Yoshiyuki Kakehi

Active Surveillance for Low-Risk Prostate Cancer Worldwide: The PRIAS Study

BACKGROUND Overdiagnosis and subsequent overtreatment are important side effects of screening for, and early detection of, prostate cancer (PCa). Active surveillance (AS) is of growing interest as an alternative to radical treatment of low-risk PCa. OBJECTIVE To update our experience in the largest worldwide prospective AS cohort. DESIGN, SETTING, AND PARTICIPANTS Eligible patients had clinical stage T1/T2 PCa, prostate-specific antigen (PSA) ≤ 10 ng/ml, PSA density <0.2 ng/ml per milliliter, one or two positive biopsy cores, and Gleason score ≤ 6. PSA was measured every 3-6 mo, and volume-based repeat biopsies were scheduled after 1, 4, and 7 yr. Reclassification was defined as more than two positive cores or Gleason >6 at repeat biopsy. Recommendation for treatment was triggered in case of PSA doubling time <3 yr or reclassification. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Multivariate regression analysis was used to evaluate predictors for reclassification at repeat biopsy. Active therapy-free survival (ATFS) was assessed with a Kaplan-Meier analysis, and Cox regression was used to evaluate the association of clinical characteristics with active therapy over time. RESULTS AND LIMITATIONS In total, 2494 patients were included and followed for a median of 1.6 yr. One or more repeat biopsies were performed in 1480 men, of whom 415 men (28%) showed reclassification. Compliance with the first repeat biopsy was estimated to be 81%. During follow-up, 527 patients (21.1%) underwent active therapy. ATFS at 2 yr was 77.3%. The strongest predictors for reclassification and switching to deferred treatment were the number of positive cores (two cores compared with one core) and PSA density. The disease-specific survival rate was 100%. Follow-up was too short to draw definitive conclusions about the safety of AS. CONCLUSIONS Our short-term data support AS as a feasible strategy to reduce overtreatment. Clinical characteristics and PSA kinetics during follow-up can be used for risk stratification. Strict monitoring is even more essential in men with high-risk features to enable timely recognition of potentially aggressive disease and offer curative intervention. Limitations of using surrogate end points and markers in AS should be recognized. TRIAL REGISTRATION The current program is registered at the Dutch Trial Register with ID NTR1718 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1718).

Constitutive Activation of Mitogen-activated Protein (MAP) Kinases in Human Renal Cell Carcinoma

Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAPK cascade, which includes MEK (also known as MAP kinase kinase), Raf-1, and Ras. In this study, we examined whether constitutive activation of the MAPK cascade was associated with the carcinogenesis of human renal cell carcinomas in a series of 25 tumors and in corresponding normal kidneys. Constitutive activation of MAPKs in tumor tissue, as determined by the appearance of phosphorylated forms, was found in 12 cases (48%), and this activation was confirmed by a direct in vitro kinase assay of immunoprecipitate using myelin basic protein as the substrate. The phosphorylation of MEK and of Raf-1, as monitored by a mobility shift in SDS-PAGE, which is reportedly associated with the activation of these kinases, occurred in 9 of 18 cases (50%) and in 6 of 11 cases (55%) respectively. The activation of MAPKs was correlated with MEK activation (P = 0.0045) and with Raf-1 activation (P = 0.067). Furthermore, overexpression of MEK was found in 13 of 25 cases (52%) by Western blot analysis, and this overexpression was associated significantly with MAPK activation (P = 0.034). No mutations were noted in H-,K-, or N-ras genes by PCR direct sequencing in any of the 25 tumor samples. Of the patients studied, 8 of 18 (44%) stage pT2 patients and four of six (67%) stage pT3 patients showed MAPK activation. The single stage pT1 patient did not evidence MAPK activation. Furthermore, one of seven (14%) grade 1 patients, 9 of 13 (69%) grade 2 patients, and two of five (40%) grade 3 patients showed MAPK activation (grade 1 versus grades 2 and 3, P = 0.046). Our results suggest that constitutive activation of MAPKs may be associated with the carcinogenesis of human RCCs.

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.

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.

Effective treatment of advanced solid tumors by the combination of arsenic trioxide and L-buthionine-sulfoximine.

AbstractClinical application of anticancer agents has been often hampered by toxicity against normal cells, so the achievement of their cancer-specific action is still one of the major challenges to be addressed. Previously, we reported that arsenic trioxide (As2O3) could be a promising new drug against not only leukemia but also solid tumors. The cytotoxicity of As2O3 occurred through the generation of reactive oxygen species (ROS), thus inhibiting radical scavenging systems would enhance the therapeutic efficacy of As2O3 provided that normal cells were relatively resistant to such a measure. Here, we report that the combination therapy of As2O3 with L-buthionine-sulfoximine (BSO), which inhibits a critical step in glutathione synthesis, effectively enhanced in vitro growth inhibition effect of As2O3 on all 11 investigated cell lines arising from prostate, breast, lung, colon, cervix, bladder, and kidney cancers, compared with As2O3 treatment alone. Furthermore, this combination enhanced cytotoxicity to cell lines from prostate cancer with less toxicity to those from normal prostate. In vitro cytotoxic assay using ROS-related compounds demonstrated that hydrogen peroxide (H2O2) is a major cytotoxic mediator among ROS molecules. Biochemical analysis showed that combined use of As2O3 and BSO blocked H2O2-scavenging systems including glutathione, catalase, and glutathione peroxidase, and that the degree of this blockade was well correlated with intracellular ROS levels and sensitivity to this treatment. Finally, the effectiveness of the combination therapy of As2O3 with BSO was demonstrated with an orthotopic model of prostate cancer metastasis. We propose that the combination therapy of As2O3 with BSO is a valid means of blockade of H2O2-scavenging system, and that the combination of a ROS-generating agent with an inhibitor of major scavenging systems is effective in terms of both efficacy and selectivity. Furthermore, because the effective doses of both compounds are within clinically achievable range, this report will lead to immediate benefit for the development of a new cancer therapy.

Measurement of multidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance.

We measured the levels of messenger RNA of the human multidrug-resistance gene (MDR1) in 25 urogenital tumors before chemotherapy. Many of the renal cell carcinomas continued to express MDR1 gene at high levels, reflecting the increased expression of MDR1 RNA in normal kidneys. In other urogenital tumors, the MDR1 RNA levels were low reflecting low MDR1 RNA levels in normal bladder, prostate and testis. For comparative purposes, we performed in vitro chemosensitivity testing on many tumor samples using soft agar culture techniques. Vinblastine sensitivity in vitro inversely correlated with MDR1 RNA levels (p less than 0.01). Moreover, mean sensitivity of seven renal cell carcinomas to vinblastin was significantly lower than that of the other seven cancers (p less than 0.05). As for doxorubicin, mean sensitivity of six renal cell carcinomas was lower than the others (p less than 0.1). These results suggest that the high MDR1 RNA levels in renal cell carcinomas are associated with intrinsic multidrug-resistance.

Symptomatic and asymptomatic benign prostatic hyperplasia: Molecular differentiation by using microarrays

Benign prostatic hyperplasia (BPH) is a disease of unknown etiology that significantly affects the quality of life in aging men. Histologic BPH may present itself either as symptomatic or asymptomatic in nature. To elucidate the molecular differences underlying BPH, gene expression profiles from the prostate transition zone tissue have been analyzed by using microarrays. A set of 511 differentially expressed genes distinguished symptomatic and asymptomatic BPH. This genetic signature separates BPH from normal tissue but does not seem to change with age. These data could provide novel approaches for alleviating symptoms and hyperplasia in BPH.

Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti‐apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non‐specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA‐synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 °C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA‐degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA‐phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.

Biochemical and molecular characterization of a novel choline-specific glycerophosphodiester phosphodiesterase belonging to the nucleotide pyrophosphatase/phosphodiesterase family

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that release nucleoside 5′-monophosphate from a variety of nucleotides and nucleotide derivatives. The mammalian NPP family comprises seven members, but only three of these (NPP1–3) have been studied in some detail. Previously we showed that lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) to produce lysophosphatidic acid, is identical to NPP2. More recently an uncharacterized novel NPP member (NPP7) was shown to have alkaline sphingomyelinase activity. These findings raised the possibility that other members of the NPP family act on phospholipids. Here we show that the sixth member of the NPP family, NPP6, is a choline-specific glycerophosphodiester phosphodiesterase. The sequence of NPP6 encodes a transmembrane protein containing an NPP domain with significant homology to NPP4, NPP5, and NPP7/alkaline sphingomyelinase. When expressed in HeLa cells, NPP6 was detected in both the cells and the cell culture medium as judged by Western blotting and by enzymatic activity. Recombinant NPP6 efficiently hydrolyzed the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5′-monophosphate. In addition, NPP6 hydrolyzed LPC to form monoacylglycerol and phosphorylcholine but not lysophosphatidic acid, showing it has a lysophospholipase C activity. NPP6 showed a preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids. It also hydrolyzed glycerophosphorylcholine and sphingosylphosphorylcholine efficiently. In mice, NPP6 mRNA was predominantly detected in kidney with a lesser expression in brain and heart, and in human it was detected in kidney and brain. The present results suggest that NPP6 has a specific role through the hydrolysis of polyunsaturated LPC, glycerophosphorylcholine, or sphingosylphosphorylcholine in these organs.

Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: Evidence of persistent oxidative stress in cancer

Data regarding oxidatively modified DNA bases suggest that cancer cells are more exposed to oxidative stress than adjacent non‐tumorous tissue. This novel concept may contribute to the understanding of certain aspects of tumor biology such as activated transcription factors, genetic instability, chemotherapy‐resistance and metastasis. We therefore tested this concept in human renal‐cell carcinomas (RCCs) by evaluating the expression of hMTH1, an enzyme preventing the misincorporation into DNA of 8‐oxo‐dGTP (8‐oxo‐7,8‐dihydrodeoxyguanosine triphosphate), an oxidized form of dGTP in the nucleotide pool. The expression of hMTH1 messenger RNA (mRNA) in tumorous kidney. Moreover, advanced‐stage tumors showed significantly higher hMTH1 mRNA expression than early‐stage tumors, and there was a modest linear correlation between hMTH1 expression and c‐myc expression. The results provide logical support for the concept of “persistent oxidative stress in cancer” and suggest a role of hMTH1 mRNA level as a prognostic marker.