Afsaneh Keyhani

Repeated intravesical instillations of an adenoviral vector in patients with locally advanced bladder cancer: A phase I study of p53 gene therapy

PURPOSE We investigated the feasibility, safety, and biologic activity of adenovirus-mediated p53 gene transfer in patients with locally advanced bladder cancer. PATIENTS AND METHODS Patients with measurable, locally advanced transitional-cell carcinoma of the bladder who were not candidates for cystectomy were eligible. On a 28-day cycle, intravesical instillations of INGN 201 (Ad5CMV-p53) were administered on days 1 and 4 at three dose levels (10(10) particles to 10(12) particles) or on either 4 or 8 consecutive days at a single dose level (10(12) particles). RESULTS Thirteen patients received a total of 22 courses without dose-limiting toxicity. Specific transgene expression was detected by reverse transcriptase polymerase chain reaction in bladder biopsy tissue from two of seven assessable patients. There were no changes in p53, p21waf1/cip1, or bax protein levels in bladder epithelium evident from immunohistochemical analysis of 11 assessable patients. Outpatient administration of multiple courses was feasible and well tolerated. A patient with advanced superficial bladder cancer showed evidence of tumor response. CONCLUSION Intravesical instillation of Ad5CMV-p53 is safe, feasible, and biologically active when administered in multiple doses to patients with bladder cancer. Observations from this study indicate that this treatment has an antitumor effect in superficial transitional-cell carcinoma. Improvements in the efficiency of gene transfer and the levels of gene expression are required to develop more effective gene therapy for bladder cancer.

Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progenitor cells

Detection of karyotypic clonal abnormalities are prognostically useful in patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS), but cytogenetic methods are not sensitive enough to detect low numbers of residual leukemic cells in patients who have achieved complete remission (CR). Fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) were used to investigate the frequency and presence of minimal residual disease (MRD) in AML and MDS patients (n = 28) with monosomy of chromosomes 7, 17 and 18 and trisomy of chromosomes 6, 8, 9 and 10 in CR. MRD was detected in all patients with monosomy 7 (n = 10) and followed by relapse in eight patients after 4.8 ± 3.1 months. In contrast, persistent leukemic cells occurred in 11/12 patients with trisomy 8, but only three of them relapsed after 7.7 ± 4.0 months. Cox regression analysis showed that cytogenetic class and levels of clonal cells at CR were related to time to relapse (P = 0.001). The level of MRD identified patients at high and low risk of relapse. High absolute levels of proliferating residual leukemic cells correlated with monosomy 7 and high risk of relapse.

Increased CD38 expression is associated with favorable prognosis in adult acute leukemia

CD38 is expressed in acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) blasts and its prognostic significance is unknown. We investigated CD38 expression in 304 AML and 138 ALL patients. CD38 was lower in AML-M3 compared to other FAB subtypes (5% vs. 41%; P < 0.001), but was similar among ALL subtypes (56.6%; P = 0.69). Ph + ALL and AML with t(15; 17) patients showed lower CD38 expression than the other cytogenetic groups. Overall survival favored AML and ALL patients with higher CD38 levels. Multivariate analysis revealed CD38 expression to be an independent outcome predictor in AML, but not in ALL.

Characterization of a panel of cell lines derived from urothelial neoplasms: genetic alterations, growth in vivo and the relationship of adenoviral mediated gene transfer to coxsackie adenovirus receptor expression.

PURPOSE Cell lines have become an essential component for the investigation of cancer. We have developed a panel of cell lines derived from human urothelial cancers and we describe some of their important characteristics. MATERIALS AND METHODS Ten human urothelial cancer cell lines were characterized by their growth in athymic nude mice, CAR expression and their susceptibility to adenoviral mediated transfer of the green fluorescence protein gene. TP53 mutation status and immunochemical analysis of p53, pRB and p16 were also examined. RESULTS Five cell lines rapidly produced tumors in athymic nude mice. Two cell lines produced tumors in 1 month, 1 produced them in 3 months and 2 were nontumorigenic. The cell lines varied in CAR expression and in their susceptibility to adenoviral mediated gene transduction. There was no direct correlation between CAR expression and susceptibility to adenoviral mediated gene transduction. Seven cell lines had TP53 mutations, of which 2 had large deletions and did not express p53 protein by immunostaining. All cell lines expressed abnormal pRB by immunochemical analysis (3 had no staining and 7 had homogenously strong staining) and 8 did not express p16 (7 showed homogeneously strong pRB staining). CONCLUSIONS Our panel of 10 human urothelial cell lines differed in genetic alterations, growth in nude mice, susceptibility to adenoviral mediated gene transduction, and expression of p53, p16 and pRB. The availability of various urothelial cancer cell lines with differing genotypic and phenotypic features will facilitate further research into bladder cancer.

Adenoviral p53 gene transfer in human bladder cancer cell lines: cytotoxicity and synergy with cisplatin

Mutations of the tumor suppressor gene p53 are common in bladder cancer. To determine whether p53 gene transfer would lead to decreased viability of bladder cancer cells, we studied the effect of p53 gene transfer in human bladder cancer cell lines with either mutant or wild-type p53. Bladder cancer cell lines 5637 and J82 (which express only mutant p53) and 253J-BV (which expresses wild-type p53) were transduced with vectors containing the beta-galactosidase gene (Ad5-lacZ), wild-type human p53 gene (Ad5CMV-p53), or no foreign gene (DL312 or Ad5-polyA). X-gal staining of cells exposed to Ad5-lacZ showed that the adenoviral vector was capable of transducing each of the cell lines. Increases in p53, p21(waf1/cip1) and bax protein were demonstrated following exposure to Ad5CMV-p53, and there was a dose-dependent increase in the number of apoptotic cells. Cell viability was decreased in all three cell lines, although J82 was less sensitive than either 5637 or 253J-BV. To determine whether cisplatin increases sensitivity of J82 cells to Ad5CMV-p53, we performed median effect analysis for cisplatin combined with Ad5CMV-p53 or DL312. The combination index for cisplatin plus Ad5CMV-p53 revealed synergy, whereas cisplatin and DL312 were only additive. These results suggest that forced p53 gene expression is cytotoxic to human bladder cancer cells with either p53 mutant or wild-type background, and that combination with cisplatin is a potential method for overcoming resistance.

Phagocyte Transport as Mechanism for Enhanced Therapeutic Activity of Liposomal Amphotericin B

Liposomal amphotericin B (L-AmB) is emerging as one of the most attractive new antifungal agents. We have attempted to show that phagocytic cells circulating in blood play an important role in transport and accumulation of L-AmB at inflammatory sites in vivo. Free AmB or L-AmB was injected intravenously to mice, and the amount of AmB in peritoneal exudate cells was quantitated by high-performance liquid chromatography. Higher levels of AmB were detected in a higher number of mice injected with L-AmB. The presence of L-AmB in inflammatory peritoneal cells after intravenous administration of fluorescence-labeled L-AmB also suggested that macrophages play an important role in the transport of intravenously administered L-AmB to inflammatory sites.

Effective cytotoxicity against human leukemias and chemotherapy-resistant leukemia cell lines by N-N-dimethylsphingosine

We evaluated the cytotoxicity of dimethylsphingosine (DMS) against four human leukemia cell lines: two acute (HL60 and a multi-drug resistance MDR-positive derivative HL60-dox) and two blast crisis chronic myelogenous leukemias (JFP1, from a treatment refractory patient and K562), and against blasts isolated from 11 leukemia patients. Cell line viability decreased proportionally to DMS concentration and treatment time (P<0.001). HL60-dox and JFP1 were the most sensitive, indicating DMS efficacy against human leukemia MDR. Importantly, leukemia samples showed a similar sensitivity to DMS as that of the cell lines, firstly demonstrating PKC-independent sphingolipid activity against fresh human tumor specimens. DMS-based chemotherapy may improve leukemia treatment.

Minimal residual disease in acute myelogenous leukaemia and myelodysplastic syndromes: a follow‐up of patients in clinical remission

The majority of patients with acute myelogenous leukaemia (AML) and myelodysplastic syndromes (MDS) relapse, especially those with unfavourable cytogenetics.

Dexrazoxane in combination with anthracyclines lead to a synergistic cytotoxic response in acute myelogenous leukemia cell lines

In an attempt to improve current therapeutic strategies for acute myelogenous leukemia (AML), we studied the effects of a commercially available drug, dexrazoxane (DEX), which protects against anthracycline-induced cardiotoxicity. The rationale was that DEX would permit higher doses of cardiotoxic drugs to be given. The drug itself may have therapeutic potential as well. Finally, there are concerns that the drug may, as a protective agent, diminish the effectiveness of various chemotherapeutics. To help resolve the question about potential drug antagonism, we undertook a series of in vitro analyses of DEX and various combinations with anthracyclines and other agents. Colony-forming assays were used to evaluate stem-cell renewal of myeloid cells in vitro, and median-effect analysis was used to evaluate antagonism, synergism, and additivity. The anthracyclines doxorubicin, daunorubicin, and idarubicin were individually combined with DEX to study in vitro effects in leukemic myeloid cell lines. In the hope, we could extend the findings to non-anthracyclines, etoposide and cytosine arabinoside were also evaluated in combination with DEX using the same in vitro model and method. We found that the effects of DEX in combination with any of the anthracyclines were schedule dependent. The antitumor effect was greater for each combination than for any anthracycline alone except when DEX was administered 24h before doxorubicin or daunorubicin. These data were corroborated through median-effect analysis. Etoposide in combination with DEX was synergistic for all combinations and schedules, and the combination of cytosine arabinoside and DEX was effective depending on the schedule used. DEX appears to be a promising drug in the treatment of AML and warrants further clinical study involving novel drug combinations.

Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer: A cross-sectional study.

Abstract Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples. Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland–Altman plot analysis, regression analysis, and Kendall &tgr; were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers. Although the Bland–Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs. Our study suggests that the measurement results for certain biomarkers of bladder urothelial carcinoma obtained from TMA-based samples can be susceptible to systematic bias, and the lack of correlation between biomarkers cannot be avoided as it is in whole mount tissue preparations. Virtual TMAs can help identify systematic bias and establish a better sampling strategy prior to performing high-throughput TMAs for biomarker studies.