Masahiko Hosaka

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: Relationship to clinicopathological parameters

To delineate more precisely the somatic von Hippel‐Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA‐SSCP followed by sequencing, methylation‐specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear‐cell or variant‐type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear‐cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear‐cell RCCs. The VHL gene therefore seems to be inactivated in a two‐hit manner by intragenic mutation or hypermethylation plus allelic loss in clear‐cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7–19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear‐cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (≦ 55 years) than that in older (≧ 56 years) patients. These data suggested that the inactivation of the VHL tumor‐suppressor gene is a specific genetic change in clear‐cell RCC, and that it may occur at an early or first step in the clear‐cell tumorigenic pathway rather than as a late event.

Telomerase Activity in Primary Prostate Cancer

PURPOSE Telomerase activity has been detected in a wide variety of human tumor types. We analyzed the telomerase activity in association with the acquisition of prostate cancer. MATERIALS AND METHODS Telomerase activity in prostate tissues was examined by PCR-based telomeric repeat amplification protocol (TRAP) assay. RESULTS Among 31 primary prostate cancers, 28 tissue samples (90%) displayed telomerase activity. The relative level of telomerase activity was associated with the pathological differentiation. High levels of telomerase activity were more frequently detected in poorly differentiated prostate cancer. None of the 10 samples taken from prostates with benign prostatic hyperplasia (BPH) or normal prostates expressed telomerase activity. In another 10 BPH samples obtained from prostate tissue adjacent to cancerous tissue, one of 10 samples (10%) showed weak telomerase activity. Furthermore, we investigated this activity in human prostate cancer cell lines (PC-3, LNCaP, and DU145) and all showed very high activity compared to normal human tissue samples. Four lymph nodes and one bone metastasis also exhibited extremely high telomerase activity. CONCLUSIONS The present results indicate that telomerase activity might be a marker for detecting malignancy of the prostate and evaluating the malignant potential of prostate cancer.

TRANSARTERIAL EMBOLIZATION AND ABLATION OF RENAL ARTERIOVENOUS MALFORMATIONS: EFFICACY AND DAMAGES IN 30 PATIENTS WITH LONG-TERM FOLLOWUP

PURPOSE We evaluate the long-term efficacy and side effects of transarterial embolization and ablation for renal arteriovenous malformations. MATERIALS AND METHODS A total of 30 patients with cirsoid arteriovenous malformations causing massive hematuria underwent 34 procedures of embolization or ablation. We confirmed the ratios of occluded arteriovenous malformation areas on angiograms and those of infarcted areas on computerized tomography. All patients were followed for 4.1 to 15.0 years (mean 8.0 +/- 2.8) after the initial procedures. RESULTS Hematuria ceased in all patients after the initial procedures, including partial embolization or ablation of the arteriovenous malformations in 8. Massive hematuria recurred in 4 patients, who had undergone absorbable gelatin sponge (2), embolization, combined alcohol and subselective absorbable gelatin sponge embolization (1) and polyvinyl alcohol particles embolization (1). In these 4 cases total ablation of the arteriovenous malformations with alcohol was successful. In 29 patients, including aforementioned 4, no hematuria recurred after 5 years following total or partial ablation with alcohol. Large nontarget embolization with reflux of subselectively infused absorbable gelatin sponge caused a nonfunctioning kidney in 1 patient. The remaining 33 procedures caused 6.3 to 48.0% (mean 15.7 +/- 6.9%) areas of renal infarction. Polyvinyl alcohol embolization caused pulmonary embolism and renin dependent hypertension. CONCLUSIONS Partial or total transarterial ablation of arteriovenous malformations with alcohol proved effective for long-term cessation of hematuria. However, this procedure as well as transarterial embolization has the potential risk of nontarget infarction.

PTEN/MMAC1/TEP1 mutations in human primary renal-cell carcinomas and renal carcinoma cell lines.

Extensive allelotyping studies have implicated several tumor‐suppressor loci on chromosomes 3p, 5q, 6q, 8p, 9pq, 10q, 11q, 14q, 17p, 18q and 19p in human kidney tumorigenesis. The PTEN (also called MMAC1 and TEP1) gene, a candidate tumor suppressor located at chromosome 10q23.3, is mutated in a variety of sporadic malignancies as well as in patients with Cowden disease. To investigate the potential role of the PTEN gene in renal tumorigenesis, we searched for abnormalities of the gene in 68 primary renal‐cell carcinomas (RCCs) as well as in 17 renal carcinoma–derived cell lines, using DNA‐SSCP, sequencing and microsatellite analysis. Five of 68 (7.5%) primary RCCs exhibited intragenic mutations (3 missense, 1 deletion and 1 splice‐site), and 1 of 17 (5.9%) cell lines had an insertion mutation. Loss of heterozygosity of the PTEN gene occurred in 25% of primary RCCs, including the 3 cases with intragenic mutation and the 1 PTEN‐mutated cell line. Clinical and histopathological examinations revealed that 4 of the 5 primary tumors with PTEN mutation were high‐grade, advanced clear‐cell RCCs with distant metastases or renal vein tumor invasions, resulting in poor prognostic courses. The other was a low‐stage papillary/chromophilic RCC. Our data suggest that PTEN mutation is observed in a subset of RCCs and that, especially in clear‐cell RCCs, it occurs as a late‐stage event and may contribute to the invasive and/or metastatic tumor phenotype.

c-erbb-2 gene amplification as a prognostic marker in human bladder cancer

OBJECTIVES To evaluate c-erbB-2 gene amplification and its prognostic significance in transitional cell carcinoma of the bladder. METHODS Alterations in the gene copy number of c-erbB-2 were detected in 57 bladder tumor samples using a method based on the polymerase chain reaction. RESULTS Eighteen tumors (32%) showed gene amplification of c-erbB-2, which correlated with tumor grade and stage. A strong association of c-erbB-2 amplification with patient survival was also found. The amplification resulted in a significantly poorer prognosis among the patients with high-grade and/or invasive tumors. Multivariate analysis revealed that c-erbB-2 amplification and tumor grade were independent prognostic factors. CONCLUSIONS Our data indicate a possible role of the c-erbB-2 gene in the development of aggressive behavior in bladder tumors. Moreover, the use of c-erbB-2 gene amplification, together with tumor grade and stage, could provide an accurate basis for determining the prognosis of bladder cancer.

GnRH analog, leuprorelin acetate, promotes regeneration of rat spermatogenesis after severe chemical damage

Future fertility is a major concern for cancer patients who undergo intensive chemotherapy. There has been controversy about whether hormonal treatments may have protective effects against the severe spermatogenic damage caused by chemotherapy or irradiation. Recently, it has been proposed that gonadotrophin‐releasing hormone (GnRH) analogs administered after testicular damage stimulate the recovery of spermatogenesis. In this study, we have investigated the effects of GnRH agonist, leuprorelin, on the damage to spermatogenesis induced by busulfan.

Computerized tomographic ureteroscopy for diagnosing ureteral tumors.

PURPOSE Helical computerized tomography (CT) image acquisition has led to the availability of improved data sets for CT endoscopic imaging that represent virtual endoscopy using CT. We assessed the usefulness of CT ureteroscopic imaging for diagnosing ureteral tumors. MATERIALS AND METHODS A total of 16 patients with ureteral stenosis underwent surface rendering CT ureteroscopy after the intravenous administration of contrast material and furosemide. To distinguish ureteral tumors from ureteral strictures 2 observers blinded to other patient history and evaluation data independently and prospectively evaluated CT ureteroscopy with reformatted CT ureterograms in these 16 patients. CT ureteroscopic images were then correlated with surgical and pathological findings, which served as the gold standard. RESULTS Surgical and pathological findings in the 16 patients revealed 16 ureteral tumors, including carcinoma in 10 (carcinoma in situ in 1, fibroepithelial polyps in 2 and hyperplastic polypoids in 4), inflammatory intrinsic stricture in 2 and extrinsic stricture in 4 caused by retroperitoneal fibrosis in 2 and lymph node metastasis in 2. CT ureteroscopy correctly detected all lesions except 1 carcinoma in situ, 1 polypoid carcinoma and 1 hyperplastic polypoid. The sensitivity and specificity of CT ureteroscopy for detecting ureteral tumors and carcinoma were 81% and 100%, and 80% and 75%, respectively, when tumors without stalks were considered carcinoma. CONCLUSIONS CT ureteroscopy is useful for visualizing the complex morphology of ureteral tumors and distinguishing tumor from ureteral stricture.

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

In spite of the general recognition of von Hippel-Lindau (VHL) as a tumor suppressor gene, the physiological and pathological importance of VHL protein in cell growth regulation and tumorigenesis remains unclear. Here we show that in normal human renal proximal tubule epithelial cells (RPTEC), the steady-state amount of VHL protein is strictly regulated by cell density. The cellular VHL content is more than 100-fold higher in dense cultures than in sparse cultures. The increase in VHL protein at high cell density was also observed for NIH3T3 fibroblasts, suggesting the generality of the phenomenon. The growth rates of renal cell carcinoma cells lacking an intact VHL gene and their derivatives with wild-type or mutant VHL expression vector do not differ significantly when they are growing in log-phase. Importantly, however, there is a difference when they reach confluency: cells lacking wild-type VHL grew continuously, while cells expressing exogenous VHL protein showed relatively limited cell growth. Using an ecdysone-inducible VHL expressing cell line, we also show that the growth inhibition at high cell density can be released by attenuating the VHL expression. Taken together, we propose that VHL protein functions as a growth suppressor at high cell density, and this might be the basis of the tumor suppressor function of VHL.

Loss of Heterozygosity at the p53, RB, DCC and APC Tumor Suppressor Gene Loci in Human Bladder Cancer

PURPOSE Allelic losses within several tumor suppressor genes have been detected frequently in various types of human cancer. We investigated the roles and possible interactions of the tumor suppressor genes p53, Rb, DCC and APC in bladder cancer. MATERIALS AND METHODS Loss of heterozygosity (LOH) of these 4 genes was examined in 45 human bladder cancers by polymerase chain reaction and restriction fragment length polymorphism assay. RESULTS Of the evaluated cases, LOH was seen at P53 in 38%, at Rb in 22%, at DCC in 36% and at APC in 6% of tumors. Loss of heterozygosity at p53 and Rb was predominantly observed in high grade (grade 3) and/or invasive (T2 or greater) tumors, whereas LOH at DCC was present irrespective of tumor grade and stage. Allelic losses at either p53, Rb, DCC or APC were seen in 82% of high grade tumors, but in only 21% of low grade (grade 1 and 2) tumors (p<0.005). Similarly, 71% of invasive tumors had LOH at one or more loci compared with 20% of superficial (Ta and T1) tumors (p<0.005). Interestingly, p53-LOH and Rb-LOH were often observed simultaneously in the same tumor. CONCLUSIONS These results suggest that loss of the p53, Rb and/or DCC genes is involved in most of the late and some of the early steps of bladder carcinogenesis.

Differential expression of protooncogenes in human germ cell tumors of the testis

Background. It has been suggested that tumorigenesis of the germ cell tumor of the testis includes abnormal and developmentlike differentiation of primordial germ cells to several mature type tumors.