurrence of prostate cancer after initial radiotherapy.

Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer.

The fibroblast growth factor receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was >or=11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of 15 non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC.

1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a selective tyrosine kinase inhibitor of fibroblast growth factor receptor-3 (FGFR3), inhibits cell proliferation of bladder cancer carrying the FGFR3 gene mutation along with up-regulation of p27/Kip1 and G1/G0 arrest.

Activating mutation of the fibroblast growth factor receptor-3 (FGFR3) gene is known as a key molecular event in both oncogenesis and cell proliferation of low-grade noninvasive human bladder urothelial carcinoma (UC), which is characterized by frequent intravesical recurrence. In this study, we investigated the antitumor potentiality of 1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a small-molecule FGFR3-selective tyrosine kinase inhibitor (TKI), as a therapeutic modality using eight UC cell lines. In our in vitro cell proliferation assay, PD173074 suppressed cell proliferation remarkably in two cell lines, namely, UM-UC-14 and MGHU3, which expressed mutated FGFR3 protein. In contrast, the other six cell lines expressing wild-type FGFR3 or without FGFR3 expression were resistant to PD173074 treatment. Cell cycle analysis revealed the growth inhibitory effect of PD173074 was associated with arrest at G(1)-S transition in a dose-depending manner. Furthermore, we observed an inverse relationship between Ki-67 and p27/Kip1 expression after PD173074 treatment, suggesting that up-regulation of p27 recruited UC cells harboring activating FGFR3 mutations in G(1) that was analogous with the other receptor TKIs acting on the epidermal growth factor receptors. In the mouse xenograft models using subcutaneously transplanted UM-UC-14 and MGHU3, orally administered PD173074 suppressed tumor growth and induced apoptotic changes comparable with the results of our in vitro assay. These findings elucidated the effectiveness of molecular targeted approach for bladder UC harboring FGFR3 mutations and the potential utility to decrease the intravesical recurrence of nonmuscle invasive bladder UC after transurethral surgical resection.

Salvage Radiotherapy Versus Hormone Therapy for Prostate-specific Antigen Failure After Radical Prostatectomy: A Randomised, Multicentre, Open-label, Phase 3 Trial (JCOG0401)†.

BACKGROUND: No standard therapy has been established for localised prostate cancer patients with prostate-specific antigen (PSA) failure after radical prostatectomy (RP). OBJECTIVE: To determine whether radiotherapy ± hormone therapy is superior to hormone therapy alone in such patients. DESIGN, SETTING, AND PARTICIPANTS: This study is a multicentre, randomised, open-label, phase 3 trial. Patients with localised prostate cancer whose PSA concentrations had decreased to <0.1 ng/ml after RP, and then increased to 0.4-1.0 ng/ml, were randomised to the salvage hormone therapy (SHT) group (80 mg bicalutamide [BCL] followed by luteinising hormone-releasing hormone agonist in case of BCL failure) or the salvage radiation therapy (SRT) ± SHT group (64.8 Gy of SRT followed by the same regimen as in the SHT group in case of SRT failure). From May 2004 to May 2011, 210 patients (105 in each arm) were registered, with the median follow-up being 5.5 yr. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was time to treatment failure (TTF) of BCL. RESULTS AND LIMITATIONS: TTF of BCL was significantly longer in the SRT ± SHT group (8.6 yr) than in the SHT group (5.6 yr; hazard ratio 0.56, 90% confidence interval [0.40-0.77]; one-sided p = 0.001). Thirty-two of 102 patients (31%) in the SRT ± SHT group did not have SRT treatment failure. However, clinical relapse-free survival and overall survival did not differ between the arms. The most frequent grade 3-4 adverse event was erectile dysfunction (83 patients [80%] in the SHT group vs. 76 [74%] in the SRT ± SHT group). Limitations include the short follow-up periods and surrogate endpoint setting to allow definitive conclusions. CONCLUSIONS: Initial SRT prolongs TTF of BCL in patients with post-RP PSA failure, indicating that SRT ± SHT is more beneficial than SHT alone. PATIENT SUMMARY: Patients who have prostate-specific antigen failure after radical prostatectomy benefit from salvage radiation therapy prior to salvage hormone therapy.

siRNA-mediated knockdown of the heme synthesis and degradation pathways: modulation of treatment effect of 5-aminolevulinic acid-based photodynamic therapy in urothelial cancer cell lines.

Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT)n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT)n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT)n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1.

[PROSTATE CANCER DETECTION ON INITIAL TRANSRECTAL PROSTATE BIOPSY IN RELATION TO PROSTATE-SPECIFIC ANTIGEN LEVELS].

(Objective) The prostate cancer detection rates on initial transrectal ultrasound guided prostate biopsies were investigated and the clinicopathological features of prostate cancer patients were examined. (Methods) A retrospective review was performed. From 2003 to 2015, 2,246 patients received initial prostate biopsy in Tochigi Cancer Center. Prostate cancer detection rates and clinicopathological features of the prostate cancer patients in relation to prostate-specific antigen levels were evaluated. (Results) Of the biopsies, 1,294 cases (57.6%) were diagnosed as prostate cancer. The prostate cancer detection rates were 27.8%, 39.8%, 53.6%, 67.4%, 88.4%, 100% in the groups with PSA levels 0.0-3.0, 3.1-4.0, 4.1-10.0, 10.1-20.0, 20.1-100.0, >100.0 ng/ml, respectively. Of all 1,294 cases in prostate cancer, patients rates with the low risk were 0%, 30%, 22%, 0%, 0%, 0% in the groups with PSA levels 0.0-3.0, 3.1-4.0, 4.1-10.0, 10.1-20.0, 20.1-100.0, >100.0 ng/ml, respectively. (Conclusions) From the results of the prostate cancer detection rates and the low risk rates, it seems difficult to determine diagnosis and treatment of prostate cancer only with PSA values.

Pneumoscrotum: a rare manifestation of perforation associated with therapeutic colonoscopy.

Pneumoscrotum is uncommon and also rarely reported as a complication associated with colonic perforation. A case of colonic perforation in delayed fashion associated with EMR, revealed by pneumoscrotum, is reported and the associated literatures are reviewed. A 52-year-old male received piecemeal EMR for a laterally spreading tumor 35 mm in size in our hospital. He complained of enlargement of the scrotum and revisited our hospital the day after the procedure. A diagnosis of pneumoscrotum was made, and as most such cases have been reported to be associated with pneumoperitoneum, colonic perforation was suspected. Free air but no fluid collection was found by abdominal computed tomography, and delayed colonic perforation was diagnosed. However, as there were no clinical signs of peritoneal irritation, conservative treatment was administered and the patient recovered uneventfully. Pneumoscrotum could be a sign of colonic perforation after EMR, and treatment should be carefully chosen.

[Prognostic factors for PSA relapse of prostate cancer after endocrine therapy].

PURPOSE: Advanced prostate cancer responds well to endocrine therapy initially, but soon becomes refractory and has a poor prognosis. We analyzed the prognostic factors of prostate cancer responding well initially to endocrine therapy with lowering of serum prostate specific antigen (PSA) level but later showing PSA relapse. MATERIALS AND METHODS: In prostate cancer patients newly diagnosed from January 1992 to December 2004 at our institution, there were 93 patients in that the PSA level of 10 ng/ml or more before therapy initially dropped below 10 ng/ml by endocrine therapy, but showed PSA relapse thereafter. We investigated the relationship between clinical stage, pathological differentiation, initial PSA, duration between initiation of therapy and PSA nadir, the value of PSA nadir, duration between initiation of therapy and PSA relapse, PSA doubling time (PSA-DT) at relapse, PSA response three months after initiation of second line therapy and prognosis after PSA relapse. RESULTS: In Kaplan-Meier method, between all or some categories investigated showed significant difference in prognosis after PSA relapse. In multivariate analysis, the factors that significantly affected prognosis after PSA relapse were clinical stage, pathological differentiation, PSA nadir value, duration between initiation of therapy and PSA relapse and PSA response three months after initiation of second line therapy. CONCLUSION: We investigated the prognostic factors refractory to endocrine therapy. These results are useful in planning the therapy, and in explaining the status or future prospective of the disease to patients and families.

[PSA-related parameters in prostate cancer relapsed after endocrine therapy].

PURPOSE: Prostate cancer is generally controlled by endocrine therapy even in an advanced state, but relapse may occur in many cases. Generally, the prognosis of a relapsed case is poor, but the prognosis differs case by case. We experienced 74 cases of prostate cancer relapsed after effective endocrine therapy, and investigated the relationship between the PSA-related parameters, clinical stage and prognosis. PATIENTS AND METHODS: We investigated 74 prostate cancer patients whose PSA declined 10 ng/ml or lower by the treatment consisting of endocrine therapy, but relapsed later. Pre-treatment PSA, the value of PSA nadir, the period from the start of treatment to PSA nadir, the period from the start of treatment to relapse, PSA doubling time (PSA-DT) at relapse and PSA response to the second line therapy at relapse were calculated, and compared with the clinical stage and prognosis. The relationship between each PSA parameter and clinical stage was tested using the Kruskal-Wallis test and chi 2 test. Cancer-specific survival after relapse in stage D patients was calculated by the Kaplan-Meier method and differences in prognosis were tested using the Logrank test. RESULTS: Pre-treatment PSA was significantly (p < 0.01) high, while the period from the start of treatment to relapse (p < 0.05) and PSA-DT at relapse (p < 0.01) was significantly short as the stage progressed. According to PSA response to the second line therapy at relapse, the rate of CR + PR was significantly (p < 0.05) high in clinical stage B + C group compared to clinical stage D group. The prognosis after relapse was significantly poorer in patients with relapse within 10 months after start of treatment than in those with relapse later, and in patients whose PSA-DT at relapse was shorter than 2 months than in those with a longer PSA-DT. CONCLUSIONS: The period from the start of treatment to relapse, and PSA-DT at relapse were useful PSA-related parameters for predicting prognosis after relapse, and for determining the strategy of cancer therapy after relapse. Using these data, the physician can inform the family and the patient of the prognosis more accurately, so that they can adjust future plans.

Unique DNA methylation patterns distinguish noninvasive and invasive urothelial cancers and establish an epigenetic field defect in premalignant tissue.

Urothelial cancer (UC) develops along two different genetic pathways, resulting in noninvasive or invasive tumors. However, it is unknown whether there are also different epigenetic pathways in UC. UC is also characterized by a high rate of recurrence, and the presence of a field defect has been postulated. In this study, we compared the DNA methylation patterns between noninvasive and invasive UC and the DNA methylation patterns between normal-appearing urothelium from bladders with cancer and urothelium from cancer-free bladders. We used the Illumina GoldenGate methylation assay at 1,370 loci in 49 noninvasive urothelial tumors, 38 invasive tumors with matched normal-appearing urothelium, and urothelium from 12 age-matched UC-free patients. We found distinct patterns of hypomethylation in the noninvasive tumors and widespread hypermethylation in the invasive tumors, confirming that the two pathways differ epigenetically in addition to genetically. We also found that 12% of the loci were hypermethylated in apparently normal urothelium from bladders with cancer, indicating an epigenetic field defect. X-chromosome inactivation analysis indicated that this field defect did not result in clonal expansion but occurred independently across the urothelium of bladders with cancer. The hypomethylation present in noninvasive tumors may counterintuitively provide a biological explanation for the failure of these tumors to become invasive. In addition, an epithelium-wide epigenetic defect in bladders with cancer might contribute to a loss of epithelial integrity and create a permissible environment for tumors to arise.

Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping.

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis.

Curative surgery for local pelvic recurrence of rectal cancer.

BACKGROUND/AIMS: Local pelvic recurrence of rectal cancer after radical resection has been associated with morbidity and cancer-related death. This study retrospectively evaluated outcome following curative resection for rectal cancer recurring after surgery on the basis of prognosis, type of procedure and perioperative morbidity. METHODS: A total of 85 consecutive patients with local pelvic recurrence of rectal cancer were evaluated. Of these, 43 underwent microscopic curative surgery for local recurrence. Among the 43 patients, 23 underwent surgery alone and 17 received preoperative radiotherapy (40 Gy) (XRT group) in addition to the surgery. Of the 43 patients, 26 were asymptomatic. RESULTS: Curative resection was higher in the recurrences that were associated with implantation, incomplete surgical margin clearance, and intrapelvic lymph node metastasis than in other types of recurrence. With regard to surgical procedure, abdominoperineal resection (APR), with or without sacral resection, was standard following previous sphincter-preserving surgery, while total pelvic exenteration (TPE), with or without sacral resection, was common following previous APR. Local excision was not considered appropriate surgery. There was a high incidence of perioperative morbidity (64%) in patients receiving TPE. Re-recurrence was observed in 18 patients (50%) after curative surgery. After a follow-up of 2 years or more, the local re-recurrence rate was 28%. The overall 5-year survival rate for patients receiving curative resection was 39%, for patients in the XRT group, 51%, and for patients in the surgery-alone group, 24% (p = 0.07). The survival rate in 26 asymptomatic patients was higher than in 17 patients with symptoms, with 5-year survival rates of 62 and 23% (p < 0.05), respectively. The cumulative local control in the preoperative radiotherapy plus en bloc surgery group (XRT group) was significantly better than in the surgery-alone group (p < 0.01), and survival in the XRT group tended to be better than in surgery alone. CONCLUSIONS: These results suggest that careful patient selection according to the pattern of recurrence, area of invasion and presence of symptoms is important for successful curative surgery. Aggressive surgery with adjuvant therapy may lead to an improved salvage rate.