Tomonori Habuchi

Single nucleotide polymorphisms of microRNA-machinery genes modify the risk of renal cell carcinoma

Purpose: MicroRNAs (miRNA) are a class of small noncoding RNA molecules that have been implicated in a wide variety of basic cellular functions through posttranscriptional regulations on their target genes. Compelling evidence has shown that miRNAs are involved in cancer initiation and progression. We hypothesized that genetic variations of the miRNA machinery genes could be associated with the risk of renal cell carcinoma. Experimental Design: We genotyped 40 single nucleotide polymorphisms (SNP) from 11 miRNA processing genes (DROSHA, DGCR8, XPO5, RAN, DICER1, TARBP2, AGO1, AGO2, GEMIN3, GEMIN4, HIWI) and 15 miRNA genes in 279 Caucasian patients with renal cell carcinoma and 278 matched controls. Results: We found that two SNPs in the GEMIN4 gene were significantly associated with altered renal cell carcinoma risks. The variant-containing genotypes of Asn929Asp and Cys1033Arg exhibited significantly reduced risks, with odds ratios (OR) of 0.67 [95% confidence interval (95% CI), 0.47-0.96] and 0.68 (95% CI, 0.47-0.98), respectively. Haplotype analysis showed that a common haplotype of GEMIN4 was associated with a significant reduction in the risk of renal cell carcinoma (OR, 0.66; 95% CI, 0.45-0.97). We also conducted a combined unfavorable genotype analysis including five promising SNPs showing at least a borderline significant risk association. Compared with the low-risk reference group with one unfavorable genotype, the median-risk and high-risk groups exhibited a 1.55-fold (95% CI, 0.96-2.50) and a 2.49-fold (95% CI, 1.58-3.91) increased risk of renal cell carcinoma, respectively (P for trend < 0.001). Conclusions: Our results suggested that genetic polymorphisms of the miRNA-machinery genes may affect renal cell carcinoma susceptibility individually and jointly.

Metachronous multifocal development of urothelial cancers by intraluminal seeding

To investigate the clonal origin of multifocal urothelial tumours, we analysed the p53 tumour-suppressor gene in 3 cases with bladder tumours developing after treatment for a renal pelvic or ureteral tumour and in 1 case with a ureteral tumour after treatment for a bladder tumour. For each case, identical p53 gene mutations were detected in all primary and recurrent tumours. The results suggest that heterotopic recurrence by intraluminal seeding from the original tumour is common in urothelial cancer. The data also support the view that multifocal urothelial tumours are derived from a single progenitor cell.

Somatic mutation of PTEN in bladder carcinoma

SummaryThe tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (≥ pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23.

Mutation of the 9q34 gene TSC1 in sporadic bladder cancer

Deletions involving chromosome 9 occur in more than 50% of human bladder cancers of all grades and stages. Most involve loss of the whole chromosome or of an entire chromosome arm but some small deletions are found which can be used to define critical regions which may contain tumour suppressor genes. We have localized such a critical region of deletion at 9q34 between the markers D9S149 and D9S66, an interval which contains the Tuberous Sclerosis gene TSC1. Single strand conformation polymorphism (SSCP) and sequence analysis of TSC1 in bladder tumours and cell lines with 9q34 loss of heterozygosity (LOH) has identified five mutations in retained TSC1 alleles. Our results support the hypothesis that TSC1 can act as a bladder tumour suppressor gene.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O‐glycans

The O‐glycan branching enzyme, core2 β‐1,6‐N‐acetylglucosaminyltransferase (C2GnT), forms O‐glycans containing an N‐acetylglucosamine branch connected to N‐acetylgalactosamine (core2 O‐glycans) on cell‐surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT‐expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT‐expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK‐activating receptor, NKG2D, by its tumour‐associated ligand, Major histocompatibility complex class I‐related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT‐expressing bladder tumour cells, poly‐N‐acetyllactosamine was present on core2 O‐glycans on MICA, and galectin‐3 bound the NKG2D‐binding site of MICA through this poly‐N‐acetyllactosamine. Galectin‐3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT‐expressing bladder tumour cells, resulting in tumour metastasis.

Hyperplasia and Carcinomas in Pten-Deficient Mice and Reduced PTEN Protein in Human Bladder Cancer Patients

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePten(flox/flox) (FPten(flox/flox)) mice]. Histologic examination revealed that all FPten(flox/flox) mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPten(flox/flox) mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPten(flox/flox) mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPten(flox/flox) urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms.

Increased risk of prostate cancer associated with AA genotype of cyclin D1 gene A870G polymorphism

CCND1 mRNA is alternatively spliced to produce 2 transcripts, and the splicing pattern may be modulated by a frequent A870G single‐nucleotide polymorphism within the conserved splice donor site of exon 4. Several studies have suggested a significant association between the CCND1 genotype and onset or progression of various cancers. To investigate the correlation of the polymorphism with genetic susceptibility to PCa and its disease status, we examined the polymorphism in 214 cases of PCa, 234 cases of BPH and 254 male controls. The CCND1 A allele was more frequently observed in the PCa group (p = 0.015) and the BPH group (p = 0.018) than the control group. Men with the AA genotype had an increased risk of PCa (aOR = 1.93, 95% CI 1.13–3.30, p = 0.016) and BPH (aOR = 1.84, 95% CI 1.09–3.09, p = 0.023) compared to those with the GG genotype. No significant association was observed when men with the AG genotype were compared to those with the GG genotype (PCa: aOR = 1.00, 95% CI 0.65–1.54, BPH: aOR = 0.91, 95% CI 0.60–1.39). The risk of PCa associated with the AA genotype appeared to be stronger in men aged 73 years or younger (aOR = 2.89, 95% CI 1.38–6.01, p = 0.005), whereas no association was found in men older than 73 years (aOR = 1.02, 95% CI 0.44–2.34). No significant difference in genotype frequency was found among patients with low‐, intermediate‐ and high‐grade tumors (p = 0.730) or between patients with localized and metastatic PCa (p = 0.679). However, in patients with high‐grade or metastatic PCa, a significantly increased risk associated with the AA genotype compared to controls was observed, while no significant results were found in those with low/intermediate or localized PCa. The A allele of the CCND1 A870G polymorphism was recessively associated with susceptibility to PCa and BPH in a Japanese population, giving a 2‐fold increased risk of PCa and BPH in men with the AA genotype compared to those with the GG genotype. Although the risk of PCa associated with the AA genotype appeared to contribute especially to men aged 73 years or younger and the A allele may be associated with disease status of PCa, these conjectures require validation in future studies on a larger number of subjects.

Impact of IGF-I and CYP19 Gene Polymorphisms on the Survival of Patients With Metastatic Prostate Cancer

PURPOSE The prognosis of metastatic prostate cancer significantly differs among individuals. While various clinical and biochemical prognostic factors for survival have been suggested, the progression and response to treatment of those patients may also be defined by host genetic factors. In this study, we evaluated genetic polymorphisms as prognostic predictors of metastatic prostate cancer. PATIENTS AND METHODS One hundred eleven prostate cancer patients with bone metastasis at the diagnosis were enrolled in this study. Thirteen genetic polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism or an automated sequencer with a genotyping software. RESULTS Among the polymorphisms, the long allele (over 18 [CA] repeats) of insulin-like growth factor-I (IGF-I) and the long allele (over seven [TTTA] repeats) of cytochrome P450 (CYP) 19 were significantly associated with a worse cancer-specific survival (P = .016 and .025 by logrank test, respectively). The presence of the long allele of either the IGF-I or CYP19 polymorphisms was an independent risk factor for death (P = .019 or .026, respectively). Furthermore, the presence of the long allele of both the IGF-I and CYP19 polymorphisms was a stronger predictor for survival (P = .001). CONCLUSION The prognosis of metastatic prostate cancer patients is suggested to be influenced by intrinsic genetic factors. The IGF-I (CA) repeat and CYP19 (TTTA) repeat polymorphisms may be novel predictors in prostate cancer patients with bone metastasis at the diagnosis.

Clinical and genetic risk factors for posttransplant diabetes mellitus in adult renal transplant recipients treated with tacrolimus.

Background. The present study investigated the incidence of posttransplant diabetes mellitus (PTDM) and calculated the risk of developing PTDM under a tacrolimus-based immunosuppression based on clinical characteristics, tacrolimus pharmacokinetics, and genetic polymorphisms related to tacrolimus pharmacokinetics or diabetes mellitus. Methods. Seventy nondiabetic adult kidney recipients were studied. Patients with continuous high plasma glucose levels, over 6.5 mg/dl of hemoglobin A1c, or requiring insulin and/or oral antidiabetic agents for more than 3 months after transplantation 6 months postoperatively were diagnosed as having PTDM. Twelve genomic polymorphisms were assessed. Results. Six months after transplantation, 10 recipients (14.3%) developed PTDM. Positive risk factors were age (P=0.019) and body mass index (P=0.038). There were no significant differences in acute rejection rate, total steroid doses, tacrolimus pharmacokinetics or its related to genetic polymorphisms between the two groups. The frequency of PTDM was significantly higher in patients with the vitamin D receptor (VDR) TaqI t allele than in those with the TT genotype (P=0.013). On multivariate analysis, age over 50 years (odds ratio 9.28, P=0.003) and the presence of the VDR TaqI t allele (odds ratio 7.05, P=0.048) were correlated with the development of PTDM. Conclusion. The incidence of PTDM was 14.3% in our cohort. Age over 50 years was a risk factor. The presence of the VDR TaqI t allele may also be a risk factor for PTDM, suggesting that genotyping of diabetes-related polymorphisms is a possible method of predicting a patients risk for developing PTDM and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals.

Laparoscopic Radical Nephroureterectomy: A Multicenter Analysis in Japan

BACKGROUND Laparoscopic nephroureterectomy (LNUx) is prevalent in Japan and throughout the world, but long-term outcome data remain limited. OBJECTIVE To understand the present state of LNUx in Japan, we conducted a multicenter analysis of clinical outcome and long-term cancer control for patients who underwent the procedure. DESIGN, SETTING, AND PARTICIPANTS Between January 1995 and December 2005, 1003 patients with urothelial cancer in the upper urinary tract were treated with LNUx at 51 institutions in Japan, and patient data were collected retrospectively. MEASUREMENTS Patient profiles were gathered and analyzed for survival, intravesical recurrence, and risk factors influencing them. RESULTS AND LIMITATIONS Median operative time was 320 min. Median bleeding volume was 232 ml. Complications occurred in 93 cases (9.3%) intraoperatively and in 107 cases (10.7%) postoperatively. Overall survival rate was 70% at 5 yr. Grade 3, pT3 or pT4, multifocal tumor, lymph-node metastasis, and previous or coexistent bladder tumor were independent risk factors for overall survival. Intravesical recurrence rate was 43% at 5 yr. Intravesical recurrence occurred more frequently in males, in patients with multifocal tumors, in patients with previous or coexistent bladder tumors, and in patients who underwent the hand-assisted approach. CONCLUSIONS Our report represents the largest multicenter analysis of LNUx reported to date. Male sex and the use of the hand-assisted approach were shown for the first time to be risk factors for recurrence-free survival and intravesical recurrence. To further analyze the effectiveness of LNUx, a long-term outcome comparison with risk stratification must be made between LNUx and open nephroureterectomy.