Irradiated allogeneic cells enhance umbilical cord blood stem cell engraftment in immunodeficient mice.

Umbilical cord blood (UCB) is a readily available source of hematopoietic stem cells for transplantation. UCB hematopoietic SCT for average- and large-sized patients is often limited by the number of cells available in a single unit. To address this limitation, we performed experiments to determine if adjunctive therapy with third-party human allogeneic cells enhances the engraftment of human UCB in immunodeficient mice. UCB cells with or without sequential infusion of irradiated third-party allogeneic cells were used in transplantation studies of NOD/SCID and NOD/SCID-IL2Rγ null mice. We studied the impact of irradiated allogeneic cells on colony formation in vitro using long-term culture assays also. Our studies demonstrate that short- and long-term UCB engraftment of immunodeficient mice is enhanced by irradiated allogeneic cells. Secondary transplants demonstrate the durability of engraftment. These preclinical studies support the further development of irradiated allogeneic cells as an adjunct to single UCB transplantation when limiting numbers of cells are available.

Accelerated R-COP: a pilot study for the treatment of advanced low grade lymphomas that has a high complete response rate.

This pilot study tested the hypothesis that dose intensity/dose density treatment may improve the response rate and remission duration in patients with advanced low grade lymphomas. ten patients with low grade lymphomas: follicular lymphoma grades I and II, marginal zone lymphoma, and small cell lymphocytic lymphoma with progressive disease were studied. Patients had an ECOG performance of 0-2, and Stage III and IV disease. Both untreated and previously treated patients with progressive disease were eligible. Patients received a combination of rituximab 375 mg/m(2), cyclophosphamide 1000 mg/m(2), and vincristine 1.4 mg/m(2) (up to a maximal dose of 2 mg), administered by intravenous infusion every two weeks, for ten treatments. Prednisone 50 mg was administered every other day orally for thirty days and then tapered over the next thirty days. Granulocyte colony stimulating factor (G-CSf) was administered on days seven to ten following each cycle of chemotherapy. After 5 and 10 cycles, patients were evaluated for response that included imaging with Ct and pet scans. A total of 10 patients (7 untreated and 3 previously treated) were enrolled into this pilot study between may 2003 and July 2004. Untreated patients received an average of 8.3 cycles of therapy (range 5 to 10 cycles). Seven of 7 untreated patients achieved a complete response (CR), and 5 had not relapsed as of 32-43 months later. Previously treated patients received an average of 9.3 cycles of therapy (range 6 to 12 cycles). One of three previously treated patients achieved a complete response and has no evidence of relapse at 29 months. the other two heavily pretreated patients achieved partial responses, lasting 2 and 5 months. Toxicity was mild consisting mainly of parasthesias requiring attenuation of the vincristine dose. There were no instances of neutropenic fever requiring hospitalization. This program is well tolerated with a high CR rate, and may serve as a basis for future trials.

Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells.

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1alpha,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 microM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 microM), VD3 (1 nM), or NaB (100 microM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < or = 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.

Adenovirus-mediated cytotoxicity of chronic lymphocytic leukemia cells.

We have studied adenovirus-mediated cytotoxicity after infection of malignant cells obtained from patients with chronic lymphocytic leukemia (CLL). Our studies indicate that adenoviruses can infect primary CLL cells and that infection of CLL cells with a replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity. Adenovirus-mediated cytotoxicity was also seen after infection of CLL cells with a variety of viruses attenuated by mutations in the adenovirus early region 1 (E1) or early region 2 (E2). Even viruses attenuated by deletion of the entire E1 region resulted in cytotoxicity after infection of the CLL cells obtained from some patients. Although there was variability in the degree of cytotoxicity induced by different viruses in different patients cells, a virus with a mutation in the E1B 19K gene resulted in the greatest degree of cytotoxicity in most of the CLL samples tested. These studies demonstrate that infection of CLL cells by attenuated adenoviruses with specific mutations in the E1 or E2 region results in cell death. Attenuated adenoviruses should be developed further as therapeutic agents for patients with CLL.

Characterization and use of a recombinant retroviral system for the analysis of drug resistant HIV.

A recombinant retroviral system was used for the analysis of early HIV breakthrough infection in the presence of antiviral drugs. The use of replication-defective HIV allowed a quantitative analysis of a single cycle of infection. This report characterizes this recombinant HIV system and demonstrates it's validity in comparison to standard assays. It is demonstrated that the protease inhibitor XM323 inhibits both early and late events in the HIV life-cycle, while dextran sulphate inhibits only early events. In addition, it is shown that this system can be used for detecting and quantitating drug resistant HIV. Thus, the use of this system may provide both novel information about the stage of the viral life-cycle inhibited and a preliminary assessment of the mechanism(s) responsible for breakthrough infection in the presence of antiretroviral drugs.

Primary extramedullary leukemia of the prostate: case report and review of the literature.

We present the case of a 67-year-old male with primary extramedullary leukemia of the prostate gland, the first reported case in the literature to the best of our knowledge. His initial symptoms consisted of episodes of urinary retention. He underwent transurethral resection of the prostate, and a diagnosis of high-grade lymphoma was rendered. He then received a course of doxorubicin-based lymphoma chemotherapy regimen. However, based on a panel of immunocytochemical stains, a diagnosis of extramedullary leukemia or chloroma was confirmed. His bone-marrow examination at this point was normal. He underwent radiation therapy to the prostate with a total dose of 3960 cGy. Seven months after his initial presentation, he progressed to acute nonlymphocytic leukemia (ANLL), M2 by FAB classification. He was successfully treated with induction and consolidation chemotherapy with Ara-C and idarubicin, and was maintained in complete remission up to 19 months of follow-up. Eight other cases of prostatic leukemia reported in the literature are presented. Five cases occurred in association with ANLL, 2 cases as sites of ANLL relapse, and 1 case in association with myelodysplasia. The use of immunohistochemical stains has aided us in diagnosis of extramedullary leukemia. Surgery, radiation therapy, and chemotherapy play complementary roles in the treatment of prostatic extramedullary leukemia.

Human immunodeficiency virus replication in the presence of antiretroviral drugs: analogies to antineoplastic drug resistance.

There are many analogies between antineoplastic therapy and antiviral therapy. For each there may be sanctuary sites in which the drug is ineffective because of decreased accumulation of the active form of the drug or increased competition by naturally occurring inhibitors. These sanctuaries may be restricted to anatomic or biochemical subsets of the population. A knowledge of these sanctuaries is essential to an understanding of the failure of therapy and for the design of more effective treatments. Eradication of these sanctuary sites may be important because they may be responsible for the viral replication or tumor cell division that continues to generate the diversity that drives clonal evolution. Ultimately, diversity as a consequence of the accumulation of mutations results in the selection of resistant viral or tumor cell variants and the failure of drug therapy. Maximizing therapy in an attempt to diminish the rate of generation of this diversity may result in better clinical outcomes, including a delay in the generation of variants with genetic drug resistance.

Pseudoleukemia after granulocyte colony-stimulating factor therapy.

Therapy with myeloid colony-stimulating factors has been safely and effectively used in a wide variety of situations associated with neutropenia. We present a case of pseudoleukemia occurring in a patient with lymphoma and pancytopenia after 2 days of treatment with granulocyte colony-stimulating factor (G-CSF). Bone marrow aspirate and flow cytometry study results were consistent with acute myelomonocytic leukemia but were normal after G-CSF was discontinued for 4 days. As previous phase I studies of bone marrow morphology after G-CSF use have not described the extreme myeloid immaturity seen in this patient, it seems likely that the action of G-CSF was enhanced by factors associated with the patient's illness. We emphasize the clinical importance of this case in light of the widespread use of G-CSF.

Sanctuary growth of human immunodeficiency virus in the presence of 3'-azido-3'-deoxythymidine.

Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.

Biochemical modulation of 5-fluorouracil with brequinar: results of a phase I study.

Biochemical modulation can increase the efficacy of 5-fluorouracil (5-FU). Pizzorno et al. have previously shown that brequinar, a de novo pyrimidine synthesis inhibitor, enhances the antitumor effect of 5-FU in vivo [Cancer Res 52: 1660-1665, 1992]. On the basis of their data, we conducted a phase I study of brequinar in combination with 5-FU in patients with refractory solid tumors. The initial dose (100 mg/m2) of brequinar was raised in 100-mg/m2 increments in cohorts of three assessable patients. The initial dose of 5-FU was 500 mg/m2, but escalation was allowed in patients who showed no significant toxic reaction. Brequinar was administered over 1 h and 5-FU over 2 h starting 18-20 h after the initiation of infusion of brequinar. Treatments were repeated weekly. Responses were evaluated after 4 weeks (one course) and then every 8 weeks thereafter. Pharmacokinetics of brequinar and determination of plasma uridine levels were performed in at least three patients at each dose level. Of the 25 patients registered in the study, 21 were assessable for toxicity studies. The dose of brequinar was escalated up to 600 mg/m2. In addition, the dose of 5-FU was increased to 600 mg/m2 as a result of a lack of a significant toxic reaction in the first nine patients. No objective responses were observed. One patient developed grade 3 stomatitis, and one developed grade 3 esophagitis at the 400 and 600 mg/m2 dose of brequinar, respectively. Brequinar produced a dose-dependent decrease in plasma uridine levels at doses up to 500 mg/m2. No additional decrease in plasma uridine occurred with higher doses of brequinar, thus suggesting a plateau effect. This observation prompted us to terminate the study before reaching the maximum tolerated dose of brequinar. Our data indicate that brequinar in doses > or = 400 mg/m2 results in significant biochemical modulation. The lack of toxicity seen at these doses of brequinar suggests that the initial dose of the effector agent 5-FU should be increased in future studies.

Percutaneous hepatic vein isolation and high-dose hepatic arterial infusion chemotherapy for unresectable liver tumors.

PURPOSE: This prospective, nonrandomized trial evaluated a percutaneous isolated chemotherapy perfusion approach for treating advanced primary and metastatic liver tumors. Chemotherapy was administered via hepatic artery catheter and hepatic venous blood isolated by a novel percutaneous double-balloon inferior vena cava (IVC) catheter was passed through a detoxification/filtration cartridge in a venovenous bypass circuit. PATIENTS AND METHODS: Among 23 patients enrolled onto the study, 58 procedures were performed on 21 patients. Twelve patients received dose escalations of fluorouracil (5-FU) (1,000 mg/m2 to 5,000 mg/m2), and nine received dose escalations of doxorubicin (50 mg/m2 to 120 mg/m2). Pharmacokinetic studies included drug accumulation in the liver, extraction by detoxification filters, systemic exposure, and alterations of half-life. Each patient received two treatments at 3-week intervals. Those showing stabilization or response received additional treatments. RESULTS: There was a direct relationship between dose and peak concentration of drug entering the hepatic veins. The system functioned efficiently throughout the dose range, with extraction efficiencies ranging from 64% to 91% (P < .001). The hepatic vein drug levels showed a sixfold increase in 5-FU with dose escalation from 1,000 to 5,000 mg/m2, and a twofold increase in dox with dose escalation from 50 to 120 mg/m2 (P < .001, filter-mediated drug extraction). The treatments were accomplished with only an overnight hospital stay and no mortality. The common procedure-related toxicity was transient hypotension (grade I to II), due to catecholamine depletion by the filter. Dose-limiting toxicity (leukopenia) was observed in patients receiving 5-FU at a dose of 5,000 mg/m2 and doxorubicin at a dose of 120 mg/m2. Significant tumor response (> 95% reduction) was obtained in two patients receiving doxorubicin at 90 mg/m2 and 120 mg/m2. CONCLUSION: The use of a double-balloon catheter to isolate and detoxify hepatic venous blood during intraarterial therapy is technically feasible, safe, and allows administration of large doses of intrahepatic chemotherapy at short intervals. This approach should allow new dose-intensification strategies to increase tumor responses in primary and metastatic liver tumors.

Recombinant retroviral systems for the analysis of drug resistant HIV.

Two recombinant retroviral systems are described that can be used to analyze antiretroviral drug activity and HIV breakthrough (replication in the presence of the drug). The first system utilizes a recombinant HIV encoding beta-galactosidase as a reporter gene (HIV-LacZ). The defective HIV-LacZ virus is produced in COS cells after co-transfection of a plasmid encoding the HIV-LacZ genome with a plasmid encoding HIV proteins necessary for packaging and infectivity. Subsequent infection of CD4+ target cells, followed by assay for LacZ expression, permits the rapid identification of individual virus-infected cells. This system can be used to quantitate the inhibition of early events in the HIV replicative cycle and is suitable for the screening of compounds for anti-HIV activity. However, this system cannot be used to analyze HIV drug resistance because of the limited genetic heterogeneity of the virus that is produced in COS cells. To circumvent this problem, a second system has been developed in which heterogenous recombinant HIV is produced by rescue with replication-competent 'helper' HIV. This system required the production of CD4+ cell lines containing defective proviruses encoding either LacZ or guanosine phosphoribosyl transferase (gpt). The defective proviruses are rescued by infection of the cell lines with 'helper' HIV and used to infect target cells in the presence of antiretroviral agents. Subsequent reporter gene assay is used to identify virus-infected cells. This system has been used to detect rare HIV breakthrough infection of cells in the presence of the non-nucleoside reverse transcriptase inhibitor TIBO R82150. Similar analyses with other antiretroviral agents, alone and in combination, may help identify therapeutic strategies that minimize breakthrough replication of HIV.

Percutaneous isolated liver perfusion for treatment of hepatic malignancy: preliminary report.

Chemotherapy for primary or metastatic hepatic malignancy is limited by poor tumor response and dose-related systemic toxicity. As an alternative to chemotherapy infusion by vein or by the hepatic artery, the authors have developed a percutaneous technique of isolated liver perfusion that allows the regional delivery of high-dose chemotherapy to the liver with little systemic toxicity. After placement of a hepatic artery infusion catheter, an 18-F double-balloon catheter is placed into the inferior vena cava through the opposite femoral vein. Balloons are inflated above and below the hepatic veins, thus isolating hepatic venous outflow. The effluent passes through fenestrations in the catheter and is pumped through charcoal hemoperfusion filters where the drug is removed. The filtered blood is returned to the patient through the internal jugular vein. Fifteen treatments have been conducted in eight patients in a phase I dose-escalation study with use of 5-fluorouracil (5-FU). While it is premature to assess tumor response to isolated liver perfusion, the data demonstrate that the procedure is safe and is tolerated by patients. Pharmacokinetic studies show a 5-FU extraction of up to 85%, with minimal drug leakage into the systemic circulation. This technique shows potential for improving liver tumor response while decreasing systemic toxicity.

Construction and uses of cell lines containing integrated adenovirus E2 promoters.

The adenovirus E1a gene encodes polypeptides which regulate the expression of adenovirus early genes as well as a variety of cellular genes. Although it is likely that the E1a encoded polypeptides regulate the expression of these genes by interaction with a variety of cellular transcription factors, the precise mechanism by which this occurs is currently unknown. This report describes the development of cell lines which contain integrated copies of the E2 promoter driving the expression of the Tn5 neo gene or the Escherichia coli beta-galactosidase gene. In each case phenotypic changes concurrent with expression of the E1a 289 amino acid polypeptide are demonstrated. The use of these cell lines to detect rare events in the activation of the E2 promoter is demonstrated in transfection experiments. These cell lines are also used to study the effects of c-myc expression on integrated E2 promoters.

Retroviral mediated transfer and expression of exogenous genes in primary lymphoid cells: assaying for a viral transactivator activity in normal and malignant cells.

In this report we describe the use of recombinant retroviruses to characterize the activity of an exogenous promoter in primary cells obtained from patients with lymphoproliferative disorders. The infection of a variety of cultured and primary lymphoid cells with a recombinant retrovirus containing a histone promoter-driven beta-galactosidase gene is shown to result in the expression of beta-galactosidase in 50% to 100% of the cells. A similar infection with a recombinant retrovirus containing the beta-galactosidase gene with an adenovirus E2 promoter, results in beta-galactosidase activity in a limited number of cultured and primary cells. Since the adenovirus E2 promoter has been well characterized and is known to be regulated by transactivators encoded by many viruses, the activity of this promoter in specific cell types is discussed in reference to both the biology of the cell and the possible presence of as yet undetected viral gene products.

Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker.

Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.