Safety and efficacy of decitabine in combination with temozolomide in metastatic melanoma: a phase I/II study and pharmacokinetic analysis.

BACKGROUND: Temozolomide (TMZ) is widely used for chemotherapy of metastatic melanoma. We hypothesized that epigenetic modulators will reverse chemotherapy resistance, and in this article, we report studies that sought to determine the recommended phase 2 dose (RP2D), safety, and efficacy of decitabine (DAC) combined with TMZ. PATIENTS AND METHODS: In phase I, DAC was given at two dose levels: 0.075 and 0.15 mg/kg intravenously daily × 5 days/week for 2 weeks, TMZ orally 75 mg/m(2) qd for weeks 2-5 of a 6-week cycle. The phase II portion used a two-stage Simon design with a primary end point of objective response rate (ORR). RESULTS: The RP2D is DAC 0.15 mg/kg and TMZ 75 mg/m(2). The phase II portion enrolled 35 patients, 88% had M1c disease; 42% had history of brain metastases. The best responses were 2 complete response (CR), 4 partial response (PR), 14 stable disease (SD), and 13 progressive disease (PD); 18% ORR and 61% clinical benefit rate (CR + PR + SD). The median overall survival (OS) was 12.4 months; the 1-year OS rate was 56%. Grade 3/4 neutropenia was common but lasted >7 days in six patients. CONCLUSIONS: The combination of DAC and TMZ is safe, leads to 18% ORR and 12.4-month median OS, suggesting possible superiority over the historical 1-year OS rate, and warrants further evaluation in a randomized setting.

Interferon Alfa-2b Adjuvant Therapy of High-Risk Resected Cutaneous Melanoma: The Eastern Cooperative Oncology Group Trial EST 1684.

PURPOSE: Interferon alfa-2b (IFN alpha-2b) exhibits antitumor activity in metastatic melanoma and on this basis has been evaluated as an adjuvant therapy following surgery for deep primary (T4) or regionally metastatic (N1) melanoma. METHODS: A randomized controlled study of IFN alpha-2b (Schering-Plough, Kenilworth, NJ) administered at maximum-tolerated doses of 20 MU/m2/d intravenously (i.v.) for 1 month and 10 MU/m2 three times per week subcutaneously (SC) for 48 weeks versus observation, was conducted by the Eastern Cooperative Oncology Group (ECOG) in 287 patients. RESULTS: A significant prolongation of relapse-free survival (P = .0023, one-sided) and prolongation of overall survival (P = .0237, one-sided) was observed with IFN alpha-2b therapy in this trial, which is now mature with a median follow-up time of 6.9 years. The impact of treatment on relapse rate is most pronounced early during the treatment interval. The overall benefit of treatment in this trial was analyzed stratified by tumor burden and the presence or absence of microscopic nonpalpable and palpable regional lymph node metastasis. The benefit of therapy with IFN alpha-2b was greatest among node-positive strata. Toxicity of IFN alpha-2b required dose modification in the majority of patients, but treatment at > or = 80% of the scheduled dose was feasible in the majority of patients through the IV phase of treatment, and for more than 3 months of SC maintenance therapy. Discontinuation of treatment due to toxicity was infrequent after the fourth month of therapy. CONCLUSION: IFN alpha-2b prolongs the relapse-free interval and overall survival of high-risk resected melanoma patients. The increment in median disease-free survival (from 1 to 1.7 years) and overall survival (from 2.8 to 3.8 years) that results from this therapy is associated with a 42% improvement in the fraction of patients who are continuously disease-free after treatment with IFN (from 26% to 37%) in comparison to observation. IFN alpha-2b is the first agent to show a significant benefit in relapse-free and overall survival of high-risk melanoma patients in a randomized controlled trial.

Inhibition of DNA repair with MGMT pseudosubstrates: phase I study of lomeguatrib in combination with dacarbazine in patients with advanced melanoma and other solid tumours.

BACKGROUND: The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) reverses the O6-methylguanine (O6-meG) lesion induced by dacarbazine. Depletion of MGMT can be achieved using O6-meG pseudosubstrates. Herein, we report the first phase I experience of the novel O6-meG pseudosubstrate lomeguatrib, combined with dacarbazine. METHODS: This is a phase I dose-escalation study to determine the maximum tolerated dose and recommended phase II dose (RP2D) of lomeguatrib combined with a single dose of dacarbazine on a 21-day schedule. RESULTS: The vast majority of the 41 patients enrolled had metastatic melanoma (36/41) and most had no previous chemotherapy (30/41). The most frequent non-hematological adverse events (AEs) were nausea (52%), and fatigue (42%). The most frequent AEs of grade 3-4 severity were neutropaenia (42%), leukopaenia (17%), and thrombocytopaenia (12%). Only 1 patient had a partial response and 10 patients had stable disease. CONCLUSION: The RP2D of lomeguatrib was 40 mg orally twice daily for 10 days combined with 400 mg m(-2) of dacarbazine IV on day 2. Oral administration of lomeguatrib substantially increases the haematological toxicity of dacarbazine consistent with experience with other O6-meG pseudosubstrates.

SAGE and antibody array analysis of melanoma-infiltrated lymph nodes: identification of Ubc9 as an important molecule in advanced-stage melanomas.

Although patients diagnosed with melanoma of

Serial studies of autologous antibody reactivity to melanoma. Relationship to clinical course and circulating immune complexes.

The low titer and incidence of autologous antibody to melanoma has hampered its evaluation. Through acid dissociation and ultrafiltration of serum, we have been able to augment the autologous immune response in 9 of 10 patients studied. This result suggests that autologous antibody is present in most patients with melanoma, but is obscured by circulating antigen and the formation of immune complexes. Because native antibody and antibody derived from circulating immune complexes are produced by the host against physiologically relevant antigens, correlations can be made to clinical course. Serological studies of three patients with melanoma were performed with serum samples obtained over many months; these studies demonstrated correlations with tumor progression and clinical course. Serial serologic studies may yet provide one of the better ways to evaluate these relationships. They have the advantage of detecting transient events that may occur with the inception of metastatic disease or autoimmune phenomena, and of avoiding the difficulties encountered in comparing antibody responses between different individuals.

Prognostic role of antibody reactivity to melanoma.

Antibody reactivity against cultured allogeneic melanoma Y-Mel 81:180 was studied in 43 patients who participated in an adjuvant trial for stage I and II melanoma. Serum samples were obtained at trial entry within 2 mo of definitive surgery. At the time of serum acquisition, all patients were free of disease by physical examination and routine radiologic and laboratory parameters. Serum antibody reactivity was tested for by protein A hemadsorption before and after acid dissociation and ultrafiltration of serum. We have previously shown that this technique for immune complex dissociation augments autologous antibody reactivity. Results of serum antibody reactivity were scored by an investigator blinded to the patient's clinical status. Of the 43 patients studied, 15 relapsed and 28 remained disease-free. At study entry, there were 25 stage I patients and 18 stage II patients. Breslow depth was 3.25 +/- 2.5 mm in relapse patients and 1.67 +/- 1.1 mm in disease-free patients. The presence and titer of antibody directed against melanoma in either native serum or serum dissociated from immune complexes was found to be associated with eventual relapse (P = 0.0001). When results were subgrouped by stage of disease, Breslow depth, and hypopigmentation, antibody reactivity was still correlated with eventual relapse. The incidence and titer of antibody reactivity against melanoma appears to be a new prognostic factor in predicting eventual disease recurrence.

Isolation and partial characterization of melanoma-associated antigens identified by autologous antibody.

The study of the autologous immune response to cancer avoids the difficulties encountered in the use of xenoantisera and may identify antigens of physiological relevance. However, the low titer and incidence of autologous antibody to melanoma have hampered further evaluation. By utilizing acid dissociation and ultrafiltration of serum, we have been able to augment the detectable autologous immune response to melanoma in the majority of patients studied. In autologous system Y-Mel 84:420, serum S150 demonstrated a rise in titer from 1:32 in native sera to 1:262,044 after dissociation. The antigen detected by S150 was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma, and head and neck carcinoma cell lines. It did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, or autologous cultured lymphocytes. Using polyacrylamide gel electrophoresis, S150 detects a 66,000-mol wt antigen in spent tissue culture media and serum ultrafiltrate. In cell lysate two bands between 20,000 and 30,000 mol wt are detected by S150. The 66,000-mol wt antigen is sensitive to trypsin digestion and but is resistant to pepsin and heat inactivation. Exposure of spent media to trypsin results in the development of a 24,000-mol wt band that appears to correspond to the antigen detected in the cell lysate. The difference between the antigens detected in the cell lysate as compared with spent media and serum ultrafiltrate may be due to degradation during cell lysis. We conclude that melanoma-associated antigens are present in the serum of patients with melanoma and are shed or secreted by their tumor cells.

IL-12p70 and IL-18 gene-modified dendritic cells loaded with tumor antigen-derived peptides or recombinant protein effectively stimulate specific Type-1 CD4+ T-cell responses from normal donors and melanoma patients in vitro.

Although CD4(+) Type-1T helper (Th1) cells secreting interferon-gamma (IFN-gamma) appear to play an essential role in promoting durable antitumor immunity, we have previously shown that patients with cancer exhibit dysfunctional Th1-type responses against epitopes derived from tumor antigens, such as MAGE-A6. Here, we engineered human dendritic cells (DCs) to secrete high levels of the IFN-gamma-inducing cytokines, interleukin (IL)-12p70 and IL-18, via recombinant adenoviral infection to generate an in vitro stimulus capable of promoting previously deficient patient Th1-type responses. Dendritic cells co-infected with Ad.IL-12 and Ad.IL-18 (DC.IL-12/18) were more effective at stimulating MAGE-A6-specific Th1-type CD4(+) T-cell responses than DCs infected with either of the cytokine vectors alone, control Ad.Psi5 virus or uninfected DCs. Furthermore, we show that DC.IL-12/18 loaded with recombinant MAGE-A6 protein (rMAGE) and used as in vitro stimulators promote Th1-type immunity that is frequently directed against multiple MAGE-A6-derived epitopes. The superiority of DC.IL-12/18-based stimulations in melanoma patients was independent of disease stage or current disease status. Based on these results, we believe this modality may prove clinically useful as a vaccine platform to promote the recovery of tumor antigen-specific, Th1-type CD4(+) T-cell responses in patients with cancer.

Plasminogen activator and its inhibitor in cancer patients treated with tumor necrosis factor.

BACKGROUND: We noted the presence of plasma fibrin degradation products in patients treated with recombinant human tumor necrosis factor (TNF) in a phase I trial. PURPOSE: To further define this observation, we investigated the effects of TNF on the fibrinolytic system in patients entered in the same trial. METHODS: In the 14 patients studied, fibrinolytic parameters were measured by analyzing blood samples for tissue plasminogen activator and inhibitor at 0, 1, 2, 4, 6, and 18-24 hours after initiation of TNF treatment. We used a chromogenic substrate method to determine activity of plasminogen activator and its inhibitor and an enzyme-linked immunosorbent assay (ELISA) to determine levels of antigen (tissue-type plasminogen activator). Molecular weight was determined by zymographic assay. RESULTS: TNF treatment was associated with tissue-type plasminogen activator induction within 1 hour of TNF initiation. The plasminogen activator produced was consistent with tissue-type plasminogen activator derived from endothelium as evidenced by molecular weight analysis and ELISA. Moreover, induction of plasminogen activator inhibitor occurred following the release of tissue-type plasminogen activator, and our data suggest a dose-response effect for TNF. At high doses (i.e., 200 and 240 micrograms/m2), there was a more rapid and prolonged release of plasminogen activator inhibitor, which had an inverse relationship with the level of antigenic tissue-type plasminogen activator. Zymographic analysis showed urokinase-type plasminogen activator activity in 13 of 14 patients. In three patients, simultaneous measurements of white blood cells and tissue-type plasminogen activator revealed a temporal association between the TNF-associated rapid granulocytopenia at 30 minutes after TNF initiation and release of tissue-type plasminogen activator antigen. CONCLUSIONS: The results suggest a positive association between TNF and rapid induction of plasminogen activator activity that is consistent with an endothelial product. It is possible that, at high doses, TNF may interact directly with vascular endothelium, leading to rapid and prolonged production of plasminogen activator inhibitor. There was a dose-response effect between TNF and release of tissue-type plasminogen activator. The release of tissue-type plasminogen activator was preceded by granulocytopenia, which may indicate an association between a proposed TNF-induced granulocyte-endothelial interaction in vivo and release of tissue-type plasminogen activator. IMPLICATIONS: These findings demonstrating the effects of TNF on the fibrinolytic system can be analyzed further in experimental systems to determine the implications for use of this agent as a biological response modifier in cancer therapy.

In vivo drug sensitivity assay of clonogenic human melanoma cells and correlation with treatment outcome.

In vitro tests of tumor cell drug sensitivity have been suggested as a means of selecting more appropriate clinical strategies against human cancer and improving preclinical drug development. The lack of biotransformation in these in vitro assays precludes the meaningful assessment of several major chemotherapeutic agents, including dacarbazine and cyclophosphamide. In vivo drug exposure of tumor cells in agar diffusion chambers placed in mice offers a possible solution to problems of drug biotransformation and pharmacokinetics. We have prospectively used this system as an assay for sensitivity of clonogenic human melanoma cells. Tumor cells were tested fresh, cryopreserved, and/or cultured in vitro before or after clinical use of dacarbazine, semustine, and mitolactol in 41 patient-drug combinations in which a clinical correlation could be made. Tumor cell drug sensitivity in the assay using fresh or cryopreserved tumor cells was highly correlated with clinical response and resistance with clinical nonresponse. Cultured melanoma cells exhibited enhanced plating efficiency in comparison to both fresh and cryopreserved cells of the same tumor. Cultured cells also showed increased drug sensitivity which did not correlate with drug sensitivity of the same fresh or cryopreserved tumor or with clinical response. Tumor cell drug sensitivity assays carried out in vivo provide a possible basis for preclinical evaluation of drugs which are unsuitable for in vitro testing.

Augmentation of autologous antibody to human melanoma following acid dissociation and ultrafiltration of serum.

Sera of 22 individuals were examined for immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody to autologous cultured tumor cells. Reactive native IgM was detected by immune adherence in four, and IgG by Protein A hemadsorption in five, sera. Direct and absorption testing of these reactive sera against a range of normal and neoplastic cells revealed one each with specificity for a highly restricted melanoma cell surface antigen and common tumor-associated antigen of melanoma. Given this low prevalence of antibody, we then tested whether IgG antibody might be retrieved from circulating immune complexes in melanoma patients' sera. Acidification and ultrafiltration of sera from seven patients have enhanced detectable IgG binding to autologous cultured melanoma in six. Characterization of one reactive autologous antibody has detected a common antigen in eight of nine melanoma lines tested. This antibody also detected two neuroblastomas, one of two glioblastomas, one of two sarcomas, and one of two breast carcinomas. The common melanoma antigen detected in these tests may be related to the neuroectodermal, oncofetal differentiation antigens described by others with autologous or allogeneic IgM. Autologous antitumor antibody in circulating immune complexes may provide a source of antibody for serodiagnostic and therapeutic applications relevant to treatment modalities, such as plasmapheresis and plasma perfusion over Protein A in melanoma and other cancers.

Urinary excretion of interferon, albumin, and beta 2-microglobulin during interferon treatment.

Serum and urinary levels of albumin, beta 2-microglobulin, and interferon were determined in ten patients undergoing interferon therapy. The pharmacokinetics during a phase I trial of interferon administration intramuscularly is presented. Only trace amounts of interferon activity are found in the urine, even during peak serum interferon activity. Serum beta 2-microglobulin levels increased after interferon treatment, especially at the higher dosing levels. Urinary excretion of beta 2-microglobulin increased due to the relatively low affinity of the transport system. Saturation, competition, or inhibition of the absorption process for beta 2-microglobulin was not attained. Measurement of the urinary albumin/urinary beta 2-microglobulin ratio reveals no glomerular or tubular lesion, and we conclude that interferon therapy does not result in a clinically significant nephrotoxicity.

Comparison of pharmacokinetics of 5-fluorouracil and 5-fluorouracil with concurrent thymidine infusions in a Phase I trial.

The serum half-life of 5-fluorouracil (5-FUra) in humans is best described as a biexponential decay function, with t1/2 alpha = 7.8 +/- 2.6 (S.E.) min and t1/2 beta = 36.8 +/- 13.5 min during initial courses of this drug alone. Pharmacokinetics of 5-FUra during courses of daily therapy (for 5 days) revealed prolongation of t1/2 in both components of the decay curve, which has not been previously reported. Despite the efficacy of thymidine (dThd) given as a continous i.v. infusion of 8 g/sq m/day in prevention of high-dose methotrexate toxicity, continuous infusion of dThd at this dose does not prevent the toxicity of 5-FUra orreverse inhibition of DNA and RNA synthesis by 5-fura. On the contrary, continuous infusion of dThd appears to increase the toxicity of 5-FUra during continuous dThd infusion revealed prolongation of the 5-FUra t1/2 which remained stable through the course of 5 days of 5-FUra with dThd. This protracted t1/2 is believed to account at least in part for the increased toxicity of 5-FUra with dThd. Dose-limiting mucositis, myelosuppression, and gastrointestinal toxicity were observed at 5-FUra doses ranging from one-half to two-thirds the customarily tolerated dose of 5-FUra alone in similar courses of daily bolus therapy (for 5 days).

Interstitial hypertension in superficial metastatic melanomas in humans.

Since 1950, several investigators have demonstrated that interstitial hypertension is a pathophysiological characteristic of experimental solid tumors. To date, interstitial fluid pressure (IFP) has not been measured in human tumors in situ. In this study we measured with the wick-in-needle technique the interstitial fluid pressure in superficial melanoma metastases (n = 12) in patients (n = 10) before and during systemic therapy. In the majority of tumors the pressure was found to be almost uniform, while in others it varied severalfold. The large variations in IFP in some tumors may be due to technical or biological factors. With the data obtained before and during therapy grouped, the mean IFP in melanoma lesions varied between 2 and 41 mm Hg with an overall mean of 14.3 +/- 12.5 (SD). IFP was found to be significantly higher (P less than 0.01) in large (22.8 +/- 13 mm Hg; n = 6) than in small (5.8 +/- 2mm Hg; n = 6) lesions. This study demonstrates that IFP can be measured in human tumors using the wick-in-needle technique and that the pressure in some of the large melanomas exceeds the values measured to date in rodent tumors or human tumor xenografts. The latter result suggests that caution must be exercised in extrapolating values of pathophysiological parameters from transplanted tumors to human tumors.

NY-ESO-1 encodes DRB1*0401-restricted epitopes recognized by melanoma-reactive CD4+ T cells.

The NY-ESO-1 gene is expressed by a range of human tumors and encodes HLA-A2-restricted melanoma peptides recognized by CD8+ CTLs. Here we report that the NY-ESO-1 gene also encodes two overlapping, but non-cross-reactive, HLA-DRB1*0401-presented peptides that are recognized by CD4+ T cells. The NY-ESO-1(119-143) peptide was able to induce specific CD4+ T cells in vitro from both an HLA-DRB1*0401+ normal donor and an HLA-DRB1*0401+ patient with melanoma. Bulk and cloned CD4+ T cells produced IFN-gamma specifically in response to, and also lysed, T2.DR4 cells pulsed with peptide NY-ESO-1(119-143) and the autologous tumor cell line, but not a DRB1*0401+ melanoma cell line that does not express NY-ESO-1. Interestingly, the NY-ESO119-143 peptide contains two overlapping putative "core" epitopes recognized by non-cross-reactive anti-NY-ESO-1(119-143) CD4+ T-cell clones. Taken together, these data support the use of this novel DR4-restricted tumor peptide, NY-ESO-1(119-143), or its two "sub-epitopes" in immunotherapeutic trials designed to generate or enhance specific CD4+ T-cell responses against tumors expressing NY-ESO-1 in vivo.