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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. [ABSTRACT FROM AUTHOR]

Background: Bortezomib is active in heavily pretreated multiple myeloma patients; the dose-limiting toxicity is peripheral neuropathy (PN)., Methods: The authors retrospectively reviewed the incidence, severity, and risk factors for PN in 78 patients who received bortezomib. The median age was 57 years (range, 33-80 years), 62% of patients were men, and 37% of patients were African Americans. Seventeen patients (22%) had diabetes mellitus (DM), and 66 patients (85%) had received thalidomide. Before bortezomib treatment, 37% of the patients reported subjective, grade 1 or 2 PN. Patients received bortezomib alone (n = 10 patients) plus dexamethasone (n = 36 patients) and thalidomide (n = 20 patients) or chemotherapy (n = 12 patients). PN affected 52% of patients, including grade 3 and 4 PN in 15% and 7%, respectively., Results: Twelve patients stopped bortezomib because of side effects that included PN (n = 9 patients), diarrhea (n = 2 patients) and cytomegalovirus pneumonia (n = 1 patient); 11 patients had dose reductions because of PN. Grade 4 PN affected 6 patients (sensory, n = 4 patients; motor/sensory, n = 2 patients). The onset of grade 4 PN was sudden rather than cumulative. Factors that were predictive of PN grade were baseline PN (P = .002), prior thalidomide use (P = .03), and the presence of DM (P = .03). Multiple myeloma responses included complete, near complete, and partial responses in 5% of patients, 10% of patients, and 27% of patients, respectively. Responses were independent of PN and of whether bortezomib was combined with chemotherapy or thalidomide. Patients remained on therapy longer for a median of 5 cycles (range, 2-36 cycles) when they received bortezomib plus thalidomide versus 3 cycles (range, 1-19 cycles) for the other combinations. PN therapy was mostly supportive. It was noteworthy that 6 of 9 patients with PN who received lenalidomide as salvage therapy after bortezomib had significant improvement in their symptoms., Conclusions: The risk of bortezomib-related PN was greater in patients who had PN and DM at baseline. The authors concluded that an unexpected, symptomatic improvement of PN on lenalidomide is worth further investigation.

Flavopiridol downregulates anti-apoptotic regulators including Mcl-1, upregulates p53, globally attenuates transcription through inhibition of P-TEFb, binds to DNA, and inhibits angiogenesis. Eighteen myeloma patients were treated with 1-hour flavopiridol infusions for 3 consecutive days every 21 days. Immunoblotting for Mcl-1, Bcl-2, p53, cyclin D, phosphoRNA polymerase II and phosphoSTAT 3 was conducted on myeloma cells. Ex vivo flavopiridol treatment of cells resulted in cytotoxicity, but only after longer exposure times at higher flavopiridol concentrations than were anticipated to be achieved in vivo. No anti-myeloma activity was observed in vivo. As administered, flavopiridol has disappointing activity as a single agent in advanced myeloma.

Purpose: Bcl-2 regulates the mitochondrial apoptosis pathway that promotes chemotherapy resistance. Bcl-2 antisense oligonucleotide, G3139, targets Bcl-2 mRNA., Patients and Methods: G3139 was administered (3 to 7 mg/kg/d for 7 days) by continuous intravenous infusion. On day 4, patients started thalidomide (100 to 400 mg as tolerated) and dexamethasone (40 mg daily for 4 days) on 21-day cycles for three cycles. Stable and responding patients continued on 35-day cycles for 2 years., Results: Thirty-three patients (median age, 60 years; range, 28 to 76 years) received 220 cycles. Patients received a median of three prior regimens including thalidomide (n = 15) and stem-cell transplantation (n = 31). The regimen was well tolerated; the median number of cycles per patient was eight (range, one to 16+ cycles). Toxicities included reversible increase in creatinine, thrombocytopenia, neutropenia, fatigue, anorexia, constipation, fever, neuropathy, edema, electrolyte disturbances, and hyperglycemia. Fifty-five percent of patients had objective responses, including two complete responses (CRs), four near CRs (positive immunofixation), and 12 partial responses; six patients had minimal responses (MRs). Of patients who received prior thalidomide, seven had objective responses, and three had MRs. The median duration of response was 13 months, and estimated progression-free and overall survival times were 12 and 17.4 months, respectively. Responding patients had significant increase in polyclonal immunoglobulin M (P = .005), indicating innate immune system activation. Western blot analysis of Bcl-2 protein isolated from myeloma cells before and after G3139 demonstrated a decrease of Bcl-2 levels in three of seven patients compared with six of nine patients using reverse transcriptase polymerase chain reaction., Conclusion: G3139, dexamethasone, and thalidomide are well tolerated and result in encouraging clinical responses in relapsed multiple myeloma patients.

Apoptosis is characterized by chromatin condensation, DNA cleavage, redistribution of phosphatidylserine, and apoptotic body formation via an actin-dependent process. We describe a novel form of the execution phase of apoptosis in human multiple myeloma cells that is morphologically and mechanistically distinct from classical apoptosis, but is caspase-dependent and inhibited by IL-6 and overexpression of Bcl-2. Electron microscopic analysis of these cells demonstrated chromatin condensation without nuclear fragmentation, and 'partitioning' of cell constituents into two components: a single, large bleb containing soluble protein and free ribosomes, and a region containing the nucleus, organelles, and RER. In some cases, the bleb separated, becoming a free vesicle exhibiting random kinetic motion. These morphologic features occurred despite inhibition of the actin and tubulin cytoskeletal systems. This novel form of apoptosis, called partitioning apoptosis, was observed in a variety of tumor cell types and in primary cells. The execution phase of apoptosis can occur in a manner that is morphologically and mechanistically distinct from classical apoptosis.

Purpose: Inosine monophosphate dehydrogenase (IMPDH) inhibitors have been used to induce leukemia blast cell differentiation but have not been tested in multiple myeloma for activity. Currently, available IMPDH inhibitor, mycophenolate mofetil (MMF), which is known as an immunosuppressant, was shown to induce apoptosis in myeloma cell lines. On the basis of our preclinical studies, we designed a clinical study to test our hypothesis that MMF has antimyeloma activity., Experimental Design: A Phase I MMF dose escalation study was conducted in relapsed and refractory myeloma patients who had documented disease progression by myeloma markers or bone marrow plasmacytosis to determine the maximum tolerated dose, toxicities, and efficacy of the drug. To assess the activity of IMPDH inhibition in the myeloma cells of patients, we measured intracellular nucleotide triphosphate levels by high-performance liquid chromatography-based analysis and examined the correlation with clinical response., Results: Among the 11 study patients, MMF was generally well tolerated and was administered up to a maximum dose of 5 g/day. The most common toxicity was grade 1 fatigue (n = 4, 36%). One patient had a partial response (3 g/day), four patients had stable disease, and six patients had progression of disease. There was a statistically significant difference in the intracellular dGTP level changes between the stable disease/partial response group versus progression of disease., Conclusions: MMF at 1 to 5 g/day daily dose is well tolerated by patients with relapsed and refractory multiple myeloma patients. Positive correlation between clinical response and depletion of intracellular dGTP level was shown. Future drug development to target this enzyme maybe useful in treating myelomas.

Bcl-2 family proteins protect against a variety of forms of cell death, including acute oxidative stress. Previous studies have shown that overexpression of the antiapoptotic protein Bcl-2 increases cellular redox capacity. Here we report that cell lines transfected with Bcl-2 paradoxically exhibit increased rates of mitochondrial H(2)O(2) generation. Using isolated mitochondria, we determined that increased H(2)O(2) release results from the oxidation of reduced nicotinamide adenine dinucleotide-linked substrates. Antiapoptotic Bcl-2 family proteins Bcl-xL and Mcl-1 also increase mitochondrial H(2)O(2) release when overexpressed. Chronic exposure of cells to low levels of the mitochondrial uncoupler carbonyl cyanide 4-(triflouromethoxy)phenylhydrazone reduced the rate of H(2)O(2) production by Bcl-xL overexpressing cells, resulting in a decreased ability to remove exogenous H(2)O(2) and enhanced cell death under conditions of acute oxidative stress. Our results indicate that chronic and mild elevations in H(2)O(2) release from Bcl-2, Bcl-xL, and Mcl-1 overexpressing mitochondria lead to enhanced cellular antioxidant defense and protection against death caused by acute oxidative stress.

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.

Mcl-1 is a critical antiapoptotic survival factor for human multiple myeloma (MM). We examined the importance of IL-6 for Mcl-1 expression in five MM cell lines and in primary MM cells from 14 patients. While culture of MM.1S cells in IL-6 did induce Mcl-1 expression, four other MM cell lines exhibited high levels of Mcl-1 expression that were unaffected by IL-6. Similarly, Mcl-1 expression in 10 of 14 primary MM isolates was found to be IL-6-independent. An analysis of the mechanisms responsible for IL-6-independent Mcl-1 expression was undertaken. ERK1/2 activity did not influence Mcl-1 expression, distinct from Mcl-1 regulation that occurs during myeloid differentiation from hematopoietic progenitor cells. Inhibition of the PI3K pathway led to growth inhibition of 8226 and ANBL-6 cells without reduction of Mcl-1 levels, and high level Mcl-1 expression was maintained in the absence of activated STAT3. Analysis of the transcriptional activity of 5'-regulatory sequences from the human Mcl-1 gene in MM cells demonstrated high levels of IL-6-independent indicator gene activation as predicted. These data demonstrate that the mechanisms regulating Mcl-1 levels in MM cells are heterogeneous, and are often independent from IL-6 signaling pathways.

Even during this past year, further advances have been made in understanding the molecular genetics of the disease, the mechanisms involved in the generation of myeloma-associated bone disease and elucidation of critical signaling pathways as therapeutic targets. New agents (thalidomide, Revimid, Velcade) providing effective salvage therapy for end-stage myeloma, have broadened the therapeutic armamentarium markedly. As evidenced in Section I by Drs. Kuehl and Bergsagel, five recurrent primary translocations resulting from errors in IgH switch recombination during B-cell development in germinal centers involve 11q13 (cyclin D1), 4p16.3 (FGFR3 and MMSET), 6p21 (cyclin D3), 16q23 (c-maf), and 20q11 (mafB), which account for about 40% of all myeloma tumors. Based on gene expression profiling data from two laboratories, the authors propose 5 multiple myeloma (MM) subtypes defined by the expression of translocation oncogenes and cyclins (TC molecular classification of MM) with different prognostic implications. In Section II, Drs. Barillé-Nion and Bataille review new insights into osteoclast activation through the RANK Ligand/OPG and MIP-1 chemokine axes and osteoblast inactivation in the context of recent data on DKK1. The observation that myeloma cells enhance the formation of osteoclasts whose activity or products, in turn, are essential for the survival and growth of myeloma cells forms the basis for a new treatment paradigm aimed at reducing the RANKL/OPG ratio by treatment with RANKL inhibitors and/or MIP inhibitors. In Section III, Dr. Fenton reviews apoptotic pathways as they relate to MM therapy. Defects in the mitochrondrial intrinsic pathway result from imbalances in expression levels of Bcl-2, Bcl-XL and Mcl-1. Mcl-1 is a candidate target gene for rapid induction of apoptosis by flavoperidol. Antisense oglionucleotides (ASO) lead to the rapid induction of caspace activity and apoptosis, which was potentiated by dexamethasone. Similar clinical trials with Bcl-2 ASO molecules alone and in combination with doxorubicin and dexamethasone or thalidomide showed promising results. The extrinsic pathway can be activated upon binding of the ligand TRAIL. OPG, released by osteoblasts and other stromal cells, can act as a decoy receptor for TRAIL, thereby blocking its apoptosis-inducing activity. MM cells inhibit OPG release by stromal cells, thereby promoting osteoclast activation and lytic bone disease (by enhancing RANKL availability) while at the same time exposing themselves to higher levels of ambient TRAIL. Thus, as a recurring theme, the relative levels of pro- versus anti-apoptotic molecules that act in a cell autonomous manner or in the milieu of the bone marrow microenvironment determine the outcome of potentially lethal signals. In Section IV, Dr. Barlogie and colleagues review data on single and tandem autotransplants for newly diagnosed myeloma. CR rates of 60%-70% can be reached with tandem transplants extending median survival to approximately 7 years. Dose adjustments of melphalan in the setting of renal failure and age > 70 may be required to reduce mucositis and other toxicities in such patients, especially in the context of amyloidosis with cardiac involvement. In Total Therapy II the Arkansas group is evaluating the role of added thalidomide in a randomized trial design. While data are still blinded as to the contribution of thalidomide, the overriding adverse importance of cytogenetic abnormalities, previously reported for Total Therapy I, also pertain to this successor trial. In these two-thirds of patients without cytogenetic abnormalities, Total Therapy II effected a doubling of the 4-year EFS estimate from 37% to 75% (P <.0001) and increased the 4-year OS estimate from 63% to 84% (P =.0009). The well-documented graft-vs-MM effect of allotransplants can be more safely examined in the context of non-myeloablative regimens, applied as consolidation after a single autologous transplant with melphalan 200 mg/m(2), have been found to be much better tolerated than standard myeloablative conditioning rege conditioning regimens and yielding promising results even in the high-risk entity of MM with cytogenetic abnormalities. For previously treated patients, the thalidomide congener Revimid and the proteasome inhibitor Velcade both are active in advanced and refractory MM (approximately 30% PR). Gene expression profiling (GEP) has unraveled distinct MM subtypes with different response and survival expectations, can distinguish the presence of or future development of bone disease, and, through serial investigations, can elucidate mechanisms of actions of new agents also in the context of the bone marrow microenvironment. By providing prognostically relevant distinction of MM subgroups, GEP should aid in the development of individualized treatment for MM.

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of malignant plasma cells with slow proliferative rate but enhanced survival. MM cells express multiple Bcl-2 family members, including Bcl-2, Bcl-XL, and Mcl-1, which are thought to play a key role in the survival and drug resistance of myeloma. The cyclin-dependent kinase inhibitor flavopiridol has antitumor activity against hematopoietic malignancies, including CLL, in which induction of apoptosis was associated with reduced expression of antiapoptotic proteins. Therefore, we sought to characterize the effect of flavopiridol on the proliferation and survival of myeloma cells and to define its mechanisms of action. Treatment of MM cell lines (8226, ANBL-6, ARP1, and OPM-2) with clinically achievable concentrations of flavopiridol resulted in rapid induction of apoptotic cell death that correlated temporally with the decline in Mcl-1 protein and mRNA levels. Levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 protected MM cells from flavopiridol-induced apoptosis. Additional analysis demonstrated that flavopiridol treatment resulted in a dose-dependent inhibition of phosphorylation of the RNA polymerase II COOH-terminal domain, thus blocking transcription elongation. These data indicate that Mcl-1 is an important target for flavopiridol-induced apoptosis of MM that occurs through inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation via the ubiquitin-proteasome pathway.

The evolutionarily conserved Ras/Raf/MEK/ERK pathway is thought to be essential for proliferation of eukaryotic cells. The human multiple myeloma (MM) cell line 8226 encodes an activated K-ras allele and proliferates without requirement for the main MM growth and survival factor IL-6. Surprisingly, the addition of the MEK1/2 inhibitors PD98059 or U0126 to 8226 cultures at doses that block virtually all ERK1/2 activity had minimal effects on the rapid proliferation of this cell line. In contrast, proliferation of the IL-6-dependent MM cell line, ANBL-6 was blocked by PD98059. Levels of activated forms of the other classical MAP kinases (JNK and p38) were very low during MM cell proliferation and, therefore, do not substitute for the mitogenic activities normally regulated by ERK kinases. These data demonstrate that proliferation of 8226 cells does not require ERK1/2 activity, and suggest that IL-6-independent growth of MM may correlate with independence from a requirement for ERK activity. Other signal transduction pathways that appear to regulate cell cycle progression in these cells were examined., (Copyright 2002 Wiley-Liss, Inc.)

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow caused primarily by failure of normal homeostatic mechanisms to prevent the expansion of postgerminal center plasma cells. We have examined the molecular mechanisms that promote the survival of MM cells and have identified a key role for myeloid cell factor-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family. These experiments were initiated by the observation that MM cells were exquisitely sensitive to culture in the presence of actinomycin D: caspase activation occurred within 3 hours of treatment and cells were not protected by interleukin-6, the main MM cell growth and survival factor. Actinomycin D-induced apoptosis was blocked by proteasome inhibitors, suggesting that a labile protein was required for MM cell survival. Further analysis demonstrated that Mcl-1 was likely to be the labile factor governing MM cell survival. Mcl-1 protein levels decreased rapidly after culture in the presence of actinomycin D in concordance with effector caspase activation, but addition of proteasome inhibitors reversed the loss of Mcl-1 and maintained cell viability. The levels of other antiapoptotic proteins, including Bcl-2 and members of the inhibitors-of-apoptosis family, were unaffected by these interventions. Furthermore, Mcl-1 antisense oligonucleotides caused a rapid down-regulation of Mcl-1 protein levels and the coincident induction of apoptosis, whereas overexpression of Mcl-1 delayed actinomycin D-induced apoptosis with kinetics that correlated with expression levels of Mcl-1. These data indicate that Mcl-1 expression is required for the survival of MM cells and may represent an important target for future therapeutics.

Factors that govern host-tumor interaction play a critical role in tumor progression. In previous studies we have shown that oncogenic Ras inhibits the expression of Fas (CD95) and renders Ras-transformed cells resistant to Fas-induced death. We now demonstrate that culture of Ras-transformed cells in the presence of the farnesyltransferase inhibitor (FTI) LB42722 leads to up-regulation of Fas expression, both under basal growth conditions and in the presence of the inflammatory cytokines IFN-gamma and tumor necrosis factor alpha. This is manifested by an increase in fas mRNA, Fas cell surface expression, and Fas-induced apoptosis. Whereas FTI up-regulates expression of FAS in Ras-transformed cells, it inhibits the expression of vascular endothelial growth factor. Culture of Ras-transformed cells in the presence of the histone deacetylase inhibitor trichostatin A resulted in morphological reversion and G(1) arrest (as observed with FTI); however, no induction of Fas was observed. Furthermore, the effects of FTI on Fas-induced death were shown to be independent of RhoB. Therefore, inhibition of oncogenic Ras by FTI can result in two events that alter host-tumor interactions: up-regulation of Fas, rendering tumors more sensitive to immune cytotoxic effector cells, and down-reglation of VEGF, which may inhibit tumor angiogenesis.

The IFN-gamma-inducible proteins monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-gamma-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-gamma. In addition to depending on IFN-gamma, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-gamma R alpha and beta-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-gamma contain increased chemotactic activity for enriched human and mouse CD3(+) T cells, as well as mouse DX5(+) NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

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.

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.

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.

IL-12 is a potent immunoregulatory cytokine that has been shown to mediate tumor regression in a variety of tumor models. We describe the construction of AdCMV-IL-12, a recombinant adenovirus that encodes both subunits of IL-12 under transcriptional control of the CMV promoter. This recombinant virus efficiently infects a wide variety of cell types leading to the production of high levels of biologically active IL-12. Because the liver is a primary site of infection after i.v.-administered adenovirus, we tested the therapeutic efficacy of this virus in a murine hepatic metastasis tumor model. Systemic administration of AdCMV-IL-12 dramatically inhibited the formation of 3-day Renca hepatic metastases (mean of 16 metastases per liver) compared with the control virus AdCMV-betagal (mean of 209) or vehicle alone (mean of 272). Histologic analysis indicated that metastatic growth inhibition was accompanied by a dramatic perivascular infiltrate consisting of T cells, macrophages, and neutrophils. Therapeutic efficacy was not diminished in animals depleted of CD4+ or CD8+ T cells, or in SCID mice, even after NK cell ablation. In the latter case, a hepatic perivascular infiltrate composed of macrophages and neutrophils was observed after AdCMV-IL-12-treatment, while numerous activated Kupffer cells were noted in the hepatic parenchyma. Analysis of therapy-induced changes in hepatic gene expression demonstrated increased levels of IP-10 and Mig RNAs, but no increase in iNOS, Fas, or FasL RNA levels was observed. Our data suggest a model of metastatic growth inhibition mediated by nonlymphocyte effector cells including macrophages and neutrophils and that may involve anti-angiogenic chemokines.

Crosslinking of CD28 receptors on resting T lymphocytes by B7 costimulatory molecules expressed by antigen-presenting cells (APCs) plays a critical role in T-cell activation. Human melanomas express major histocompatibility complex (MHC)-restricted tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTL), yet they remain poorly immunogenic. One mechanism for the failure of T-cell response is the lack of expression of costimulatory molecules by human melanoma cells. We have transfected the B7-1 gene into three HLA-A2-expressing human melanoma cell lines, and studied their capacity to stimulate primary human T cells. B7-expressing melanoma cells were excellent inducers of T-cell proliferation, cytokine production, and cytolytic activity in allogeneic mixed lymphocyte cultures through a process dependent on the function of the T-cell receptor as well as interactions between B7:CD28, CD2:LFA-3, and LFA-1:ICAM-1. Subset analysis demonstrated that CD4+ T cells or addition of exogenous interleukin-2 was required for the induction of CD8+ CTL. Untransfected parental melanoma cells were inert as APCs in these cultures. Rotating stimulation of T cells with the three B7-expressing cell lines led to the generation of T-cell lines that were cytolytic for HLA-A2+ melanoma cells and other HLA-A2+ targets that were pulsed with HLA-A2-restricted MART-1 peptides. These data demonstrate that expression of B7-1 by human melanoma cells converts them into effective APCs for the in vitro induction of MHC-restricted, melanoma-specific CTL.

A number of active prediction planning and execution (APPE) systems have recently been proposed for robotic interception of moving objects. The cornerstone of such systems is the selection of a robot-object rendezvous-point on the predicted object trajectory. Unlike tracking-based systems, which minimize the state difference between the object and the robot at each control period, in this methodology the robot is sent directly to the selected rendezvous-point. A fine-motion tracking strategy would then be employed for grasping the moving object. Herein, a novel strategy for selecting the optimal (earliest) rendezvous-point is presented. For objects with predictable trajectories, this is a significant improvement over previous APPE strategies which select the rendezvous-point from a limited number of non-optimally chosen candidates.

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.

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.

Signaling through the CD28 molecule on T cells by its natural ligand, B7, on APCs has recently been shown to require the presence of an active phosphatidylinositol 3-kinase pathway to mediate some of its costimulatory activities (1-7). Using the phosphatidylinositol 3-kinase inhibitor, wortmannin (WN) (8), on human and murine T cells, we have inhibited B7-1-mediated T cell activation and induced Ag-specific tolerance. The addition of WN and/or the B7-1 antagonist, CTLA4Ig, to primary human T cell cultures stimulated with B7-1-transfected allogeneic melanoma cell lines inhibited the generation of alloantigen-specific proliferative and cytolytic responses in vitro. Subsequent examination of these WN- and CTLA4Ig-treated primary T cell cultures revealed that these lymphocyte populations were tolerized to rechallenge with the priming alloantigens in secondary cultures in the absence of additional inhibitor(s). However, reactivity to a third party allogeneic stimulator remained intact. This WN-induced tolerance was reversed by the addition of high dose IL-2, but not IL-4 or IL-7, to the primary cultures, indicating that T cell anergy, not deletion, was responsible for this phenomenon. In vivo studies using a murine graft-vs-host disease (GVHD) model demonstrated that WN treatment of allogeneic donor lymphocytes in vitro failed to generate a significant GVHD in irradiated mouse recipients compared with control allogeneic donor lymphocytes. These findings suggest potentially novel therapeutic strategies for the prevention of GVHD.

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.

We performed a prospective, randomized study to determine whether subcutaneous administration of interleukin-2 (IL-2) in combination with an autologous renal cell vaccine is feasible and can potentiate antitumor immunity. Seventeen patients with metastatic renal cell carcinoma underwent surgical resection with preparation of an autologous tumor cell vaccine. Patients were vaccinated intradermally twice at weakly intervals with 10(7) irradiated tumor cells plus bacillus Calmette-Guérin, and once with 10(7) tumor cells alone. Patients were randomized to one of three groups: no adjuvant IL-2, low-dose IL-2 (1.2 x 10(6) IU/m2), or high-dose IL-2 (1.2 x 10(7) IU/m2). IL-2 was administered subcutaneously on the day of vaccination and the subsequent 4 days. Immune response was monitored by delayed-type hypersensitivity (DTH) response to tumor cells as compared with normal autologous renal cells. Sixteen of 17 patients received vaccine therapy. Four patients developed cellular immunity specific for autologous tumor cells as measured by DTH responses; two had received no IL-2 and two had received high-dose IL-2. There were two partial responses (PR) noted, both in patients who received high-dose IL-2. One responding patient was DTH(+) and one was negative. A third patient who was DTH(+) after vaccination with no IL-2 had a dramatic PR after receiving IL-2 subcutaneously in a subsequent protocol. Prospective testing of response to recall antigens indicated that only 5 of 12 tested patients were positive, including both clinical responders. These data suggest that subcutaneously administered adjuvant IL-2 does not dramatically augment the immunologic response to autologous renal cell vaccines as determined by the development of tumor-specific DTH response.

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.

The adoptive transfer of anti-CD3-stimulated T killer (T-AK) cells was tested with different bolus and infusional interleukin-2 (IL-2) regimens, and anti-CD3 stimulation procedures to determine immunologic and antitumor effects in patients with a variety of advanced cancers. Indium-111 labeling was used to observe traffic patterns of the infused T-AK. Autologous peripheral blood mononuclear cells were obtained by leukapheresis. Cyclophosphamide (300 mg/m2) was given to most patients immediately after leukapheresis. The harvested cells were activated ex vivo with anti-CD3 overnight or for 4 days, at which time cells were reinfused and an IL-2 regimen was begun. Treatment was repeated 28 days later. This treatment regimen induced significant increases in leukocytes, lymphocytes, and eosinophils in patients in most treatment cohorts. Circulating lymphocytes were predominantly CD3+ T cells with preferential expansion of the CD8+ subset. Patients receiving cells stimulated in vitro for 4 days had significant T-cell lymphocytosis with either infusional or bolus plus infusional IL-2 regimens. T-cell viability was decreased in culture after a second 4-day stimulation with anti-CD3 at day 28; this decrease could be prevented by adding IL-2 to the culture media. Cells stimulated overnight required both bolus and infusional IL-2 to show an atypical lymphocytosis in vivo. Overnight-stimulated T-AK did not show decreases in in vitro viability at the day 28 restimulation. Indium-III-labeled cells trafficked to the liver, spleen, and bone marrow. No increase in uptake was observed in tumor deposits. There were 2 patients with partial responses, 5 with minor responses, 19 with stable disease, and 88 with progressive disease. The length of in vitro anti-CD3 stimulation, and the dose and timing of IL-2 administration in vivo results in different circulating leukocyte populations after adoptive T-AK infusion. Generally, the CD8+ T-cell subset was preferentially expanded by this treatment approach. Repeated ex vivo stimulation with anti-CD3 may cause cell death.

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.

CD28 activation by antibody-mediated ligation has been shown to provide an important co-stimulatory signal for T cell adhesion to purified protein ligands. However, the effect of CD28 ligation by one of its natural ligands, B7.1, on T cell adhesion to other cells has not been studied. Therefore, in the present manuscript, we characterized the adhesive interactions between human T cells and B7.1-transfected major histocompatibility complex class II+ and class II- melanoma cells. In our studies, human T cells and T cell clones adhered to B7.1-transfected melanoma cells, but not to untransfected parental cells. The adhesive reaction in this model was rapid, occurring within 15 min, and was inhibited by anti-B7.1 antibody and soluble CTLA-4 immunoglobulin. Antibody inhibition studies demonstrated that adhesion between T cells and B7.1-transfected melanoma cells was mediated by interactions between LFA-1:ICAM-1 and CD2:LFA-3. Inhibition by pharmacological agents demonstrated that the CD28-induced adhesion required specific intracellular signaling events. A protein kinase C inhibitor, staurosporin, significantly inhibited T cell binding to transfected melanoma cells, while cyclosporin A and wortmannin, an inhibitor of phosphatidylinositol-3-kinase, did not. These results suggest that the presence of B7 on various cell populations may activate lymphocytes to adhere better, thus promoting activation, cytolysis, and migration.

Background: Interleukin-2 (IL-2) has been used successfully in the treatment of some patients with metastatic renal cell carcinoma and melanoma, with a partial response rate of 15%-20%. It produces a well documented spectrum of side effects. Autoimmune diseases have been associated with IL-2 immunotherapy and the development of autoimmune thyroiditis may correlate with antitumor clinical response., Methods: A patient with metastatic renal cell carcinoma is described who developed a polymyositis-like myopathy after an autologous tumor vaccine and IL-2 therapy., Results: The patient had a delayed response for 15 months after developing this previously unreported toxicity., Conclusions: To the authors' knowledge, this represents the first reported case of necrotizing myositis in association with IL-2 therapy. Subsequent continuous partial response of the advanced malignancy was observed for 15 months. In this case, IL-2 may have broken tolerance to both normal muscle cells and tumor cells.

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.

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.

The spectrum of MAGE gene expression in the human melanoma cell line DM150 was examined using reverse transcription polymerase chain reaction and cDNA cloning. We have isolated five full-length cDNAs from DM150 which were identified as MAGE-1, MAGE-3, MAGE-12 and two previously undescribed MAGE genes, MAGE-3b and MAGE-X2. DNA sequence analysis of the coding regions of the MAGE-3b and MAGE-X2 genes revealed 83% and 88% identity with MAGE-1, while MAGE-3b was 98% homologous with the full length MAGE-3 clone. The predicted amino acid sequences of MAGE-X2 and MAGE-3b contain consensus HLA-A1 peptide binding motifs, suggesting that, like MAGE-1, they may code for tumor-associated antigens. In addition, a nonamer peptide encoded by both the MAGE-3 and MAGE-12 genes was shown by direct binding studies to contain an aggretope for HLA-A2.

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.

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.

Background: Thrombocytopenia is a frequent side effect of cancer chemotherapy and commonly limits attempts to escalate drug doses. To determine whether interleukin-1 alpha could ameliorate carboplatin-induced thrombocytopenia, we combined it with high-dose carboplatin in 43 patients with advanced neoplasms., Methods: High-dose carboplatin (800 mg per square meter of body-surface area) was administered alone to a control group. Subsequent patients were randomly assigned to receive the same dose of carboplatin with interleukin-1 alpha, administered either before or after carboplatin. Interleukin-1 alpha was given intravenously at a dose of 0.03, 0.1, or 0.3 microgram per kilogram of body weight per day for five days., Results: Carboplatin alone consistently produced thrombocytopenia with a median nadir of 19,000 platelets per cubic millimeter and a median of 10 days with less than 100,000 platelets per cubic millimeter. All 15 patients receiving interleukin-1 alpha before carboplatin had similar findings. In contrast, 5 of the 15 patients given one of the two higher doses of interleukin-1 alpha after carboplatin had minimal thrombocytopenia (nadir, 91,000 to 332,000 platelets per cubic millimeter). In the 10 patients given 0.3 microgram of interleukin-1 alpha per kilogram after carboplatin treatment, the platelet count recovered to 100,000 per cubic millimeter significantly earlier than in either the control group (P = 0.002) or the patients who received interleukin-1 alpha before carboplatin (P = 0.003), with the median times to recovery in the three groups being 16, 21, and 23 days, respectively. At the highest dose of interleukin-1 alpha, toxicity was substantial (but reversible), requiring inpatient support for hypotension, supraventricular arrhythmias, and pulmonary-capillary leak., Conclusions: Interleukin-1 alpha can accelerate the recovery of platelets after high-dose carboplatin therapy and may be clinically useful in preventing or treating thrombocytopenia induced by chemotherapy.

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.

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.

Objective: Based upon their individual clinical activity and combined effects in animal models or in vitro, we wished to evaluate a regimen of cisplatin, interferon-alpha, and IL-2 in patients with metastatic melanoma., Design: Phase II pilot study., Setting: Referral-based US Government clinical research unit., Patients: Nine patients with metastatic malignant melanoma., Intervention: Cisplatin 75-100 mg/m2 was administered intravenously over 30 minutes on days 1 and 8. Interferon-alpha 2a 5 Mu/m2 body surface area (BSA) was given subcutaneously for 4 days beginning 1 day before each dose of cisplatin. Beginning on day 15 and day 22, IL-2 was administered by intravenous continuous infusion at 3 Mu/m2 BSA/d for 96 hours and by daily intravenous bolus concurrent with daily subcutaneous doses of interferon-alpha 2a., Main Outcome Measures: Antitumor response and toxicities., Results: The study was stopped due to renal and hematopoietic toxicity and severe, delayed nausea and vomiting associated with the cisplatin-interferon treatment. Three of 9 patients achieved a partial response (duration 2.5, 4, 14+ months), and an additional patient had a 50% reduction in measurable tumor volume before undergoing resection of residual disease. Overall response rate was 45%., Conclusion: This regimen was associated with excessive toxicity, and the lack of complete responses in a patient cohort with favorable characteristics for response (good performance status, predominance of skin and lymph node metastatic sites) suggests that it had no advantage over less toxic treatment regimens., Registration: National Cancer Institute/Cancer Therapy Evaluation Program T89-0137.

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.