Phase I study of pazopanib and vorinostat: a therapeutic approach for inhibiting mutant p53-mediated angiogenesis and facilitating mutant p53 degradation.

BACKGROUND: We carried out a phase I trial of the vascular endothelial growth factor inhibitor pazopanib and the histone deacetylase inhibitor vorinostat to determine the safety and efficacy. Because these agents are known to target factors activated by TP53 mutation and facilitate mutant p53 degradation, a subgroup analysis may be interesting in patients with TP53 mutant malignancies. PATIENTS AND METHODS: Patients with advanced solid tumors (n = 78) were enrolled following a 3 + 3 design, with dose expansion for those with responsive tumors. Hotspot TP53 mutations were tested when tumor specimens were available. RESULTS: Adverse events of ≥grade 3 included thrombocytopenia, neutropenia, fatigue, hypertension, diarrhea and vomiting. Overall, the treatment produced stable disease for at least 6 months or partial response (SD ≥6 months/PR) in 19% of the patients, median progression-free survival (PFS) of 2.2 months, and median overall survival (OS) of 8.9 months. In patients with detected hotspot TP53 mutant advanced solid tumors (n = 11), the treatment led to a 45% rate of SD ≥6 months/PR (1 PR and 3 SD ≥6 months), median PFS of 3.5 months, and median OS of 12.7 months, compared favorably with the results for patients with undetected hotspot TP53 mutations (n = 25): 16% (1 PR and 3 SD ≥6 months, P = 0.096), 2.0 months (P = 0.042), and 7.4 months (P = 0.1), respectively. CONCLUSION: The recommended phase II dosage was oral pazopanib at 600 mg daily plus oral vorinostat at 300 mg daily. The preliminary evidence supports further evaluation of the combination in cancer patients with mutated TP53, especially in those with metastatic sarcoma or metastatic colorectal cancer. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov, NCT01339871.

Melanoma patients in a phase I clinic: molecular aberrations, targeted therapy and outcomes.

BACKGROUND: The purpose of the study was to assess the outcome of patients with advanced melanoma treated with matched molecularly targeted therapy. PATIENTS AND METHODS: We reviewed 160 consecutive patients with metastatic melanoma treated in the phase I program (N = 35 protocols). Treatment was considered to be 'matched' (N = 84) if at least one drug in the regimen was known to inhibit the functional activity of at least one of the patient's mutations. RESULTS: Of 160 patients, 134 (83.7%) had adequate tissue for molecular analysis; 69% (110 of 160) had ≥1 mutation: 61.2% (82 of 134), BRAF; 20.7% (23 of 111), NRAS; 2.6% (2 of 77), KIT; 2.3% (1 of 44), KRAS; 20% (1 of 5), GNAQ; 11.1% (1 of 9), P53 and 2.6% (1 of 39), coexisting mutations in BRAF and PIK3CA. Eighty-four patients (52.4%) were treated with matched-targeted agents, most of whom had BRAF mutations (N = 74). Twenty-six percent of patients (41 of 160) achieved a complete or partial remission (CR/PR) [40% (34 of 84)) on a matched phase I protocol versus 9.2% (7 of 76) for those on a non-matched study (P ≤ 0.0001)]. The median progression-free survival (PFS) (95% CI) was longer for patients treated on a matched phase I trial than on their prior first standard treatment [5.27 (4.10, 6.44) versus 3.10 (1.92, 4.28) months, P = 0.023], but not on non-matched phase I treatment. Multivariable analysis showed that matched therapy was an independent predictor of higher CR/PR rates, prolonged PFS and survival. CONCLUSIONS: For melanoma patients, especially those with BRAF mutations, administering molecularly matched agents can be associated with better outcomes, including longer PFS compared with their first-line systemic therapy.

A T cell-independent antitumor response in mice with bone marrow cells retrovirally transduced with an antibody/Fc-gamma chain chimeric receptor gene recognizing a human ovarian cancer antigen.

In order to treat common cancers with immunotherapy, chimeric receptors have been developed that combine the tumor specificity of antibodies with T-cell effector functions. Previously, we demonstrated that T cells transduced with a chimeric receptor gene against human ovarian cancer were able to recognize ovarian cancer cells in vitro and in vivo. We now report that recipients of bone marrow cells transduced with these genes exhibited significant antitumor activity in vivo. Moreover, in vivo depletion of T cells in reconstituted mice did not affect antitumor activity, suggesting that other immune cells expressing the chimeric receptor gene may play an important role in tumor rejection.

Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Lysis of ovarian cancer cells by human lymphocytes redirected with a chimeric gene composed of an antibody variable region and the Fc receptor gamma chain.

To expand the spectrum of recognition of effector lymphocytes and to redirect them towards predefined targets, we have altered the specificity of human tumor-infiltrating lymphocytes (TIL) through stable modification with chimeric receptor genes consisting of single-chain antibody variable regions linked to the gamma subunit common to the immunoglobulin (Ig)G and IgE Fc receptors. Using either hapten or ovarian carcinoma-specific monoclonal antibodies, we constructed chimeric receptor genes and retrovirally introduced them into CD8+ TIL. Redirected TIL specifically lysed trinitrophenyl-labeled Daudi or a human ovarian carcinoma cell line (IGROV-1), and secreted granulocyte/macrophage colony-stimulating factor upon stimulation with the appropriate antigen. This strategy may allow new approaches towards the adoptive immunotherapy of cancer in humans.

Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective against established pulmonary metastases.

Dendritic cells (DCs) are bone marrow-derived leukocytes that function as potent antigen presenting cells capable of initiating T cell-dependent responses from quiescent lymphocytes. DC pulsed with tumor-associated antigen (TAA) peptide or protein have recently been demonstrated to elicit antigen-specific protective antitumor immunity in a number of murine models. Transduction of DCs with TAA genes may allow stable, prolonged antigen expression as well as the potential for presentation of multiple, or unidentified, epitopes in association with major histocompatibility complex class I and/or class II molecules. To evaluate the potential efficacy of retrovirally transduced DCs, bone marrow cells harvested from BALB/c mice were transduced with either a model antigen gene encoding beta-galactosidase (beta-gal) or a control gene encoding rat HER-2/neu (Neu) by coculture with irradiated ecotropic retroviral producer lines. Bone marrow cells were differentiated into DC in vitro using granulocyte/macrophage colony-stimulating factor and interleukin-4. After 7 d in culture, cells were 45-78% double positive for DC phenotypic cell surface markers by FACS(R) analysis, and DC transduced with beta-gal were 41-72% positive for beta-gal expression by X-gal staining. In addition, coculture of beta-gal transduced DC with a beta-gal-specific T cell line (CTLx) resulted in the production of large amounts of interferon-gamma, demonstrating that transduced DCs could process and present endogenously expressed beta-gal. DC transduced with beta-gal and control rat HER-2/neu were then used to treat 3-d lung metastases in mice bearing an experimental murine tumor CT26.CL25, expressing the model antigen, beta-gal. Treatment with beta-gal-transduced DC significantly reduced the number of pulmonary metastatic nodules compared with treatment with Hank's balanced salt solution or DCs transduced with rat HER-2/neu. In addition, immunization with beta-gal-transduced DCs resulted in the generation of antigen-specific cytotoxic T lymphocytes (CTLs), which were significantly more reactive against relevant tumor targets than CTLs generated from mice immunized with DCs pulsed with the Ld-restricted beta-gal peptide. The results observed in this rapidly lethal tumor model suggest that DCs transduced with TAA may be a useful treatment modality in tumor immunotherapy.

Cytokines and angiogenic factors in patients with metastatic renal cell carcinoma treated with interferon-alpha: association of pretreatment serum levels with survival.

BACKGROUND: To correlate serum cytokine and angiogenic factor (CAF) levels with overall survival (OS) in metastatic renal cell carcinoma (mRCC) treated with interferon-alpha (IFN-alpha). PATIENTS AND METHODS: Serum CAF levels were measured in 103 patients treated on a randomized trial with IFN-alpha 0.5 million units (MU) twice daily or 5 MU daily. Concentrations of 17 analytes were determined by multiplex bead immunoassays [vascular endothelial growth factor A (VEGF(A)) and several cytokines] or enzyme-linked immunosorbent assay (basic fibroblast growth factor). We used proportional hazards models to evaluate the effect of CAF levels and clinical factors on OS. RESULTS: Pretreatment serum interleukin (IL) 5, IL-12 p40, VEGF(A), and IL-6 levels and Memorial Sloan-Kettering Cancer Center risk grouping independently correlated with OS, with hazard ratios of 2.33, 2.00, 2.07, 1.82, and 0.39, respectively (concordance index = 0.69 for the combined model versus 0.60 for the CAF model versus 0.52 for the clinical model). Based on an index derived from these five risk factors (RFs), patients with 0-2 RF had a median OS time of 32 months versus 9 months for patients with 3-5 RF (P < 0.0001). CONCLUSIONS: Serum CAF profiling contributes to prognostic evaluation in mRCC and helps to identify a subset of patients with 20% 5-year OS.

Management of non-neuronopathic Gaucher disease with special reference to pregnancy, splenectomy, bisphosphonate therapy, use of biomarkers and bone disease monitoring.

Enzyme replacement was introduced as treatment for non-neuronopathic Gaucher disease more than 15 years ago. To ensure the best use of this costly ultra-orphan agent, a systematic disease management approach has been proposed by an international panel; this includes the development, by consensus, of achievable treatment goals. Here we critically review these goals and monitoring guidelines and incorporate emerging experience of the disease in the therapeutic era, as well as contemporary clinical research. This review makes recommendations related specifically to the management of pregnancy; the appropriate use of splenectomy and bisphosphonate treatment; the relevance of biochemical markers to disease monitoring; and the use of semi-quantitative methods for assessing bone marrow infiltration. In addition, we identify key areas for development, including the requirement for a validated index of disease severity; the need to correlate widely used biomarkers with long-term disease outcomes, and the desirability of establishing agreed standards for monitoring of bone disease particularly in infants and children with Gaucher disease.

Antitumor activity of dual-specific T cells and influenza virus.

Activation and expansion of T cells are important in disease resolution, but tumors do not usually satisfy these immune requirements. Therefore, we employed a novel strategy whereby dual-specific T cells were generated that could respond to both tumor and influenza virus, reasoning that immunization with influenza virus would activate and expand tumor-specific cells, and inhibit tumor growth. Dual-specific T cells were generated by gene modification of influenza virus-specific mouse T cells with a chimeric gene-encoding reactivity against the erbB2 tumor-associated antigen. Dual-specific T cells were demonstrated to respond against both tumor and influenza in vitro, and expanded in vitro in response to influenza to a much greater degree than in response to tumor cells. Following adoptive transfer and immunization of tumor-bearing mice with influenza virus, dual-specific T cells expanded greatly in numbers in the peritoneal cavity and spleen. This resulted in a significant increase in time of survival of mice. However, tumors were not eradicated, which may have been due to the observed poor penetration of tumor by T cells. This is the first demonstration that the potent immunogenic nature of an infectious agent can be utilized to directly impact on T-cell expansion and activity against tumor in vivo.

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.

CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner.

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens.

BACKGROUND: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. METHODS: In phase I studies, 54 patients received escalating doses (between 10(7) and 10(11) plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. RESULTS: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. CONCLUSIONS: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.

Retroviral transduction of human dendritic cells with a tumor-associated antigen gene.

Dendritic cells (DCs) are potent antigen-presenting cells that can activate quiescent T lymphocytes. When pulsed with tumor-associated antigen (TAA) peptide or protein, murine DCs can provide antitumor immunity. We reasoned that DCs retrovirally transduced with TAA genes might have important advantages over peptide- or protein-pulsed DCs, including long-term TAA presentation in vivo, and presentation of important but undefined epitopes. Therefore, we attempted to retrovirally transduce human DCs with a melanoma TAA gene (MART-1) and determine whether these transduced DCs could raise a specific antitumor response from quiescent autologous T lymphocytes. After retroviral transduction, human CD34+ cells were differentiated into DCs in vitro using granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor. This method consistently yielded a population of DCs as analyzed by morphology, phenotype, and MLR. Flow cytometric analysis revealed that 22-28% of cells expressing the DC phenotype also expressed a transduced marker gene. When DCs were transduced with the gene encoding MART-1, they stimulated much higher levels of cytokine release by MART-1-specific tumor-infiltrating lymphocytes than control DCs transduced with an irrelevant gene. In vitro stimulation using MART-1-transduced DCs but not control-transduced DCs raised specific antitumor CTLs from autologous quiescent T cells. These results provide evidence that human DCs can be retrovirally transduced with a TAA gene and that these transduced cells can raise a specific antitumor immune response in vitro. Transduced DCs may be useful for in vivo immunization against TAA.

In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes.

In an effort to broaden the applicability of adoptive cellular immunotherapy toward nonmelanoma cancers, we have designed chimeric antibody/T-cell receptor genes composed of the variable domains from mAbs joined to T-cell receptor-signaling chains. We have demonstrated that T cells retrovirally transduced with these genes can recognize antibody-defined antigens and that this recognition leads to T-cell activation, specific lysis, and cytokine release. In this study, we have examined the in vivo activity of murine T cells transduced with a chimeric receptor gene (MOv-gamma) derived from the mAb MOv18, which binds to a folate-binding protein overexpressed on most human ovarian adenocarcinomas. Nude mice that were given i.p. implants of human ovarian cancer (IGROV) cells were treated 3 days later with i.p. murine tumor-infiltrating lymphocytes (TIL) derived from an unrelated tumor. Mice treated with MOv-gamma-transduced TIL (MOv-TIL) had significantly increased survival compared to mice treated with saline only, nontransduced TIL, or TIL transduced with a control anti-trinitrophenyl chimeric receptor gene (TNP-TIL). In another model, C57BL/6 mice were given i.v. injections of a syngeneic methylcholanthrene-induced sarcoma transduced with the folate-binding protein (FBP) gene. Three days later, mice were treated i.v. with various transduced murine TIL (derived from an unrelated tumor), followed by low-dose systemic interleukin 2. Eleven days after tumor injection, mice were sacrificed, and lung metastases were counted. In multiple experiments, mice receiving MOv-TIL had significantly fewer lung metastases than did mice treated with interleukin 2 alone, nontransduced TIL, or TNP-TIL. These studies indicate that T cells can be gene modified to react in vivo against tumor antigens, defined by mAbs. This approach is potentially applicable to a number of neoplastic and infectious diseases and may allow adoptive immunotherapy against types of cancer not previously amenable to cellular immunotherapy.

Antiangiogenic gene therapy of cancer utilizing a recombinant adenovirus to elevate systemic endostatin levels in mice.

Gene therapy represents a possible alternative to the chronic delivery of recombinant antiangiogenic proteins to cancer patients. Inducing normal host tissues to produce high circulating levels of these proteins may be more effective than targeting antiangiogenic genes to tumor tissue specifically. Previously reported gene therapy approaches in mice have achieved peak circulating endostatin levels of 8-33 ng/ml. Here we report plasma endostatin levels of 1770 ng/ml after administration of a recombinant adenovirus. Growth of MC38 adenocarcinoma, which is relatively resistant to adenoviral infection, was inhibited by 40%. These findings encourage gene delivery approaches that use the host as a "factory" to produce high circulating levels of antiangiogenic agents.

Costimulation enhances the active immunotherapy effect of recombinant anticancer vaccines.

Activation of T lymphocytes in the absence of a costimulatory signal can result in anergy or apoptotic cell death. Two molecules capable of providing a costimulatory signal, B7-1 (CD80) and B7-2 (CD86), have been shown to augment the immunogenicity of whole-tumor cell vaccines. To explore a potential role for costimulation in the design of recombinant anticancer vaccines, we used lacZ-transduced CT26 as an experimental tumor and beta-galactosidase (beta-gal) as the model tumor antigen. Attempts to augment the function of a recombinant vaccinia virus (rVV) expressing beta-gal by admixture with rVV expressing murine B7-1 were unsuccessful. However, a double recombinant vaccinia virus engineered to express both B7-1 and the model antigen beta-gal was capable of significantly reducing the number of pulmonary metastases when administered to mice bearing tumors established for 3 or 6 days. Most important, the double recombinant vaccinia virus prolonged the survival of tumor-bearing mice. These effects were antigen specific. The related costimulatory molecule B7-2 was found to have a similar, although less impressive enhancing effect on the function of a rVV expressing beta-gal. Thus, the addition of B7-1 and, to a lesser extent, B7-2 to a rVV encoding a model antigen significantly enhanced the therapeutic antitumor effects of these poxvirus-based, therapeutic anticancer vaccines.

In vivo sensitivity of human melanoma to tumor necrosis factor (TNF)-alpha is determined by tumor production of the novel cytokine endothelial-monocyte activating polypeptide II (EMAPII).

Tumor necrosis factor (TNF)-alpha is a potent anticancer agent that seems to selectively target tumor-associated vasculature resulting in hemorrhagic necrosis of tumors without injury to surrounding tissues. The major limitation in the clinical use of TNF has been severe dose-limiting toxicity when administered systemically. However, when administered in isolated organ perfusion it results in regression of advanced bulky tumors. A better understanding of the mechanisms of TNF-induced antitumor effects may provide valuable information into how its clinical use in cancer treatment may be expanded. We describe here that the release of a novel tumor-derived cytokine endothelial-monocyte-activating polypeptide II (EMAPII) renders the tumor-associated vasculature sensitive to TNF. EMAPII has the unique ability to induce tissue factor production by tumor vascular endothelial cells that initiates thrombogenic cascades, which may play a role in determining tumor sensitivity to TNF. We demonstrate here that constitutive overexpression of EMAPII in a TNF-resistant human melanoma line by retroviral-mediated transfer of EMAPII cDNA renders the tumor sensitive to the effects of systemic TNF in vivo, but not in vitro. This interaction between tumors and their associated neovasculature provides an explanation for the focal effects of TNF on tumors and possibly for the variable sensitivity of tumors to bioactive agents.

Immunization against endogenous retroviral tumor-associated antigens.

Endogenous retroviral gene products have been found in some human tumors, and therefore, may serve as antigens for immunotherapy approaches. The murine colorectal carcinoma CT26 and melanoma B16 have recently been found to express the endogenous retroviral gene products gp70 and p15E, respectively, that can serve as antigens recognized by T cells. To date, though, there has been no demonstration of tumor treatment using an endogenous retroviral protein. In this study, we demonstrate that mice immunized with recombinant vaccinia encoding the gp70 H2-L(d)-restricted minimal determinant were protected from CT26 tumor challenge. Splenocytes from mice immunized with vaccinia gp70 specifically secreted IFN-gamma in response to gp70 peptide-pulsed stimulators. Although this strategy could protect against subsequent tumor challenge, it was ineffective against established tumors. Therefore, to investigate the treatment of established CT26 or B16 lung metastases, mice were treated with cultured dendritic cells (DCs) pulsed with gp70 or p15E peptide. Significant inhibition of established lung metastases required immunization with peptide-pulsed DCs pretreated with CD40 ligand that has been demonstrated to increase the T-cell stimulatory activity of DCs. The ability to immunize against endogenous retroviral tumor antigens may have relevance in the induction of antitumor immunity for some human cancers.

Prospective randomized trial of the treatment of patients with metastatic melanoma using chemotherapy with cisplatin, dacarbazine, and tamoxifen alone or in combination with interleukin-2 and interferon alfa-2b.

PURPOSE: The combination of chemotherapy with immunotherapeutic agents such as interleukin-2 and interferon alfa-2b has been reported to provide improved treatment results in patients with metastatic melanoma, compared with the use of chemotherapy alone. We have performed a prospective randomized trial in patients with metastatic melanoma, comparing treatment with chemotherapy to treatment with chemoimmunotherapy. PATIENTS AND METHODS: One hundred two patients with metastatic melanoma were prospectively randomized to receive chemotherapy composed of tamoxifen, cisplatin, and dacarbazine or this same chemotherapy followed by interferon alfa-2b and interleukin-2. Objective responses, survival, and toxicity in the two groups were evaluated at a median potential follow-up of 42 months. RESULTS: In 52 patients randomized to receive chemotherapy, there were 14 objective responses (27%), including four complete responses. In 50 patients randomized to receive chemoimmunotherapy, there were 22 objective responses (44%) (P2 = .071), including three complete responses. In both treatment groups, the duration of partial responses was often short, and there was a trend toward a survival advantage for patients receiving chemotherapy alone (P2 = .052; median survival of 15.8 months compared with 10.7 months). Treatment-related toxicities were greater in patients receiving chemoimmunotherapy. CONCLUSION: With the treatment regimens used in this study, the addition of immunotherapy to combination chemotherapy increased toxicity but did not increase survival. The use of combination chemoimmunotherapy regimens is not recommended in the absence of well-designed, prospective, randomized protocols showing the benefit of this treatment strategy.