Hideyasu Matsuyama

Overexpression of Polo-Like Kinase 1 (PLK1) and Chromosomal Instability in Bladder Cancer

Polo-like kinase 1 (PLK1) participates in bipolar spindle formation and entry into mitosis. Chromosomal instability (CIN) is caused by abnormalities in spindle formation and chromosome segregation. In this study, we investigated the relationship of PLK1 overexpression to CIN, and compared the PLK1 status with clinicopathological parameters in 101 human urothelial carcinomas of the urinary bladder. Expression of PLK1 and the number of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9 and 17 spots that allowed estimation of CIN were evaluated by fluorescence in situhybridization, and DNA ploidy was assessed by laser scanning cytometry. Cancers with a large intercellular variation in centromere copy number were defined as CIN cancers.Tumors with PLK1 overexpression were associated more frequently with CIN (p < 0.0001), DNA aneuploidy (p = 0.0007) and centrosome amplification (p = 0.0013) than those without. Overexpression of PLK1 was significantly related to higher pathological grade (p = 0.0024), multiple tumors (p = 0.0241) and positive urine cytology (p = 0.0192). These data suggest that a high level expression of PLK1 confers tumor progression advantages to urothelial cancer cells, although other factors are also involved.

Centrosome Hyperamplification Predicts Progression and Tumor Recurrence in Bladder Cancer

Purpose: Recent studies have reported that centrosome hyperamplification (CH) is closely related to chromosomal instability in bladder cancer. In this study, we investigated whether CH could be used as a prognostic biomarker for patients with bladder cancer. Experimental Design: CH was evaluated by immunohistochemistry in 50 bladder cancers (≤pT1: 43; ≥pT2: 7). In addition, numerical aberrations of chromosomes 7, 9, and 17 and gain of 20q13, on which the Aurora-A gene is located, were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed. Preliminary experiments on eight bladder cancer cell lines found that six had over 5% of CH cells associated with a gain of 20q13 and overexpression of Aurora-A; therefore, CH-positive cases (CH+) were defined as those having over 5% of cells with ≥3 centrosomes per cell. Results: CH+, 20q13 gain, chromosomal instability, and DNA aneuploidy were detected in 30 (60%), 18 (36%), 22 (44%), and 19 (38%) patients, respectively. There were significant differences in tumor number, grade, recurrence, and progression between the CH+ and CH− groups. The later had significantly higher recurrence-free and progression-free survivals than the former (P = 0.0028 and P = 0.0070, respectively, log-rank test). Multivariate analysis revealed that CH+ was the strongest predictor for tumor recurrence in nonmuscle invasive (pTa and pT1) bladder cancer (hazard ratio, 1.882; 95% confidence interval, 1.161–3.325; P = 0.0094). Conclusions: Detection of CH may provide crucial prognostic information about tumor recurrence in bladder cancer.

Gain of 5p15.33 Is Associated with Progression of Bladder Cancer

Objective: To search for a biological marker to distinguish low-risk from high-risk bladder cancer indicating disease progression. Methods: The whole genome-wide copy numbers were screened in 18 patients with bladder cancer using array comparative genomic hybridization (CGH) consisting of 4,030 bacterial artificial chromosome clones. Results: Gain of 5p15.33, including TPPP (tubulin polymerization-promoting protein)and ZDHHC11 (zinc finger DHHC domain-containing protein 11) genes, was detected in 5 of 9 (55.6%) high-grade bladder cancers and no (0%; n = 9) low-grade bladder cancer. To confirm the preliminary data, 5p15.33 gain was studied by fluorescence in situhybridization (FISH) in 100 patients, and the results were compared with biological characteristics. In FISH analysis, gain of 5p15.33 was significantly correlated with higher histological grade (p < 0.0001) and advanced pathological stage (p = 0.0284). Tumors with a gain of 5p15.33 had a significantly higher progression-free survival rate than those without (p = 0.0006, log-rank test). Multivariate analysis revealed that gain of 5p15.33 was a predictor for disease progression in bladder cancer (hazard ratio: 1.887, 95% confidence interval: 1.215–2.968, p = 0.0050). Conclusion: These data suggest that gain of 5p15.33 (TPPP and ZDHHC11) may become a potential biomarker identifying high-risk patients with disease progression in bladder cancer.

Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology.

Varicocele is the most common and treatable cause of male infertility. Studies of a rat experimental left varicocele model and human testicular biopsy samples have shown the involvement of various factors in its pathophysiology. Among them, oxidative stress plays a major role in impairing spermatogenesis and sperm function. Therefore, in addition to palpation, scrotal ultrasonography and color Doppler ultrasound, evaluation of testicular oxidative stress (e.g. scrotal temperature is a surrogate parameter) is recommended to enable diagnosis and suitable treatment of varicocele. Varicocelectomy increases the fertilization, pregnancy and live birth rates, indicating improved sperm function; it is therefore important even in couples undergoing intracytoplasmic sperm injection. Routine sperm‐function tests are warranted to monitor the sperm quality after varicocelectomy and consequent improvement in the outcomes of assisted reproductive technology. Furthermore, the indications of varicocelectomy in assisted reproductive technology should be widened.

Single nucleotide polymorphisms in DNA repair genes might be prognostic factors in muscle-invasive bladder cancer patients treated with chemoradiotherapy.

DNA repair enzymes repair DNA damaged by platinum agents and ionising radiation. Single nucleotide polymorphisms (SNPs) in DNA repair genes modulate the repair capacity and might affect response and prognosis following platinum-based chemoradiotherapy (CRT). We investigated associations between the functional SNPs in DNA repair genes and response and survival in muscle-invasive bladder cancer patients treated with CRT to determine the predictive value of the SNPs in patient selection for bladder conservation therapy. The study group comprised 78 patients who underwent CRT for transitional cell carcinoma of the bladder. Single nucleotide polymorphisms in xeroderma pigmentosum complementation groups C (Lys939Gln, A/C), D (XPD; Lys751Gln, A/C), and G (Asp1104His, G/C), and X-ray repair cross-complementing groups 1 (XRCC1; Arg399Gln, G/A) and 3 (Thr241Met, T/C) genes were genotyped. Combined genotypes with at least one variant allele in XPD or XRCC1 were significantly associated with improved cancer-specific survival compared with remaining groups (P=0.009). In multivariate analysis, only the combined XPD and XRCC1 genotypes were independently associated with cancer-specific survival (P=0.04). The association was stronger in stage T3/T4 patients (P=0.0008). These results suggest that combined XPD and XRCC1 genotypes might be prognostic factors in muscle-invasive bladder cancer patients treated with CRT.

Biological Characteristics in Bladder Cancer Depend on the Type of Genetic Instability

Purpose: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. Experimental Design: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. Results: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. Conclusions: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter is associated with conventional renal cell carcinoma

Matrix metalloproteinase (MMP)‐1 is associated with tumor cell invasion and metastasis, and its promoter polymorphism has been shown to influence the transcriptional level. Our study explored the association between this polymorphism and renal cell carcinoma in a Japanese population. DNA was extracted from peripheral blood and normal tissue of 119 patients with conventional renal cell carcinoma (RCC) and from 210 age‐ and sex‐matched healthy volunteers. Genotyping was carried out using direct sequencing. In the MMP‐1 gene promoter polymorphism, it was demonstrated that the frequency of the 2G/2G genotype, which is associated with higher enzyme activity, was significantly higher in patients with RCC than in controls (p = 0.0015; OR = 2.082; 95% CI 1.317–3.293). When stratified for gender, only men showed a significant association of the polymorphism with RCC (p = 0.0028; OR = 2.307; 95% CI 1.333‐3.990). However, no significant association was observed between the 2G/2G genotype and clinicopathologic parameters including age, gender, tumor grade and pathologic stage. Our present data suggest that the MMP‐1 promoter polymorphism may be linked to susceptibility for conventional RCC.

Links between genetic and environmental factors and prostate cancer risk

Genetic polymorphisms and expression of steroid receptors may explain why some individuals are more at risk of developing prostate cancer. Some risk factors often discussed are androgen stimulation, and vitamin A and D deficiency. Long CAG‐repeats in exon 1 of the androgen receptor (AR) gene on the X chromosome seem to have a protective role against androgen overstimulation. Likewise, long vitamin D receptor alleles in the poly‐A tract may prevent vitamin D stimulation.

Human chorionic gonadotrophin treatment prior to microdissection testicular sperm extraction in non-obstructive azoospermia

BACKGROUND Despite the improved success rate of sperm retrieval by microdissection testicular sperm extraction (micro-TESE), methods to stimulate spermatogenesis in men with non-obstructive azoospermia (NOA) remain unexplored. The aim of this study was to evaluate the effects of hCG-based hormonal stimulation in men with NOA on the success of sperm retrieval by micro-TESE. METHODS Forty-eight men with NOA who had negative sperm retrieval results by the micro-TESE procedure were included. A second micro-TESE was subsequently performed on these men: 20 were not treated by any hormonal therapy, and 28 subjects received daily subcutaneous injections of hCG for 4-5 months prior to the second micro-TESE. Recombinant FSH was added if endogenous gonadotrophin levels decreased during the hCG stimulation. The sperm retrieval rate at the second micro-TESE; the levels of gonadotrophins, testosterone and estradiol; and the effects of hormonal therapy on testicular histology were evaluated. RESULTS Among the 28 men with hCG stimulation, 15 (54%) showed decreased LH and FSH levels (0.67 ± 0.10 and 0.96 ± 0.14 mIU, mean ± SEM, respectively) due to elevated serum testosterone (9.5 ng/dl). Sperm were obtained at the second micro-TESE from six men who had received hormonal therapy (21%), whereas no sperm were retrieved from untreated men (P < 0.05). Success at the second micro-TESE was more likely if histology at the first micro-TESE showed hypospermatogenesis. CONCLUSIONS The Leydig cells of the testis can respond positively to exogenous hCG even under hypergonadotropic conditions. HCG-based hormonal therapy prior to a second micro-TESE attempt is effective in men with hypospermatogenesis.

Two putative tumor suppressor genes on chromosome arm 8p may play different roles in prostate cancer

Although loss of heterozygosity (LOH) on the short arm of chromosome 8 has been frequently observed in human prostate cancer, the relationship between LOH and clinical background is poorly understood. Fluorescence in situ hybridization (FISH) was employed to evaluate the chromosomal deletion on 8p in 42 prostate cancers using a centromeric probe for chromosome 8, in combination with 4 cosmid probes spanning 8p12 to 8p22. Deletions for at least one locus on the 8p were observed in 29 (69.0%) tumors. The most frequently deleted regions were 8p22 (54.8%) and 8p21.3 (52.4%), in almost the same frequency. The second most frequently deleted region was 8p21.1-p21.2 (38.1%). Deletions of 8p22 and 8p21.3 significantly correlated with tumor grade (P=0.0034, Fishers exact probability test). A significantly higher frequency of the deletion on 8p21.1-p21.2 was observed in advanced prostate cancer (beyond capsular penetration or positive nodal metastases) than in localized prostate cancer (P=0.0033). In particular, deletion of 8p21.1-p21.2 was more frequently observed in the cases with lymph node metastases than without them (P=0.0029). No clinicopathological parameters had significant relation to deletions on 8p12. These results suggest that deletions on 8p22-p21.3 play an important role in tumor differentiation, while an 8p21.1-p21.2 deletion plays a role in the progression of prostate cancer.