Tzong-Shin Tzai

Low-Dose Etoposide Enhances Telomerase-Dependent Adenovirus-Mediated Cytosine Deaminase Gene Therapy through Augmentation of Adenoviral Infection and Transgene Expression in a Syngeneic Bladder Tumor Model

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.

Postoperative immuno-gene therapy of murine bladder tumor by in vivo administration of retroviruses expressing mouse interferon-gamma.

The murine MBT-2 bladder tumor model in syngeneic C3H/HeN mice was used to investigate the feasibility of gene therapy based on the delivery of interferon-γ (IFN-γ) in vivo by retroviral vectors. We constructed a recombinant retroviral vector pRUFneo/IFN-γ, which was transfected into a retroviral packaging cell line ψCRE, to produce ψCRE/pRUFneo/IFN-γ cells. The expressions of the neo and IFN-γ genes were verified by reverse transcription-polymerase chain reaction and IFN-γ was detected in the culture supernatant from ψCRE/pRUFneo/IFN-γ cells. After receiving MBT-2 cells admixed with retroviral pRUFneoIFN-γ supernatant, C3H/HeN mice exhibited lower tumor incidence, lower tumor mass, and higher survival rate, as well as higher antitumor responses compared to those injected with MBT-2 cells admixed with control retroviral supernatant. Moreover, the retroviral pRUFneoIFN-γ supernatant was able to suppress the growth of rechallenged tumors in postoperated mice. Although the IFN-γ protein secreted from ψCRE/pRUFneo/IFN-γ cells partly contributes to the antitumor effect of retroviral pRUFneoIFN-γ supernatant, the retroviruses carrying the IFN-γ gene transduced MBT-2 cells in vivo, which may result in enhancing local IFN-γ production from tumor cells. Because bladder is suitable for the intravesical instillation of therapeutic agents, in vivo administration of retroviral vectors encoding IFN-γ may be explored for the treatment of bladder cancer. Cancer Gene Therapy (2001) 8, 73–81

Enhancement of antitumor immune response by targeted interleukin-12 electrogene transfer through antiHER2 single-chain antibody in a murine bladder tumor model.

Interleukin-12 (IL-12), despite exerting antitumor activity, has limited therapeutic uses due to its systemic toxicity. Since HER2 (also known as ErbB-2, neu, and HER2/neu) is frequently overexpressed on cancer cells, HER2-targeted delivery of IL-12 to tumors may be a promising strategy for enhancing antitumor immunity. Here we showed that intramuscular electrogene transfer of an expression vector encoding a fusion protein antiHER2scFv-IL12, which consists of antiHER2 single-chain variable fragment (scFv) and single-chain IL-12, significantly retarded tumor growth and prolonged the survival in a syngeneic bladder tumor model. Elevated IL-12 and interferon-gamma (IFN-gamma) levels, increased infiltration of CD4(+) and CD8(+) T cells, and reduced vascular endothelial growth factor (VEGF) expression in the tumors, as well as enhanced cytolytic activity of splenocytes were noted in the treated mice. Our results suggest that this approach may be effective for the treatment of HER2-overexpressing tumors.

Prognostic Relevance of Prothymosin-α Expression in Human Upper Urinary Tract Transitional Cell Carcinoma

OBJECTIVESnTo investigate the prognostic role of prothymosin-alpha (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC).nnnMETHODSnParaffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored.nnnRESULTSnThe expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up.nnnCONCLUSIONSnThe results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-γ, IL-1β, and tumor necrosis factor-α proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Aggressive Treatment of Testicular Choriocarcinoma with Lung Metastasis and Pulmonary Hemorrhage

Choriocarcinoma is not common and is frequently associated with a poor prognosis among testicular tumos. From 1988to1999, three cases of testicular choriocarcinoma were found in our hospital. The ages of all 3 patients were in the second or third decades. One case was pure choriocarcinoma;the other 2 were of mixed types(teratoma with choriocarcinoma and seminoma with choriocarcinoma, respectively). All 3 patients had a markedly enlarged testicular mass with elevated beta-human chorionic gonadotropin(B-hCG)at onset and fulfilled the definition of choriocarcinoma. syndrome. Two were suffering from hemothorax initially. All 3 patients recaived cisplatin-based chemotherapy after radical orchiectomy. One patient died of respiratory failure and hemothorax resulting from pulmonary metastases within l wk after treatment. Another patient died of septic shock, hemoptysis 3 mo after therapy because of both myelosuppressive and pulmonary toxicity from chemotherapeutic agents. The third case is still alive after a total of 6 courses of cisplatin-based chemotherapy and after receiving palliative pulmonary wedge resection for active hemorrhage. Despite some residual pulmonary and retroperitoneal nodules, the serum s-hCG level declined from more than 5000 mIU/ml to an undetectable level after chemotherapy. Subsequent surgical intervention of residual pulmonary or retroperitoneal nodules, for histology will be done. From our experience, early pulmonary metastases with fatal pulmonary hemorrhage in patients with choriocarcinoma contributed to treatment failure, even to death. We present our experience and review the literature.

599. Low-dose etoposide enhances tumor-specific adenovirus-mediated cytosine deaminase gene therapy through induction of integrin |[alpha]|v|[beta]|3 and |[alpha]|v|[beta]|5 overexpression and upregulation of human telomerase reverse transcriptase promoter

Purpose: The human telomerase reverse transcriptase (hTERT) promoter is used to restrict adenoviral expression of suicide or proapoptotic genes in telomerase-positive cancer cells. Etoposide can enhance intratumoral transgene expression in mice immunized with adenoviral vectors. The aim of this study is to evaluate whether greater therapeutic benefit can be achieved by combination treatment of low-dose etoposide and replication-incompetent adenoviral vectors encoding cytosine deaminase (CD) driven by the hTERT promoter for murine bladder carcinoma.

Incidental Renal Cell Carcinoma：Experience at National Cheng Kung University Hospital

OBJECTIVES: As ultrasonography and computed tomography have become commonplace, renal rumors are increasingly being detected incidentally. To assess the characteristics of incidentally discovered renal cell carcinoma, we retrospectively reviewed 92 cases of pathologically proven disease. nMATERIALS AND METHODS: From June1998 to January 2001, totally 90 cases of pathologically proven renal cell carcinoma (RCC) were reviewed at NCKUH. Charts were analyzed to record the presenting symptoms, image modality used, tumor stage, tumor size, local tumor recurrence or distant metastasis, and outcome. nRESULTS: Of the 92 patients, there were 61 males and 1 females with a male to female ratio of 1.97. Patients’ ages ranged from 19 to 87 with a mean of 57.8 years. Sixty-three patients (68.5%) presented with clinical symptoms and/or signs related to renal pathology. Twenty-nine patients (31.5%) were incidentally diagnosed during image studies for non-renal symptoms or routine health examinations. The overall detection of organ-confined disease (stages I and II) was 72.4% of the ”incidental” and 30.1% of the “symptomatic” group. Smaller tumor size was also found in the “incidental” group (5.17 vs. 8.72 cm). The overall 5-year survival rate for patients with incidentally detected RCC was 92.3%, and was 52.5% in those with symptomatic tumors. Postoperatively, there was local tumor recurrence or distal metastasis in 6.9% of patients with incidental RCC and 30.2% of symptomatic patients. nCONCLUSIONS: With the widespread use of non-invasive image modalities, the detection of smaller, lower-stage renal tumors will significantly improve the outcome of this disease.

Efficacy of Combined Finasteride and α1-blocker Treatment for Benign Prostatic Hyperplasia after Initial Unsatisfactory Response to α1-blocker

OBJECTIVE: The purpose of our study was to assess the efficacy of a combination of finasteride and alphal-adrenergic antagonist(α1-blocker)for treatment of benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Charts of 69 patients with symptoms of BPH treated at our department from January 1995 to December 1998 with α1-blocker first then in combination with finasteride (ProscarR 5 mg/day) owing to the unsatisfactory subjective response to the initial α1-blocker were retrospectively reviewed. Data were collected with regards to prostate volume, uroflow rate, and PSA levels before and after finasteride. Changes in prostate volume, PSA level, and maximum uroflow rate were recorded and analyzed for each patient RESULTS: There was a statistically significant decrease in prostate volume and prostate specific antigen level after combination therapy. A mean decrease in prostate volume of 20%±14% was achieved in the first year and 17%±17% in the second year of combination therapy. Mean reductions of 47%± 48% and 57%±34% in serum PSA levels were achieved in the first and second years, respectively. For maximal uroflow rate (Qmax), a significant increase from combination therapy was observed only in patients with baseline PSA values greater than 4 ng/ml and prostate volumes greater than 35cm3 in the first year of treatment (mean 1.1±3.0 and 1.8±3.3ml/s increase, respectively). However, all patients showed improvement in symptoms and quality of life. CONCLUSIONS: Based on our observations, patients with higher baseline PSA levels and larger prostate volumes benefited the most from a combination of finasteride and α1-blocker for treatment of BPH.

The Role of Percent Free-Prostate Specific Antigen (PSA) in Patients with Total PSA Levels Between 4.0 and 10.0 ng/ml

To explore the role of percent free-prostate specific antigen(PSA) on the differentiation between benign and malignant prostatic diseases in patients with serum total PSA levels between 4.0 and 10.0 ng/ml. Forty-three out of the 221 screened men who had total PSA levels between 4.0 and 10.0 ng/ml were retrospectively enrolled into this study. in addition to digital rectal examination(DRE), transrectal ultrasonography(TRUS), and total PSA, they received either TRUS-guided biopsies(n=27) or transurethral resection of the prostate (TUR-P) (n=16). All sample sera were then stored at -70 °C after the initial measurement of total PSA levels using an immunoenzymometric assay(Tandem-E®, Hybritech). Thereafter, all stored sera were quantified for free-PSA levels and total PSA levels using a different radioimmunometric assay(PSA-RJACT, CIS). Two values of percent free-PSA were obtained for each individual by dividing the same free-PSA level by 2 different total PSA values as measured by 2 different assays. Among the 43 patients, there were 34(79.1%) patients with benign prostatic hyperpla-sia(BPH), 5(11.6%) with prostatitis, and 4(9.3%) with prostate cancer(PC). Using these 2 different total PSA assays, the percent free-PSA in patients with BPH, prostatitis, and PC were 28.0%±12.2%, 23.6%±9.1%, 8.7%±1.3% (FPSA-RJACT/Tandem-E®) and 29.6%±13.1%,28.5%±13.1%, 9.7%±1.1% (FPSA-RJACT/PSA-RIACT), respectively. Patients with PC had significantly lower values of percent free-PSA (less than 12%) while those with either BPH or prostatitis had values greater than l2%(p values, 0.015 and 0.003, unpaired t-test). In contrast, the percent free-PSA values were not statistically different between BPH and prostatitis patients in both assays (p=0. 44, unpaired t-test). Besides, the correlation between the 2 values of percent free-PSA was good(r2 =0.90), based on 2 different assays for total PSA levels. Our study implies that percent free-PSA can help differentiate prostate cancer from benign prostatic diseases, and thus greatly reducing unnecessary biopsies. There is no need to measure the total PSA levels again while determining the percent free-PSA.