Regulation of intracellular actin polymerization by prenylated cellular proteins.

Posttranslational modification by covalent attachment of polyisoprene intermediates to a carboxyterminal CAAX-box motif is required for the biologic function of proteins such as p21ras, the supergene family of ras-related proteins, nuclear lamins, and subunits of heterotrimeric G-proteins. Cells grown in the presence of lovastatin, which inhibits HMG-CoA reductase and prevents synthesis of intermediates required for protein prenylation, develop a round, refractile morphology. Our data indicate that this is due to the selective loss of actin cables without gross changes in the microtubular lattice or intermediate filament structure. Microinjection of a competitive peptide inhibitor of protein prenyltransferases into the cytoplasm of cells induces an identical change in morphology with loss of actin cables. Mevalonate (MVA) reverses the lovastatin-induced morphologic change by inducing a rapid repolymerization of actin cables with coincident reversion to the flat morphology. Furthermore, microinjection of farnesyl-pyrophosphate or geranylgeranyl-pyrophosphate into lovastatin-treated cells also results in rapid morphologic reversion. The morphologic reversion induced by MVA requires the presence of serum, and is independent of extracellular calcium. The addition of cycloheximide to the growth medium prevents lovastatin-induced loss of actin cables, and causes morphologic reversion of lovastatin-treated cells by a mechanism that is independent of MVA. A1F4- induces morphologic reversion in a manner indistinguishable from MVA. These data indicate that prenylated protein(s) play a critical role in regulating the state of intracellular actin, and that GGPP can rescue the lovastatin-induced morphologic phenotype in the absence of upstream intermediates of cholesterol biosynthesis. We have begun to dissect the signaling events that mediate this pathway.

Isotypic exclusion of gamma delta T cell receptors in transgenic mice bearing a rearranged beta-chain gene.

The rearrangement of T cell antigen receptor beta- and gamma-chain gene segments was studied in transgenic mice that bear a functional beta-chain gene. Virtually all CD3-positive T cells derived from transgenic mice express beta chains containing the transgene-encoded V beta 8.2 variable region on their surfaces and do not express endogenous beta-chain variable regions. Expression of endogenous V beta genes is inhibited at the level of somatic recombination during thymic ontogeny. Furthermore, rearrangements of the TCR gamma-chain genes are also markedly inhibited in these transgenic animals. Hence expression of the TCR beta transgene has led to allelic exclusion of alpha beta receptors and isotypic exclusion of gamma delta T cell receptors.

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.

Induction of T-cell immunity against Ras oncoproteins by soluble protein or Ras-expressing Escherichia coli.

BACKGROUND: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported. PURPOSE: The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo. METHODS: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines. In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins. Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides. For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Ras]; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 [Leu 61 Ras]). Ten mice per group were used in each experiment. RESULTS: Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein. In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras. Immunization with heat-killed E. coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity. T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not. CONCLUSIONS: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.

Cytotoxic T-cell response and in vivo protection against tumor cells harboring activated ras proto-oncogenes.

BACKGROUND: Activated forms of the ras proto-oncogene have been found in approximately 30% of human malignancies, including pancreatic, colon, and lung adenocarcinomas. Ras oncoproteins arise by somatic mutation and contain amino acid changes at residues 12, 13, or 61, thus generating unique tumor-specific proteins that are attractive targets for cancer therapy. PURPOSE: The goal of this study was to determine whether vaccination with mutant Ras protein could lead to the generation of cytotoxic T lymphocytes (CTLs) specific for the mutant epitope and to protection against challenge with tumor cells expressing the mutant oncoprotein. METHODS: To determine a methodology for generating CTL responses following immunization with soluble protein, ovalbumin was used as a model tumor antigen. C57BL/6 mice were immunized with soluble ovalbumin administered intraperitoneally at 2-week intervals or with intravenous injection of ovalbumin or osmotically loaded splenocytes. Immunized mice were challenged with E.G7 cells (which express a transfected ovalbumin gene), and tumor growth was monitored. Generation of ovalbumin-specific CTLs was determined by 51Cr release assays. Purified wild-type or mutant H-Ras proteins (containing single amino acid substitutions at position 12 converting Gly to Arg or Val) were used to immunize BALB/c mice intraperitoneally. Ras-immunized mice were challenged with tumor cells containing Arg 12 or Val 12 mutations or not harboring mutant forms of Ras. Cytolytic and proliferative responses to mutant forms of Ras were studied, and the effects of in vivo depletion of CD4+ or CD8+ T lymphocytes were determined. RESULTS: In vivo challenge with E.G7 showed that intraperitoneal immunization with soluble ovalbumin was as effective as osmotic loading, resulting in long-term disease-free survival of some mice and the development of ovalbumin-specific CTLs. Immunization with Arg 12 Ras led to disease-free survival in nine of 10 animals challenged with tumor cells containing an Arg 12 mutation, while no protection was afforded against tumors expressing other forms of Ras or other oncogenes. Splenocytes from BALB/c mice immunized with Arg 12 Ras demonstrated cytolytic activity specific against tumor cells expressing Arg 12 Ras, with most of this activity residing in the CD8+ subset. Mutation-specific proliferation to Arg 12 Ras peptides was also observed. Immunization with Val 12 Ras did not elicit detectable Val 12-specific immunity. CONCLUSIONS: Antigen-specific CTLs can be induced following intraperitoneal immunization of mice with purified, soluble proteins. For both ovalbumin and Arg 12 Ras, specific in vivo protection against tumor cell challenge was observed.

Pilot study of interleukin-2 and lymphokine-activated killer cells combined with immunomodulatory doses of chemotherapy and sequenced with interferon alfa-2a in patients with metastatic melanoma and renal cell carcinoma.

BACKGROUND: Experiments in animal tumor models suggest that the antitumor effects of interleukin-2 (IL-2) or IL-2 in combination with lymphokine-activated killer (LAK) cells can be enhanced by chemotherapy agents such as cyclophosphamide or doxorubicin or by the biologic agent interferon alpha. PURPOSE: We determined the toxicity and clinical response rate of an IL-2-LAK cell regimen modified by the addition of moderate, immunomodulatory doses of chemotherapy and sequenced with interferon alfa-2a (IFN alpha-2a) in patients with metastatic melanoma and renal cell carcinoma. METHODS: IL-2 (3-6 million units/m2 per day) was administered by continuous infusion on days 0-5 and days 11-16. LAK cells were infused on days 11 and 12 or on days 11, 12, and 14. Low doses of cyclophosphamide (300 mg/m2) and doxorubicin (25 mg/m2) were given on day 9 before the LAK cell infusions. Following the IL-2-LAK cell infusion, IFN alpha-2a (12 million units/m2) was administered for a total of nine doses to complete a cycle of treatment. A total of 89 patients were enrolled in the study. RESULTS: For each histology, there were eight partial responses in 40 assessable patients, for an overall response rate of 20% (90% confidence interval = 10%-33%). The median response duration was 5 months, although two patients with renal cell carcinoma and one patient with metastatic melanoma had almost complete disappearance of tumor and are still responding after 26+, 22+, and 26+ months, respectively. Toxic effects were severe in patients receiving the highest dose of IL-2 administered in this study and similar to those reported with other high-dose IL-2-LAK cell regimens. Although toxic effects were completely reversible in most patients, there were four treatment-related deaths. CONCLUSIONS: This regimen is active in patients with metastatic melanoma and renal cell carcinoma and produces meaningful responses in a small percentage of these patients; however, it is not clear whether cyclophosphamide, doxorubicin, and IFN alpha-2a as used in this protocol appreciably augmented the antitumor activity of the IL-2-LAK cell regimen.

Phase II trial of interleukin 1 alpha and indomethacin in treatment of metastatic melanoma.

BACKGROUND: The rising incidence of malignant melanoma and the lack of curative therapies for metastatic disease represent a therapeutic challenge. New agents effective in treating this disease are needed. PURPOSE: Because of the additive antitumor effects of interleukin 1 alpha (IL-1 alpha) and indomethacin in vivo, we conducted a phase II trial of this combination in patients with melanoma. We used the recommended dose determined from our phase I trial to ascertain the antitumor activity of the combination. METHODS: From August 1, 1990, through July 28, 1992, 49 patients entered the study. They were stratified into two groups based on the presence of visceral (n = 14) and nonvisceral (n = 35) metastases. The patients received 7 days of both IL-1 alpha (O.1 micrograms/kg per day by intravenous bolus) infusion) and indomethacin (50 mg orally every 8 hours). At least two cycles of therapy, repeated at 21-day intervals, were planned. Additional treatment was given to those patients who had stable or responding lesions. A chi-squared test for homogeneity of proportions was used to compare groups on several measures. All P values resulted from two-sided tests. RESULTS: Fever, chills, and hypotension were among the most common side effects. None of the 14 patients with visceral metastases responded to the treatment. Of the 35 patients with non-visceral metastases, three showed a partial response for 6 months each and one showed a complete response for more than 34 months; the response rate was 11% (95% confidence interval [CI] = 5%-26%). All responding patients required phenylephrine for treatment of IL-1 alpha-induced hypotension, whereas six (19%) of 31 of the nonresponding patients with nonvisceral metastases required phenylephrine (P = .0008). The response rate in women was higher; three of 10 women (30%; 95% CI = 11%-60%) responded, whereas one of 25 men (4%; 95% CI = 0%-20%) responded (P = .029). All three women were positive for human leukocyte antigen (HLA) B7 expression (P = .011). CONCLUSIONS: The combination of IL-1 alpha and indomethacin has minimal antitumor activity in melanoma patients. All responses were confined to patients with nonvisceral metastases. IL-1 alpha-induced hypotension, gender, and HLA B7 expression were positively associated with response. IMPLICATIONS: Administration of higher doses of IL-1 alpha alone has been shown to produce hypotension in a large proportion of patients but can be given safely with phenylephrine support. Because of the association of hypotension with antitumor activity, treatment with higher IL-1 alpha doses alone may be a strategy for attaining better response rates.

Irreversible G2-M arrest and cytoskeletal reorganization induced by cytotoxic nucleoside analogues.

The mechanisms by which cytotoxic agents perturb the normal cell biology and cell cycle progression of cancer cells were explored using B16F10 cells genetically modified to express the Herpes Simplex Virus-thymidine kinase gene. Culture in the presence of the nucleoside analogue ganciclovir induced a profound morphological change that required entry of treated cells into S phase and was dependent on prenylated proteins such as those of the rho gene family. Cell cycle arrest occurred in late S phase or G2 phase due to the activation of the G2-M DNA damage checkpoint. This checkpoint control operated at the level of inhibition of the activity of Cdc2/cyclin B and occurred by two mechanisms: (a) p53-mediated up-regulation of p21CIP/WAF1 expression and its association with Cdc2/cyclin B; and (b) prevention of the dephosphorylation of tyrosine 15 of Cdc2. These events occurred in vitro and in vivo, and were shown to mediate bystander killing in this model. The mechanism of cell death seemed to be due to the irreversible cell cycle arrest at the G2-M checkpoint, rather than induction of apoptosis. These data link DNA damage checkpoints with cytoskeletal signaling pathways and the core cell cycle machinery and may represent a general mechanism of cytotoxicity of this class of nucleoside analogues.

Transcriptional targeting of recombinant adenoviruses to human and murine melanoma cells.

One potential avenue for future cancer therapy involves the specific targeting of effector genes to cancer cells throughout the body, including distant metastatic sites. As a first step toward this goal, we tested the ability of the transcriptional regulatory elements of the human and mouse tyrosinase genes to promote high levels of pigment cell-specific transcription. A construct consisting of 209 bp of the human tyrosinase promoter linked to two enhancer elements was demonstrated to drive high-level, melanoma-specific expression of a beta-galactosidase (beta-gal) reporter gene in transient transfection assays. In studies of the murine tyrosinase promoter region, constructs containing up to 2500 bp of the 5' regulatory region were found to have very low transcriptional activity in murine melanoma cells. However, as with the human system, addition of two tandem repeats of an upstream enhancer element resulted in high levels of lineage-specific transcriptional activation. The murine tyrosinase promoter-enhancer expression cassette was introduced into the E1 region of a recombinant adenovirus to generate the virus AdmTyr-beta gal. This virus grows to high titer and maintains transcriptional specificity for pigment cell lineages. Strikingly, AdmTyr-beta gal is extremely active in human melanoma cells, in some cases exceeding the transcriptional activity of a cytomegalovirus promoter-driven recombinant beta-gal virus. Tissue specificity of gene expression is maintained, with very low levels observed in tumors and primary human cells derived from other lineages. These data provide evidence that it is possible to target human melanoma cells with great efficiency and specificity using high-titer recombinant adenovirus vectors.

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

CD4+ T-cell immunity to mutated ras protein in pancreatic and colon cancer patients.

Mutated p21 ras proteins contain single substituted amino acid residues and represent cancer-specific proteins. The current study examined whether primed T cell immunity to mutant p21 ras proteins and/or peptides can be detected in patients with pancreatic or colon cancer. Studies focused on the aspartic acid substitution in amino acid position 12 (denoted D12) as the commonest mutation in gastrointestinal malignancy. Peripheral blood lymphocytes from patients or normal individuals were tested for the ability to proliferate in response to normal or mutated ras peptides or proteins. T-cell responses were defined as a stimulation index of > 2.0. Results showed that 7 of 16 (44%) pancreatic cancer patients responded to ras-D12 peptide. Responses to ras-D12 protein were studied in only the last four patients that responded to D12 peptides. Three of the 4 patients that responded to ras-D12 peptide showed a substantial response to p21 ras-D12 protein (stimulation indices of 12, 8, and 24). Specificity was validated by examining responses to normal and alternate ras peptides and proteins. T-cell responses to ras-D12 peptides were detected in only 2 of 25 (8%) colon cancer patients. None of 11 normal individuals tested had positive responses to normal or mutant ras p21 proteins and/or peptides. Thus, CD4+ T-cell immunity to the mutated segment of ras protein is present in some patients with gastrointestinal cancer.

Treatment of cancer patients with ex vivo anti-CD3-activated killer cells and interleukin-2.

PURPOSE: This study describes the physiologic and biologic effects resulting from the adoptive transfer of ex vivo anti-CD3-stimulated T-killer cells (T-AK) to patients with advanced cancer in combination with interleukin-2 (IL-2). METHODS: Autologous peripheral-blood mononuclear cells were obtained by leukapheresis and stimulated ex vivo with anti-CD3. The stimulated cells were reinfused at one of three dose levels on the next day (5 x 10(9), 7.5 x 10(9), and 1 x 10(10)). Cell administration was followed by IL-2 given by bolus and continuous infusion (1.5 x 10(6) U/m2 and 3.0 x 10(6) U/m2, respectively) for 7 days, or continuous infusion alone (3.0 x 10(6) U/m2) for 14 days. RESULTS: Pronounced leukocytosis and atypical lymphocytosis were observed with individual values as high as 80,000 and 50,000 cells/microL, respectively. The other major clinical sequelae included a marked lactic acidosis with bicarbonate levels as low as 4.0 mmol/L in some patients, and prolongation of the prothrombin time (PT) and partial thromboplastin time (PTT) due to decreases in clotting factors VII, IX, and X. Antithrombin III levels were also reduced. Hypotension associated with increased serum nitrate and neopterin levels was observed. These toxicities were accompanied by increases in hepatocellular enzymes and creatinine previously described with IL-2. These events occurred at a time when the number of circulating T-AK cells reached their peak. The amount of bolus IL-2 correlated with increases in WBC count (P = .0311), atypical lymphocytes (P = .0241), PT (P = .0006), and PTT (P = .0122). CONCLUSION: Substantial in vivo expansion of activated T lymphocytes was induced by a protocol combining ex vivo activation of peripheral-blood cells with anti-CD3 antibody followed by adoptive transfer and IL-2 administration. The synchronous expansion of these T cells superimposed on diminished liver and kidney function from IL-2 can cause profound but reversible metabolic changes.

Dose-related immunologic effects of levamisole in patients with cancer.

PURPOSE: This phase I study was conducted to determine the maximum-tolerated dose (MTD) and the immunologic properties of levamisole in cancer patients when administered alone and in combination with interferon gamma (IFN-gamma). PATIENTS AND METHODS: Twenty patients with advanced cancer and 36 patients with completely resected melanoma (n = 33) or renal cell cancer (n = 3) received levamisole orally every other day for six doses at 1.0, 2.5, 5.0, or 10.0 mg/kg. Ten days later, patients restarted levamisole and began IFN-gamma 0.1 mg/m2 by subcutaneous injection every other day. Blood samples were collected for measurement of neopterin and soluble interleukin-2 receptor (sIL-2R), and for flow-cytometric analysis. RESULTS: The MTD of levamisole was 5 mg/kg, and this was not changed by the addition of IFN-gamma. Dose-related increases in serum levels of neopterin and sIL-2R were noted. Multiple doses of > or = 5 mg/kg of levamisole were required to elicit immune changes, which were more prominent in patients with minimal tumor burdens. Increased expression of CD64 and class I and class II major histocompatibility antigens on monocytes was also observed. The combination of IFN-gamma and levamisole did not result in greater immunologic effects than those observed in previous trials of IFN-gamma alone. CONCLUSION: Levamisole induces dose-related immunologic changes in patients with large or minimal tumor burdens. These changes may be involved in the beneficial effects noted in recent adjuvant trials of levamisole. Ongoing clinical trials should correlate immune changes with response, and trials exploring different schedules of administration using higher, more immunologically active, doses of levamisole should be performed.

The toxic and hematologic effects of interleukin-1 alpha administered in a phase I trial to patients with advanced malignancies.

PURPOSE: A phase I trial was undertaken because interleukin-1 alpha (IL-1 alpha) possesses antiproliferative, immunostimulatory, antiinfection, myeloprotective, and myelorestorative properties that could be beneficial in cancer treatment. PATIENTS AND METHODS: In this phase I trial, IL-1 alpha was administered intravenously (IV) during a 15-minute period daily for 7 days to patients with advanced solid malignancies. RESULTS: The maximum-tolerated dose (MTD) of IL-1 alpha alone was 0.3 microgram/kg. A second group of patients received indomethacin plus IL-1 alpha based on preclinical studies, which indicated that indomethacin could abrogate IL-1 alpha-induced hypotension; however, the MTD of IL-1 alpha plus indomethacin was 0.1 microgram/kg lower than IL-1 alpha alone. Fever, chills, headache, nausea, vomiting, and myalgia were common but were not dose-limiting. Hypotension resulted from a marked decrease in systemic vascular resistance and required pressors at 0.3 and 1.0 micrograms/kg IL-1 alpha. Dose-limiting toxicities included hypotension, myocardial infarction, confusion, severe abdominal pain, and renal insufficiency. IL-1 alpha treatment caused a significant, dose-related increase in the total WBC count (mainly segmented neutrophils and neutrophilic bands). Bone marrow cellularity increased because of enhanced numbers of relatively mature myeloid cells and megakaryocytes. Platelet counts decreased during therapy but were significantly elevated above baseline values 1 to 2 weeks posttreatment; this may have been an effect of IL-6 that was shown to be induced by IL-1 alpha treatment. Significant increases in triglycerides, cortisol, C-reactive protein, thyroid-stimulating hormone and decreases in cholesterol, testosterone, and protein-C were observed with treatment. CONCLUSION: We conclude that at doses of IL-1 alpha that can be given safely to cancer patients, significant, potentially beneficial hematopoietic effects occur.

Phase I study of subcutaneously administered interleukin-2 in combination with interferon alfa-2a in patients with advanced cancer.

PURPOSE: Although high-dose interleukin-2 (IL-2) can produce durable remissions in a subset of responding patients with renal cell carcinoma (RCC), this occurs in the setting of significant toxicity. The purpose of this study is to define the maximum-tolerated dosage (MTD) of IL-2 and interferon alfa-2a (IFN alpha-2a) that can be administered chronically on an outpatient basis. PATIENTS AND METHODS: Fifty-three patients with advanced cancer of variable histology with good prognostic features were treated in six cohorts. Patients in cohorts one through five received IL-2 (1.5 or 3.0 x 10(6) million units (mU)/m2) Monday through Friday and IFN alpha-2a (1.5 or 3 x 10(6) mU/m2) daily for a 4-week cycle. In cohort six, IFN alpha-2a was given three times a week. Immunologic monitoring, including serum levels of soluble IL-2 receptor (sIL-2R) and neopterin, flow cytometry, and natural killer cell (NK) activity, were measured. Patients were evaluated for toxicity, response, and survival. RESULTS: Almost all patients developed grade I/II toxicities commonly associated with cytokine therapy. Symptoms were most severe with the first treatment of each week. Dose-limiting toxicities included grade III fatigue, hypotension, and creatinine elevations. The MTD was 1.5 mU/m2 daily x 5 given subcutaneously repeated weekly for IL-2 and 1.5 mU/m2 daily subcutaneously (dose level 3) for IFN. Six of 25 assessable patients with RCC (24%) achieved a partial response (PR), including four of eight patients who were previously untreated. There were no objective responses in patients with other tumors, including 12 melanoma patients. CONCLUSION: IL-2 and IFN alpha-2a can be given with tolerable toxicities on an outpatient basis and shows significant activity in patients with metastatic RCC.

Melanoma-specific cytotoxicity induced by a tyrosinase promoter-enhancer/herpes simplex virus thymidine kinase adenovirus.

The ability to specifically and efficiently express selected genes in tumor cells is an important goal for cancer gene therapy. Transcriptional targeting of adenovirus to tumor cells, thereby limiting their expression to specific cell types, represents one experimental approach to this problem. We have previously shown that a recombinant adenovirus containing the murine tyrosinase promoter coupled to a dimer of the tyrosinase-enhancer element can target the expression of beta-galactosidase cDNA to melanoma cells. We now report that this same promoter/enhancer cassette can efficiently drive the expression of the herpes simplex virus thymidine kinase gene in melanoma cells. Infection of melanoma cells with the AdmTyr-tk virus along with subsequent ganciclovir treatment induces S phase cell cycle arrest associated with a profound change in cell size and morphology. Treated cells remain viable for prolonged periods, but clonogenic assays demonstrate that the cell cycle arrest is irreversible. In contrast, nonmelanoma cells are unaffected by this treatment regimen, exhibiting normal growth kinetics, metabolic activity, and cell cycle progression. The therapeutic efficacy of the AdmTyr-tk virus was tested in vivo using a xenograft model of human melanoma. The injection of the AdmTyr-tk virus into established subcutaneous tumor nodules in combination with systemic ganciclovir administration led to a decreased tumor growth rate and to complete tumor regressions in some cases. These studies demonstrate the feasibility of selectively targeting growth-inhibitory genes to melanoma cells in vitro and in vivo.

Cellular and molecular studies in the treatment of murine renal cancer.

Antigen-nonspecific approaches to the use of BRMs for cancer treatment have resulted in only limited success to date. In particular, the use of large numbers of adoptively transferred, broadly cytotoxic LAK cells in combination with IL-2 has been effective for only small subsets of cancer patients. Recent demonstrations of T-lymphocyte-mediated antigen-specific responses against some human tumors, and the more potent effects of these cells in preclinical models, have refocused much of the dialogue for biological therapy to potentiation of T-lymphocyte-mediated antitumor effects. Our studies are using the well-characterized Renca murine renal cancer model to study the induction of antitumor T-lymphocyte-mediated responses, the mechanisms by which positive effects are achieved, and the reasons why T lymphocytes in tumor-bearing mice may not respond as predicted. One possible reason why T-lymphocyte responses may not be triggered easily by tumors could be an impairment of critical nuclear transcription factors. We also are studying two approaches for stimulating T-cells in tumor-conditioned hosts. (1) We have shown that IL-7 has potent costimulatory effects on T cells as well as some antitumor effects. (2) We are developing a comprehensive vaccine-type gene therapy approach whereby T cells and antigen-presenting dendritic cells are recruited through the use of antigen, chemokines and GM-CSF. Studies are in progress to determine whether the activity of these recruited cells can then be potentiated by Renca or fibroblast transfectants that express T-cell costimulatory cytokines (IL-2, IL-4, IL-7, or IL-12). This approach should optimize both MHC class I- and class II-dependent pathways for induction of T-lymphocyte-mediated responses to cancer, and perhaps overcome tumor-induced impairments in the T lymphocyte function.

Activity of single-agent melphalan 220-300 mg/m2 with amifostine cytoprotection and autologous hematopoietic stem cell support in non-Hodgkin and Hodgkin lymphoma.

High-dose chemotherapy using melphalan (HDMEL) is an important component of many conditioning regimens that are given before autologous hematopoietic stem cell transplantation (AHSCT). In contrast to the situation in myeloma, and to a lesser degree acute leukemia, only a very limited published experience exists with the use of HDMEL conditioning as a single agent in doses requiring AHSCT for lymphoma, both Hodgkin lymphoma (HL) and especially non-Hodgkin lymphoma (NHL). Thus, we report results of treating 26 lymphoma patients (22 with NHL and four with HL) with HDMEL 220-300 mg/m(2) plus amifostine (AF) cytoprotection and AHSCT as part of a phase I-II trial. Median age was 51 years (range 24-62 years); NHL histology was varied, but was aggressive (including transformed from indolent) in 19 patients, indolent in two patients and mantle cell in one. All 26 patients had been extensively treated; 11 were refractory to the immediate prior therapy on protocol entry and two had undergone prior AHSCT. All were deemed ineligible for other, 'first-line' AHSCT regimens. Of these 26 patients, 22 survived to initial tumor evaluation on D +100. At this time, 13 were in complete remission, including four patients who were in second CR before HDMEL+AF+AHSCT. Responses occurred at all HDMEL doses. Currently, seven patients are alive, including five without progression, with a median follow-up in these latter patients of D +1163 (range D +824 to D +1630); one of these patients had a nonmyeloablative allograft as consolidation on D +106. Conversely, 14 patients relapsed or progressed, including five who had previously achieved CR with the AHSCT procedure. Two patients, both with HL, remain alive after progression; one is in CR following salvage radiotherapy. Six patients died due to nonrelapse causes, including two NHL patients who died while in CR. We conclude that HDMEL+AF+AHSCT has significant single-agent activity in relapsed or refractory NHL and HL. This experience may be used as a starting point for subsequent dose escalation of HDMEL (probably with AF) in established combination regimens.

Hepatobiliary lymphoepithelioma-like carcinoma associated with Epstein-Barr virus.

We describe the clinical and pathologic features of a lymphoepithelioma-like carcinoma (LELC) that originated in the hepatobiliary system. A woman, aged 71 years, was first seen with a noncholangiolar adenocarcinoma with lymphoid stroma, which was discovered by open liver biopsy in 1993. In 1995, retroperitoneal and peripancreatic lymph nodes were involved by LELC. There currently is no evidence of distant metastasis outside the hepatobiliary peripancreatic region. Review of the biopsy material revealed a well-differentiated adenocarcinoma with transition into LELC. Epstein-Barr virus (EBV) transcripts were expressed in all histologic phases of the tumor by in situ hybridization using immunoalkaline phosphatase-labeled oligonucleotide probes for EBV-encoded RNA 1 on formalin-fixed, paraffin-embedded sections. Polymerase chain reaction analysis for EBV nuclear antigen 2 was consistent with EBV strain type A. The LMP-1 gene was found to be wild type by polymerase chain reaction analysis. To our knowledge, this is the first report of a primary hepatobiliary adenocarcinoma associated with EBV infection that transformed into an undifferentiated LELC.

Sequence analysis of a 1296-nucleotide plasmid from Xylella fastidiosa.

A cryptic plasmid from Xylella fastidiosa strain ATCC 35868 was cloned, sequenced, and the sequence entered into GenBank (U71220). The plasmid is 1296 nucleotides in length with 55% GC content and three open reading frames. A plasmid with sequence homology was found in only one other strain of X. fastidiosa, ATCC 35878. Searches of the GenBank reveal nucleotide sequence homology with plasmid pNKH43 from Stenotrophomonas maltophilia, and amino acid sequence homology with phage Pf3 from Pseudomonas aeruginosa, plasmid pAP12875 from Acetobacter pasteurianus, and plasmid pVT736-1 from Actinobacillus actinomycetemcomitans.