Hironobu Wakeda

Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein

DNA methylation, which is an important epigenetic event for transcriptional regulation, is regarded as a biomarker for cancer. A rapid and sensitive method for measuring DNA methylation levels in target genomic regions may enable early diagnosis of cancer. To detect DNA methylation levels conveniently, we developed a detection system for DNA methylation, designated as methylated DNA precipitation combined luciferase-fused zinc finger assay (MELZA), which uses methyl CpG-binding domain (MBD) and luciferase-fused zinc finger protein. This system comprises the following 3 steps: (1) MBD-based methylated DNA precipitation, (2) PCR amplification of the target genomic region, and (3) detection of the PCR product quantity by using luciferase-fused zinc finger protein. Using this system, we have accurately measured methylation levels of the androgen receptor gene promoter region in LNCaP, PC3, Du145, and whole blood cells. This system does not require bisulfite treatment, and all the steps can be automated. Therefore, it might be useful for measuring DNA methylation levels in clinical cancer diagnoses.

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure.

Detection of histone modification by chromatin immunoprecipitation combined zinc finger luciferase-based bioluminescence resonance energy transfer assay.

Epigenetic modification such as DNA methylation and histone modification have important roles in gene regulation. Epigenetic modification can be altered by environmental influences and are related to diseases. Therefore, epigenetic modifications may serve as biomarkers. In this study, we developed a convenient histone modification detection system by combining a chromatin immunoprecipitation (ChIP) and bioluminescence resonance energy transfer (BRET)-based homogeneous PCR product detection system using zinc finger fused to luciferase (ZF-luciferase) with DNA intercalating dye (ChIP-ZF-BRET assay). The ChIP-ZF-BRET assay comprises the following 3 steps: (1) ChIP, (2) PCR amplification of the target genomic region, which includes a zinc-finger recognition site, and (3) homogeneous detection of the PCR product by BRET using ZF-luciferase and fluorescent DNA intercalating dye. Using this system, we conveniently and accurately detected target histone modification at the androgen receptor gene promoter region in LNCaP and Du145 cells. The system can be applicable to DNA methylation detection using a methyl-CpG-binding domain protein or methylcytidine antibody instead of histone modification antibodies. Therefore, it may be useful and convenient for simultaneous detection of histone modification and DNA methylation in clinical diagnoses.

Do metastatic upper tract urothelial carcinoma and bladder carcinoma have similar clinical responses to systemic chemotherapy? A Japanese multi-institutional experience

OBJECTIVE There has been no clear evidence supporting similar chemo-responses for upper and lower urothelial carcinomas. METHODS We conducted a multicenter retrospective cohort study to analyze urothelial carcinoma patients who underwent systemic chemotherapy at 17 centers from 2004 to 2010. A total of 298 patients with either urothelial carcinoma of the bladder (N = 151) or upper tract urothelial carcinoma (N = 147) were included. Differences in tumor location (urothelial carcinoma of the bladder vs. upper tract urothelial carcinoma) were evaluated in relation to the patient backgrounds and clinical responses to systemic chemotherapy. RESULTS Overall 216 patients were treated with cisplatin-based chemo-regimens (gemcitabine and cisplatin in 92, or methotrexate, vinblastine, adriamycin and cisplatin/methotrexate, epirubicin and cisplatin in 124). Among 186 initially metastatic patients, the incidences of lung metastasis and liver metastasis were 39.2 and 34.1%, respectively, in upper tract urothelial carcinoma patients, and were significantly higher than those with urothelial carcinoma of the bladder (22.4% for lung; 8.4% for liver metastasis). Among 112 post-surgical recurrent/metastatic patients, age was significantly higher and estimated glomerular filtration rate at baseline was significantly lower in upper tract urothelial carcinoma patients than those with urothelial carcinoma of the bladder. No significant differences were observed in overall clinical response rates for systemic chemotherapy between urothelial carcinoma of the bladder (45.8%) and upper tract urothelial carcinoma (38%) in initially metastatic patients or between urothelial carcinoma of the bladder (43.2%) and upper tract urothelial carcinoma (44.1%) in post-surgical recurrent/metastatic patients. Tumor location was not independently associated with cancer-specific survival in either initially metastatic or post-surgical recurrent/metastatic urothelial carcinoma patients. CONCLUSIONS No significant difference was observed in response rates of urothelial carcinoma of the bladder and upper tract urothelial carcinoma to systemic chemotherapy, suggesting that a similar chemo-regimen can be applied to metastatic urothelial carcinoma patients regardless of tumor location (upper vs. lower).

Treatment of acute uncomplicated cystitis with faropenem for 3 days versus 7 days: multicentre, randomized, open-label, controlled trial

OBJECTIVES The increasing prevalence of resistant bacteria such as fluoroquinolone-resistant or extended-spectrum β-lactamase-producing strains in pathogens causing acute uncomplicated cystitis has been of concern in Japan. Faropenem sodium is a penem antimicrobial that demonstrates a wide antimicrobial spectrum against both aerobic and anaerobic bacteria. It is stable against a number of β-lactamases. METHODS We compared 3 and 7 day administration regimens of faropenem in a multicentre, randomized, open-label, controlled study. RESULTS In total, 200 female patients with cystitis were enrolled and randomized into 3 day (N = 97) or 7 day (N = 103) treatment groups. At the first visit, 161 bacterial strains were isolated from 154 participants, and Escherichia coli accounted for 73.9% (119/161) of bacterial strains. At 5-9 days after the completion of treatment, 73 and 81 patients from the 3 day and 7 day groups, respectively, were evaluated by intention-to-treat analysis; the microbiological efficacies were 58.9% eradication (43/73), 20.5% persistence (15/73) and 8.2% replaced (6/73), and 66.7% eradication (54/81), 6.2% persistence (5/81) and 7.4% replaced (6/81), respectively (P = 0.048). The clinical efficacies were 76.7% (56/73) and 80.2% (65/81), respectively (P = 0.695). Adverse events due to faropenem were reported in 9.5% of participants (19/200), and the most common adverse event was diarrhoea. CONCLUSIONS The 7 day regimen showed a superior rate of microbiological response. E. coli strains were in general susceptible to faropenem, including fluoroquinolone- and cephalosporin-resistant strains.

Demographics, management and treatment outcomes of benign and malignant retroperitoneal tumors in Japan

To show the demographics, type of treatment and clinical outcomes of patients with retroperitoneal tumors in Japan.

Superior vena cava syndrome due to metastasis from urothelial cancer: A case report and literature review

Superior vena cava (SVC) syndrome is caused by compression or obstruction of the SVC. We report here in a case of SVC syndrome due to lymph node metastasis from urothelial cancer to the mediastinum and lung. The origin of metastasis was determined by computed tomography (CT)-guided biopsy of metastases. After radiotherapy to the mediastinum with glucocorticoid failed, anticancer pharmacotherapy including paclitaxel, gemicitabine, and cisplatin proved effective and SVC syndrome resolved. But patient died from cerebral bleeding from newer brain metastases 10 months later.

A novel case presenting with an unusual ureteral diverticular lesion similar to adenomyomatous hyperplasia of the gallbladder

We present the first case of an unusual ureteral diverticular lesion demonstrating similarities to adenomyomatous hyperplasia of the gallbladder. A 68‐year‐old asymptomatic Japanese man with high prostate‐specific antigen levels was clinically evaluated. Left hydronephrosis and benign prostatic hyperplasia were detected. A bilateral retrograde pyelogram revealed that the upper and middle portions of the left ureter exhibited an irregular narrow lumen and some pooling of contrast material, which was compatible with ureteral pseudodiverticulosis. Although no malignant cells were seen on cytology, computed tomography detected a fusiform shaped lesion with a circumferential thick wall including multiple diverticulae. Left nephroureterectomy was performed because malignancy could not be ruled out. Pathology demonstrated that the ureteral lesion showed a localized thick wall consisting of multilocules and/or multicysts and a hyperplastic muscularis propria. The cysts were mostly seen in the muscularis propria or a deeper site. The inner layers of the cysts were lined with normal urothelium, and some cysts opened onto the mucosal surface, indicating that they were derived from invaginated mucosal epithelium. We believe that this lesion may be a novel form of diverticular disorder demonstrating similarities to adenomyomatous hyperplasia of the gallbladder.

CT evaluation of acupuncture needles inserted into sacral foramina

Objective To use CT scanning to evaluate the precision with which acupuncture needles can be inserted into sacral foramina to establish sacral nerve modulation by electroacupuncture. Methods The subjects were five adult women (mean age 71.6 years). These five cases were divided into two groups. In the first three subjects (group A) the intention was to insert acupuncture needles in the S3 and S4 foramina; in the remaining two subjects (group B) the intention was to insert acupuncture needles in the S2 and S3 foramina. Results CT scanning showed that in subject 1 of group A, the acupuncture needle intended for insertion in S3 was actually in the S4 foramen, and the acupuncture needle intended for insertion in S4 was actually distal to the sacral body. In subjects 2 and 3, the acupuncture needles were inserted accurately in the S3 and S4 foramina. In the three subjects who had acupuncture needles inserted in the S4 foramen, the tip of the acupuncture needle was an average distance of 6.0 mm from the rectum. The acupuncture needles inserted in subjects 4 and 5 of group B were inserted accurately into the S2 and S3 foramina. Conclusions Inserting acupuncture needles into the sacral foramina of S2 and S3 at an angle of about 60° has the potential to be used for sacral nerve modulation by repeated electroacupuncture stimulation. Needling may be less accurate in subjects with higher body mass index. Because of the potential risk of perforating the rectum with the needle, this technique must be used by specialists only. Trial Registration Number 2013-026

Pure Verrucous Carcinoma of the Penis: Two Cases

Verrucous carcinoma is a rare subtype of penile verruciform squamous cell carcinoma. However, in past literature, the diagnosis of verrucous carcinoma has been confused with giant condylomas, and papillary and warty carcinomas due to the absence of critical criteria in pathological diagnosis. We experienced two cases of penile verruciform tumor occurring in a 48-year-old Japanese male and an 88-year-old Japanese male. We performed partial penectomy without lymph node dissection after biopsy. Although we initially detected small palpablelymph node swelling in the latter case, unnecessary lymph node dissection could be avoided by pathological diagnosis using preoperative biopsy. The resected specimens were diagnosed in accordance with the World Health Organization (WHO) classification and current pathological reviews. Histopathological findings and immunohistochemical appearance were compatible with a diagnosis of verrucous carcinoma.