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Aims: Immunotherapy with interferon-α (IFN-α) is associated with psychiatric side-effects, including depression. One of the putative pathways underlying these psychiatric side-effects involves tryptophan (TRP) metabolism. Cytokines including IFN-α induce the enzyme indoleamine 2,3-dioxygenase (IDO), which converts TRP to kynurenine (KYN), leading to a shortage of serotonin (5-HT). In addition, the production of neurotoxic metabolites of KYN such as 3-hydroxykynurenine and quinolinic acid (QA) might increase and contribute to IFN-α-induced psychopathology. In contrast, other catabolites of KYN, such as kynurenic acid (KA), are thought to have neuroprotective properties. Methods: In a group of 24 patients treated with standard IFN-α for metastatic renal cell carcinoma (RCC), combined psychiatric and laboratory assessments were performed at baseline, 4 and 8 weeks, and at 6 months. Results: No psychopathology was observed, despite an increase in neurotoxic challenge as reflected in indices for the balance between neurotoxic and neuroprotective metabolites of KYN. Conclusions: The present hypothesis that a shift in the balance between neurotoxic and neuroprotective metabolites of KYN underlies the neuropsychiatric side-effects of IFN-α-based immunotherapy, is neither supported nor rejected.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p

Interleukin 12 (IL-12) has potential efficacy in malignant, infectious and allergic diseases. Its side-effects include activation of coagulation and fibrinolysis, as documented in chimpanzees. We assessed the coagulative and fibrinolytic response in 18 patients with renal cell carcinoma after subcutaneous injection of 0.5 microg/kg recombinant human IL-12. IL-12 induced a fibrinolytic response in 17 patients (94%): plasmin-alpha2-anti-plasmin complexes (PAPc) increased from 11.8 +/- 6.6 nmol/l (mean +/- SD) to a maximum of 18.8 +/- 7.4 nmol/l at 72 h. Baseline levels of tissue plasminogen activator (tPA) and plasminogen-activator inhibitor-I (PAI) were elevated in eight and 14 patients respectively. tPA increased from 12.6 +/- 5.2 ng/ml to a maximum of 19.0 +/- 6.7 ng/ml at 72 h. PAI decreased from 111 +/- 69 ng/ml to a minimum of 65 +/- 53 ng/ml at 8 h, thereafter remaining below baseline. Elevation of PAPc correlated with elevation of tPA and reduction of PAI. A coagulative response occurred in nine patients (50%): thrombin-anti-thrombin III complexes increased from 29 +/- 53 ng/ml to a maximum of 460 +/- 322 ng/ml at 12 h. Patients with and without a coagulative response had similar levels of recombinant human IL-12, interferon-gamma or tumour necrosis factor-alpha. We conclude that IL-12 can activate both fibrinolysis and coagulation in a significant proportion of patients with cancer. The time-frame and sequence of these activation processes differ from those known for other cytokines.

Background Tumour necrosis factor-alpha (TNFa) has been found to be very effective in the isolated limb perfusion setting for advanced extremity tumours. In a phase I study of intrapleural administration of TNFa 5 patients were followed for inflammatory response patterns. Patients and methods Malignant mesothelioma patients were treated with repeated intrapleural administration of 0·1‐0·2 mg recombinant TNFa. Samples of serum and pleural fluid were taken at different time-points before and after TNFa-administration. Levels of TNFa, interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and secretory phospholipase A2 (sPLA2) were measured using enzyme-linked immunosorbent assays (ELISAs). Alpha 1-acid glycoprotein (a1-AG) was measured by nephelometry. Results In pleural fluid TNFa and IL-8 reached peak levels, up to 50‐700 ng mL π1 and 6‐ 60 ng mL π1 , respectively, 24 h after administration of TNFa. IL-6 (peak levels up to 250 ng mL π1 ) and sPLA2 peaked after 48 h. A slower and less dramatic pattern was observed for the levels of CRP and a1-AG. In serum no detectable levels of TNFa and no IL-8 were observed, whereas serum levels of IL-6, sPLA2 and CRP showed a clear increase after intrapleural administration of TNFa. Cytokines and acute-phase proteins showed the same pattern during subsequent cycles even up to 12 cycles. Tumour regression was not observed. Conclusions In the setting of a phase I study of repetitive intrapleural administration of TNFa in mesothelioma patients, we studied the characteristics of the inflammatory response. Intrapleural administration was followed by a clear inflammatory response locoregionally. In spite of TNFa peak levels as high as 700 ng mL π1 systemic levels were never detectable. The secondary cytokine response led to very high intrapleural IL-6 and IL8 levels. Systemically IL-8 levels were never detectable whereas high IL-6 levels were induced systemically initially, with a decreased response to each intrapleural TNFa administration over time. The acute-phase response in contrast remained remarkably constant throughout the course of repeated intrapleural administrations of TNFa. Intrapleural administration of TNFa is well tolerated but associated with inconsistent and rather moderate impact on production of pleural fluid. This can be achieved by other simpler and cheaper treatment, thus we see no justification for further studies.

We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4-, 8+, 16-, 56-. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4-, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov- 1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation.

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 microM), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V. The fluorescence signal was quantitated by flow cytometry (FCM). During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one. At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present. A dose-effect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence. The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA breaks by in situ nick translation assay and DNA content by DNA-propidium iodine fluorescence in a bivariate analysis, 2) membrane integrity by dye exclusion, and 3) morphological characteristics of apoptosis. The dimly fluorescent cell population appeared to represent apoptotic cells in the early phase of the death process, as demonstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation. The brightly fluorescent cells predominantly had sub-G1 DNA content, nuclear fragmentation, leaky cell membranes, and probably represent late apoptotic cells. These results demonstrate that cytotoxic drug-induced apoptosis can be quantitated by annexin V binding and that by using this assay early and late apoptotic cells can be identified.

We treated 72 patients with metastatic renal-cell cancer according to 2 protocols consisting of two 5-week induction cycles of continuous i.v. high-dose interleukin-2 (IL-2), i.m. interferon-alpha (IFN alpha) and ex vivo IL-2-activated lymphocytes, followed for patients with stable disease (SD), partial response (PR) or complete response (CR) by four 4-week maintenance cycles of IL-2 and IFN alpha. Protocol 2 (55 patients) differed from protocol 1 (17 patients) in (i) the addition of IFN alpha to the first IL-2 infusions in both induction cycles; (ii) the use of Teceleukin IL-2, reconstituted with carrier protein, instead of Proleukin IL-2 without carrier protein. We classified 23 patients with CR and PR as responders (4 in protocol 1 and 19 in protocol 2) and 45 patients with SD and progressive disease as non-responders. Prior to immunotherapy, patients entered into protocol 2 already had higher IFN gamma serum concentrations, higher peripheral blood CD56-,3+ and CD8-,4+ lymphocyte numbers and lower NKK562 activity than those entered into protocol 1. These differences persisted during and after immunotherapy. In line with these observations, ex vivo IL-2-activated lymphocytes had larger proportions of CD56-,3+ and CD8-,4+ lymphocytes and lower NKK562 activity in protocol 2 than in protocol 1. Higher IL-2 serum concentrations were reached during the IL-2 infusion in protocol 2 than in protocol 1. In addition, the immunomodulation in protocol 2 was stronger than in protocol 1 as indicated by higher TNF alpha serum concentrations and a more pronounced eosinophilia. Differences between responders and non-responders treated according to the 2 protocols were not significant, except for the total number of lymphocytes obtained by apheresis, which was higher in responders than in non-responders.

Background. In a group of patients with metastatic melanoma treated with high dose immunotherapy, there was an unexpectedly high incidence of severe cardiac adverse effects. Methods. Sixteen patients with metastatic melanoma were treated with high dose interleukin-2 (IL-2) and alpha-interferon (α-IFN). Each treatment cycle consisted of IL-2 at a dose of 12 MIU/m2 and α-IFN at a dose of 3 MIU/m2, given as intravenous bolus injections every s hours on Days 1-5, every 3 weeks for a total of three cycles. Before treatment, careful cardiologic screening was performed, including electrocardiogram (ECG), stress test, cardiac multiple uptake-gated acquisition (MUGA) scan, and echocardiography. During therapy, patients were monitored with daily ECG and creatine phospokinase measurements. Once cardiac damage was suspected, IL-2 and α-IFN were discontinued, and echocardiography, stress test and MUGA-scan were repeated. If indicated, cardiac catheterization with endomyocardial biopsies was performed. Results. Despite pretreatment cardiac screening, seven patients (44%) exhibited myocardial injury. Acute myocardial infarction occurred in one patient, cardiomyopathy developed in four, asymptomatic ECG changes appeared in one, and 1 patient died of acute cardiac arrest. Echocardiography showed hypokinesis and decreased left ventricular ejection fraction. These abnormalities disappeared within 6 months. Cardiac catheterization in four affected patients revealed normal coronary arteries, but endomyocardial biopsies showed interstitial edema, vacuolation, and degeneration of myocytes. Electron-microscopic examination showed fragmentation of myofibrils, swelling of mitochondria and loss of mitochondrial cristae. Conclusions. This intensive treatment schedule of IL-2 and α-IFN is prohibited by severe and life-threatening cardiac toxicity.

A multicentre study of IL2 and IFN alpha has been performed in 58 patients with metastatic melanoma. The scheme consisted of IL2 3.0 BRMP MU/m2/d as a continuous infusion for 4 d combined with subcutaneous administration of IFN alpha 6 MU/m2/d, day 1 + 4. The cycle was repeated every 2 weeks for a maximum duration of 26 weeks. 54 patients were evaluable for response. One (2%) achieved a complete and 10 (19%) a partial response. 19 (35%) patients were stable and 24 (44%) showed progressive disease. Common side-effects included fever, chills, fatigue, skin rash, anorexia, nausea and diarrhoea. Hypothyroidism was noted in 10% of the patients. These results show that this regimen of IL2 and IFN alpha is active but, in contrast to what could be expected, not superior to IL2 alone possibly due to suboptimal dosing. In an ongoing study in Rotterdam and Nijmegen, a more intense schedule was chosen, consisting of three daily i.v. doses of IL2 4.5 BRMP MU/m2 and IFN alpha 3.0 MU/m2 for 5 d. This regimen is repeated at intervals of 3 weeks for a total of three cycles. Presently, nine patients have been entered. One patient achieved a complete response, four a partial response (overall 56%), three had stable disease and one progressed. Toxicity was severe and treatment was prematurely stopped in five patients: myocardial infarction (one patient), atrial fibrillation (one patient), negative T waves and myocardial hypokinesia (one patient) and psychosis (two patients). This regimen can only be justified if the therapeutic results are superb, which has yet to be awaited.

Ninety-one patients with disseminated testicular non-seminomas were treated with 3 to 4 cycles of cisplatin, vinblastine, and bleomycin (PVB) induction chemotherapy followed by cisplatin and vinblastine maintenance therapy for 1 year. The follow-up of these patients ranges from 24 to 66 months. Forty-nine (54%) patients achieved complete remission by chemotherapy alone and 14 (15%) were rendered free of tumor by surgery after chemotherapy, for a total complete remission rate of 69%. Three complete responders relapsed within 13 months, and two died. One additional complete responder died of a noncancer-related cause. One of the surgical complete responders relapsed and died. Overall, 58 (64%) patients remain free of disease. The 5-year survival is 95% for complete responders, 32% for partial responders, and 72% overall. This combination regimen has significantly improved the survival of disseminated testicular cancer patients, equaling that of Stage II patients in older literature.

Two hundred and twenty-eight patients with advanced testicular cancer were entered into a randomized study of chemotherapy comprising cis-platinum (P) 20 mg/m2-, days 1-5 every 3 weeks for four courses, bleomycin (B) 30 mg weekly for 12 weeks, and vinblastine (V) at either the low dose of 0.15 mg/kg or the high dose of 0.20 mg/kg on days 1 and 2 every 3 weeks for four courses. In this interim analysis, 64 patients were randomized to high dose PVB. Forty-five (71%) achieved a complete response, and 13 (25%) a partial response. Seventy patients received low dose PVB of whom 50 (71%) achieved a complete response and 16 (23%) a partial response. Thus there is no difference in the efficacy of this combination chemotherapy with respect to the dose of vinblastine, but the low dose schedule was less toxic (particularly to bone marrow). It was also apparent that the response rate varied with the volume of metastatic disease, irrespective of the dose of vinblastine. Patients with low volume metastases had a complete response rate (CR) of 88%, while those with high volume had a CR rate of 60%. In a second randomization, 68 patients achieving CR were randomized to receive either 1 year of further (maintenance) chemotherapy with cis-platinum and vinblastine, or no further chemotherapy. One of 37 patients (3%) receiving treatment and 2 of 31 patients (6%) not receiving treatment relapsed, with a follow-up of at least 10 months. Thus maintenance chemotherapy appears not to be necessary in the treatment of advanced testicular cancer.