[Show abstract][Hide abstract] ABSTRACT: We previously found that hypoxia-inducible factor (HIF) prolyl hydroxylase-3 (PHD3) was frequently overexpressed in renal cell carcinomas (RCCs), unlike in normal tissues, and therefore, we studied the mechanism and role of PHD3 expression in RCC.
The von Hippel-Lindau (VHL)-gene-mutant RCC cell lines SMKT-R2 and SMKT-R3 and wild-type VHL cell lines Caki-1 and ACHN were used. Associations of the expression of PHD3 with HIF-α proteins and signal transduction pathways were evaluated under normoxic conditions. The effect of PHD3 on cell proliferation was also examined by small interference RNA and cDNA transfection. Moreover, the prognostic impact of PHD3 expression in clear cell RCC (CCRCC) was evaluated using primary cancer tissues.
In SMKT-R2 and SMKT-R3, HIF-α proteins were expressed and PHD3 was highly expressed. On the other hand, ACHN had low expression of HIF-α proteins and PHD3. However, Caki-1 had high expression of PHD3 even though there was no distinct expression of HIF-α proteins. PHD3 expression was inhibited by blockade of Akt and mammalian target of rapamycin (mTOR), but not by HIF-1α and HIF-2α double knockdown. In addition, PHD3 knockdown resulted in the promotion of cell proliferation in SMKT-R2, SMKT-R3 and Caki-1. On the other hand, forced expression of PHD3 reduced cell proliferation in ACHN. In immunohistochemistry, PHD3 expression was a significant factor for better recurrence-free survival in patients with CCRCC.
PHD3 expression can be induced by the phosphatidylinositol-3 kinase/Akt/mTOR pathway in RCC independently of HIF proteins. Furthermore, PHD3 has an antiproliferative function independent of HIF protein status in RCC, indicating a novel expression mechanism and function of PHD3.
Journal of Cancer Research and Clinical Oncology 01/2014; 140(3). DOI:10.1007/s00432-014-1593-7 · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the clinical outcomes of patients who underwent radical cystectomy for bladder cancer at a single institution and compare those who had pT0 specimens with those who had residual cancer.
From January 1990 to December 2006, 186 patients underwent radical cystectomy with or without neoadjuvant chemotherapy for cT2 or higher stage urothelial carcinoma in the bladder in our hospital. We estimated the 5-year disease-free survival, cancer-specific survival and overall survival by the pathological stage.
The median follow-up of the 186 patients was 38.5 months (0-194). Of these, 51 received neoadjuvant chemotherapy. For all subjects, the 5-year disease-free survival was 54.9%, cancer-specific survival 61.0% and overall survival 57.1%. Of the 186 patients, 24 (12.9%) had no residual cancer in the bladder specimen at radical cystectomy. Of the 24 patients with pT0, only 1 (4.2%) died of bladder cancer. The 5-year disease-free survival, cancer-specific survival and overall survival rates in patients with pT0 were ∼96.0%. We found pT0 histology in 11 of the 51 patients (21.6%) with neoadjuvant chemotherapy and in 13 of the 135 patients (9.6%) with radical cystectomy alone (P = 0.047).
We demonstrated that the outcomes of patients who underwent radical cystectomy were similar to those in previous reports. Patients with pT0 showed favorable outcomes for disease-free survival, cancer-specific survival and overall survival in our study. However, they should be periodically followed up because pT0 does not always mean cure.
Japanese Journal of Clinical Oncology 01/2011; 41(1):115-20. DOI:10.1093/jjco/hyq148 · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alpha-methylacyl-CoA racemase (AMACR) is an enzyme playing an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives. High expression levels of AMACR have been described in various cancers, including prostate cancer, colorectal cancer and kidney cancer. Because of its cancer-specific and frequent expression, AMACR could be an attractive target for cytotoxic T-lymphocyte (CTL)-based immunotherapy for cancer. In the present study, we examined the induction of AMACR-specific CTLs from prostate cancer patients' peripheral blood mononuclear cells (PBMCs) and determined HLA-A24-restricted CTL epitopes.
RT-PCR and immunohistochemical analysis revealed that AMACR was strongly expressed in prostate cancer cell lines and tissues as compared with benign or normal prostate tissues. Four AMACR-derived peptides carrying the HLA-A24-binding motif were synthesized from the amino acid sequence of this protein and analyzed to determine their binding affinities to HLA-A24. By stimulating patient's PBMCs with the peptides, specific CTLs were successfully induced in 6 of 11 patients. The peptide-specific CTLs exerted significant cytotoxic activity against AMACR-expressing prostate cancer cells in the context of HLA-A24. Our study demonstrates that AMACR could become a target antigen for prostate cancer immunotherapy, and that the AMACR-derived peptides might be good peptide vaccine candidates for HLA-A24-positive AMACR-expressing cancer patients.
Journal of Translational Medicine 12/2009; 7(1):103. DOI:10.1186/1479-5876-7-103 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The clinical outcome is poor for patients who have local recurrence after radical cystectomy for bladder cancer. We investigated clinical outcomes of patients with isolated local recurrence and with both local recurrence and distant metastasis. From 1990 to 2006, 299 patients who had bladder cancer without distant metastasis underwent radical cystectomy in our hospital. We included 200 patients who had a negative surgical margin at radical cystectomy and urothelial carcinoma, and who had not had upper urinary tract cancer or other active cancer. Fifteen (7.5%) had isolated local recurrence (isolated group) and 18 (9.0%) had local recurrence and distant metastasis simultaneously (simultaneous group). The median overall survival time and that for survival after recurrence were 24 and 13 months, respectively, in the isolated group, and 18 and 5 months, respectively, in the simultaneous group. Although we did not find a significant difference in overall survival (p = 0.314), there was one for survival after recurrence (p = 0.001) between the two groups. In the isolated group, 13 died of cancer, 1 of another cause and 1 was alive without cancer. All patients in the simultaneous group died of cancer. Three patients in the isolated group underwent surgery for residual tumors after systemic chemotherapy, and 2 of the 3 survived for more than 2 years after recurrence. Some patients with isolated local recurrence benefited from multimodal therapies. In these patients, surgery for the residual tumor after systemic chemotherapy was likely to be effective if a negative margin was achieved in surgical specimens.
[Show abstract][Hide abstract] ABSTRACT: Survivin, a member of the inhibitor of apoptosis protein family, is expressed in many malignant tumors including urothelial cancer but is hardly detectable in normal, differentiated adult tissues. Previously we reported CD8-positive cytotoxic T-lymphocytes (CTLs) were successfully induced by stimulation with survivin-2B80-88 peptide in vitro. We started a phase I clinical study of survivin-2B80-88 peptide vaccination for advanced urothelial cancer patients to assess the safety and efficacy of this vaccination. Nine patients were received vaccination and were evaluated for immunological evaluation, adverse events, and clinical responses. A total of 46 vaccinations were carried out. There was no severe adverse event. HLA-A24/survivin-2B80-88 peptide tetramer analysis revealed a significant increase in the peptide-specific CTL frequency after the vaccination in five patients. Slight reduction of the tumor volume was observed in one patient. Survivin-2B80-88 peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in urothelial cancer patients.
Cancer Immunology and Immunotherapy 04/2009; 58(11):1801-7. DOI:10.1007/s00262-009-0691-x · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD8(+) CTLs have an essential role in immune response against tumor. Although tumor-associated antigens have been identified in renal cell carcinoma (RCC), few of these are commonly shared and investigated as therapeutic targets in the clinical medicine. In this report, we show that HIFPH3, a member of prolyl hydroxylases that function as oxygen sensor, is a novel tumor antigen and HIFPH3-specific CTLs are induced from peripheral blood lymphocytes of RCC patients.
Expression of HIFPH3 was examined by reverse transcription-PCR and immunostaining with anti-HIFPH3 antibody. To identify HLA-A24-restricted T-cell epitopes of HIFPH3, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. Peptide-specific CTLs were induced by stimulating peripheral blood lymphocytes of HLA-A24-positive RCC patients with these peptides in vitro. HLA-A24-restricted cytotoxicity of the CTLs against HIFPH3(+) RCC lines was assessed by chromium release assay.
HIFPH3 was overexpressed in many RCC cell lines and primary RCC tissues, whereas it was not detectable in normal adult tissues by reverse transcription-PCR. Of the eight peptides that contained HLA-A24-binding motif, HIFPH3-8 peptide (amino acid sequence, RYAMTVWYF) could induce the peptide-specific CTLs from 3 of 6 patients with HIFPH3-positive RCC. Furthermore, HIFPH3-8 peptide-specific CTLs showed cytotoxicity against HIFPH3(+) RCC cell lines in a HLA-A24-restricted manner.
HIFPH3 may be a target antigen in immunotherapy for RCC and HIFPH3-8 peptide could be used as a peptide vaccine for HLA-A*2402(+)/HIFPH3(+) RCC patients.
Clinical Cancer Research 12/2008; 14(21):6916-23. DOI:10.1158/1078-0432.CCR-08-0466 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clinical and operative features and early postoperative complications were reviewed in 26 patients 75 years old and older who were treated with radical cystectomy including pelvic lymphadenectomy and urinary diversion in our hospital from 1994 to 2005. These findings were compared with those in 170 patients younger than 75 years old who received the same surgery and in 26 patients 75 years old and older who were not surgically treated. Early postoperative complications were found in 9 elderly patients (34%), but there were no deaths in the preoperative and early postoperative periods. There was no significant difference in the rate of early postoperative complications between patients 75 years old and older and those younger than 75. Preoperative performance status (PS) and the American Society of Anesthesiologists Score (ASA score) were significantly better in elderly patients with the surgery than those without surgery. Therefore, evaluation with PS and the ASA score may allow urologists to appropriately select elderly candidates for radical cystectomy and urinary diversion. Chronological age alone is not a determinant for indicating the surgery.
[Show abstract][Hide abstract] ABSTRACT: HLA class I down-regulation in cancer cells confers immunological escape from cytotoxic T lymphocytes. We assessed the frequency of down-regulation of HLA class I antigens in a large series of prostate cancer tissues and determined the mechanism of up-regulation by investigating prostate cancer cell lines.
Immunohistochemical staining for HLA class I was done in specimens of 419 prostate cancers. We also investigated clinicopathological parameters, and the relationships between HLA class I down-regulation and the parameters. Furthermore, we examined whether HLA down-regulation was caused by epigenetic changes in vitro.
HLA class I was down-regulated in 311 prostate cancers (74.2%) and it significantly correlated with beta2-microglobulin down-regulation and a higher clinical stage. Flow cytometric analysis revealed a low level of HLA class I in LNCaP cells, which was up-regulated by the histone deacetylase inhibitor trichostatin A (Sigma). Trichostatin A up-regulated LNCaP beta2-microglobulin at the protein level. Furthermore, chromatin immunoprecipitation assay using an anti-acetylated histone H3 antibody provided direct evidence that trichostatin A up-regulated beta2-microglobulin by modulating the acetylation status of the promoter region in LNCaP cells.
The current study shows that the prevalence of HLA class I down-regulation is high in prostate cancer but histone deacetylase inhibitors can up-regulate HLA class I in LNCaP cells by up-regulating beta2-microglobulin. We suggest that the combination of an immunotherapeutic approach and histone deacetylase inhibition would accentuate the effects of current immunotherapies for prostate cancer.
The Journal of Urology 09/2007; 178(2):692-6. DOI:10.1016/j.juro.2007.03.109 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously reported that 1,2-di-O-acyl-3-O-(-D-sulfoquinovosyl)-glyceride with two stearic acids (beta-SQAG9) bound to L-selectin on the cell surface of the CD62L(+) T-cell subset and inhibited T-cell migration into lymph nodes in a rat skin allograft model. The aim of this study was to verify the efficacy of beta-SQAG9 for kidney allograft survival in miniature swine. Recipient swine underwent bilateral nephrectomy and then received renal allograft transplantation from a swine leukocyte antigen-mismatched donor. Swine were divided into 4 experimental groups. The control (n=2), 25-SQ (n=3), FK (n=3) and 10-SQ/FK (n=2) groups were treated with no immunosuppressant, 25 mg/kg beta-SQAG9, 0.1 mg/kg FK506, and a combination of 10 mg/kg beta-SQAG9 and 0.1 mg/kg FK506, respectively, for 14 days. All recipients were autopsied on the day of death to evaluate the cause of death histopathologically. In the control group, the grafts survived for 12 and 15 days. By comparison with the control, beta-SQAG9 alone did not contribute to prolongation of graft survival (9, 10 and 24 days), whereas the FK group had significantly longer graft survival (19, 20 and 68 days, p=0.0289). The 10-SQ/FK pigs died of lethal visceral hemorrhage, although the grafts were still functioning. In conclusion, our results suggest that beta-SQAG9 possesses an insufficient immunosuppressive effect for kidney allografts in miniature swine, and may affect blood coagulation and fibrinolysis. In addition, the combination of beta-SQAG9 and FK506 can potentially cause severe hemorrhagic complications.
[Show abstract][Hide abstract] ABSTRACT: To assess the expression of livin, a member of the inhibitor of apoptosis protein family, in renal cell carcinoma (RCC) and to determine its prognostic relevance.
Immunohistochemical staining for livin was performed using paraffin-embedded tissues from 45 cases of RCC. Then we assessed anti-livin antibodies (Abs) in patient sera by enzyme-linked immunosorbent assay and Western blotting. Disease-specific survival of patients was assessed, and differences between the immunohistologically livin-positive and livin-negative groups and between the anti-livin Ab-positive and anti-livin Ab-negative groups were compared with recurrence-free survival using the Kaplan-Meier method and log-rank test.
Of the 45 RCC specimens, 26 (57.8%) showed positive staining of livin immunohistochemically. In the RCC patients, anti-livin antibodies were detected and their levels were significantly higher than those in healthy volunteers. However, there was no difference in disease-specific survival between the livin-positive and livin-negative patients or between the anti-livin-positive and anti-livin-negative patients.
Although livin expression may not provide predictive information, it may be recognized as a tumor antigen by the immune system in RCC patients.
[Show abstract][Hide abstract] ABSTRACT: We determined the prognostic impact of human leukocyte antigen class I on the survival of patients with clear cell renal cell carcinoma.
Immunohistochemical staining for HLA class I was performed on specimens from 45 patients with clear cell renal cell carcinoma. We performed univariate and multivariate analyses of various factors affecting cause specific survival including HLA class I, Fuhrman grade, TNM stage and tumor size. Furthermore, we compared the survival of patients with HLA class I positive renal cell carcinoma to that of those with down-regulated HLA class I using the Kaplan-Meier method and log rank test.
HLA class I was immunohistochemically down-regulated in 17 (37.8%) clear cell renal cell carcinomas. The down-regulation had no correlation with other clinicopathological parameters such as tumor size, perirenal fat invasion, tumor thrombus, TNM stage or nuclear grade. Univariate and multivariate analyses revealed that HLA class I expression, tumor grade and TNM stage were significant factors influencing the disease specific survival of patients with renal cell carcinoma. Patients with HLA class I positive renal cell carcinoma had longer recurrence-free survival than those with down-regulated expression at 5-year followup (95.5% and 61.1%, respectively).
Our data demonstrate that down-regulation of HLA class I on tumor cells is an independent prognostic factor for clear cell renal cell carcinoma. This finding suggests that HLA class I restricted cytotoxic T lymphocytes have an important role in the suppression of renal cell carcinoma.
The Journal of Urology 05/2007; 177(4):1269-72; discussion 1272. DOI:10.1016/j.juro.2006.11.082 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There have only been a few reports about adverse events of methotrexate, vinblastine, adriamycin and cisplatin (MVAC) chemotherapy under supportive care with granulocyte stimulating factor (G-CSF) and 5-hydroxytryptamine 3 receptor (5-HT3R) antagonists. The purpose of this study was to retrospectively review the adverse events of the chemotherapy. We analyzed 59 patients with advanced bladder cancer who received MVAC chemotherapy at Sapporo Medical University hospital from January 1992 to September 2004. The adverse events were evaluated according to the Common Terminology Criteria for Adverse Events version 3.0 (Japanese edition). Thirty-one of the 59 patients (52.6%) received MVAC in the neoadjuvant setting. Two courses of chemotherapy were most frequently used in the neoadjuvant and adjuvant settings, and treatment of metastatic or recurrent lesions. More than 90% of patients experienced hematological adverse events such as some grade of leukocytopenia and neutropenia in each course of the chemotherapy. Grade 3 or 4 neutropenia was seen in 60-75% of patients. Grade 3 or 4 leukopenia and/or neutropenia in the first course of the chemotherapy was associated with patients with impaired renal function (60 mL/min< or = creatinine clearance <80 mL/min). Febrile neutropenia was found in 6 patients (5.0%), including one who died of subsequent septic shock and adult respiratory distress syndrome. Nausea was seen in 70-80% of patients. MVAC chemotherapy for advanced bladder cancer was performed with tolerable adverse events. The current results provide relevant information mainly for those who need 2 courses of chemotherapy in the neoadjuvant or adjuvant setting.
[Show abstract][Hide abstract] ABSTRACT: To examine the role of pelvic lymph node dissection (PLND) in patients who underwent radical cystectomy for bladder cancer. The diagnostic and therapeutic role of PLND is still controversial in bladder cancer. The extent of PLND and the necessary number of lymph nodes to remove have not been defined.
This retrospective review included 146 patients with refractory superficial and muscle-invasive disease treated with radical cystectomy, regional PLND (internal iliac, external iliac, and obturator nodes) and urinary diversion from January 1990 to December 2002.
Lymph node metastases were detected in 25 patients (17.1%). The average number of nodes removed in the node-positive and node-negative patients was 13.9 and 14.2, respectively. Although no difference was found in disease-specific survival in the node-negative patients when stratified by the number of nodes removed (13 or more versus less than 13), a significant survival advantage was found in the node-positive patients with 13 or more nodes removed versus less than 13 nodes removed. The patients with four or more positive nodes had a worse outcome than those with less than four positive nodes. However, even if the patients had less than four positive nodes, the survival of patients with less than 13 nodes removed was as poor as that of the patients with four or more positive nodes.
In this series, the removal of 13 or more pelvic lymph nodes was essential for more accurate pathologic examination to predict patient outcome and contributed to an increased chance of survival.
[Show abstract][Hide abstract] ABSTRACT: Various immune systems play important roles in the clinical efficacy of intravesical Bacillus Calmette-Guerin (BCG) instillation for bladder cancer. However, human leukocyte antigen (HLA) class I molecules on tumor cells and various immune system cells infiltrating to/around the tumor have not been evaluated, although many prognostic factors, including clinical, pathologic, and molecular ones, have been investigated. The aim of this study was to determine immunologic prognostic factors of BCG immunotherapy for bladder cancer.
Immunohistochemical staining for HLA class I, CD4, CD8, CD20, CD68, TIA-1, S-100, and FOXP3 was carried out on specimens from 30 patients who underwent BCG immunotherapy from whom both pretreatment and posttreatment specimens were obtained. We did univariate and multivariate analyses of factors affecting recurrence-free survival. The positive, weakly positive, and negative groups of cells that infiltrated to/around the tumor were compared with recurrence-free survival using the Kaplan-Meier method and log-rank test.
HLA class I was a significant prognostic factor both in univariate and multivariate analyses. The 5-year recurrence-free survivals of the patients with HLA class I-positive tumors and those with HLA class I-negative tumors were 55.7% and 19.1%, respectively (P = 0.019). There was a significant association between infiltration of CD8, CD20, and CD68-positive cells after BCG therapy and therapeutic effects.
Our data show that expression of HLA class I molecules on tumor cells contributes significantly to the therapeutic effect of BCG immunotherapy for bladder cancer. It is suggested that CTLs may be one of main effectors in this therapy.
Clinical Cancer Research 09/2006; 12(15):4641-4. DOI:10.1158/1078-0432.CCR-06-0595 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the expression of survivin in transitional cell carcinoma of the bladder and to study whether survivin is a transitional cell carcinoma-specific antigen that could be a target for immunotherapy. Survivin, an inhibitor of apoptosis family member, has been reported to be expressed in various cancers but not in normal adult tissues.
Immunohistochemical staining for survivin and human leukocyte antigen (HLA) class I was performed on specimens from 88 patients who underwent transurethral resection and radical cystectomy. To determine whether survivin was recognized as a tumor antigen by the host immune system, we assessed anti-survivin antibodies in the sera of 52 patients and 18 healthy volunteers with an enzyme-linked immunosorbent assay using recombinant survivin.
Survivin and HLA class I were expressed in 77 (87.5%) and 59 (67.0%) of the 88 bladder cancer specimens, respectively, and 56 (63.6%) expressed both survivin and HLA class I. The absorbance values of anti-survivin antibodies in patients with bladder cancer were significantly greater than those in the healthy volunteers. A relationship was found between the level of serum anti-survivin antibodies and the staining intensity of survivin in the specimen.
Survivin is expressed in carcinoma of the bladder with high sensitivity. It is suggested that survivin is presented on HLA class I molecules of antigen-presenting cells in 64% of patients with bladder cancer. Therapeutic targeting of survivin in bladder cancer is a future possibility.