Continuous infusion of interleukin-2 and cyclophosphamide as treatment of advanced cancers: a National Biotherapy Study Group Trial

We have evaluated the effect of Interleukin-2 [IL-2] after Cyclophosphamide (C) chemotherapy in 41 patients with metastatic cancer. IL-2 was given as a continuous infusion priming cycle 36 hours after C at 1 gm/m2 intravenously. In 39 evaluable patients, there were no complete remissions [CR], 2 partial remissions [PR], and 1 had a minor response [MR]. Stable disease for 30 days was seen in 16 patients whereas 20 progressed. The durations of partial and minor responses were brief, ranging from 1-6 months. Grade 3-4 neutropenia was seen in 41%. This was more severe than seen with IL-2 alone or IL-2 combined with lower doses of C. The marrow suppression was due to the chemotherapy. This combination of IL-2 and C appears to be reasonably well tolerated by patients, but toxicity is greater and the response rate is no better than results achieved by IL-2 alone. Responses of 26 patients with renal cancer appear to be inferior to our historical data using IL-2/LAK cells without C. Immune monitoring demonstrated changes expected with C chemotherapy (i.e., a non-selective decline in immune function). C induced no further differences in IL-2 induced changes in immune function.     

Continuous infusion interleukin-2 and tumor-derived activated cells as treatment of advanced solid tumors: a National Biotherapy Study Group Trial

Metastases from patients with solid tumors were harvested from 196 patients for the purpose of growing tumor-derived activated cells (TDAC). Cells were prepared from autologous tumor cultures by incubation with Interleukin-2 (IL-2) followed by repeated exposure to tumor antigen and/or anti-CD3 monoclonal antibody. Initial growth success was achieved in 66%; 45/56 (80%) of these early cultures were subsequently expanded for in vivo therapy. It took a mean of 69.4 +/- 24.0 days to grow TDAC for treatment. Thirty-eight patients were treated with cyclophosphamide (1 g/m2) on day one followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/day) on days 2-5 and approximately 10(11) TDAC on day 2. Patients subsequently received monthly IL-2 as a 96-hour constant infusion if their cancers were stable or regressing. Median age was 51 yrs; 58% were male. Performance status was 0-1 in 64%, 29% had lung metastases; 34% had liver metastases. The usual IL-2 toxicities were seen. Responses were seen only in 1/38 patients (3%); a partial response in a patient with lymphoma. Forty-two percent were stable 90 days post-treatment, the rest were progressive or inevaluable. We conclude that a treatment plan for IL-2/TDAC is technically difficult, costly, and not practical under these conditions. Clinical results to date are not clearly different than those obtained with other IL-2 regimens.     

Phase I study of combination recombinant interleukin-2 and interferon gamma in patients with advanced malignancies

A phase I trial of interleukin-2 and interferon gamma combination treatment in patients with advanced malignancies was performed based on preclinical in vitro and in vivo data which demonstrated synergistic antitumor effect. The toxicities, immune parameters, and tumor responses are described. The clinical and biologic maximal tolerated doses were extrapolated from these data.     

Adjuvant treatment of breast cancer: A pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha

Immune responses, including natural immunity (NI), potentiate the antitumor effects of chemotherapy. Since interferons and interleukin-2 (IL-2) augment NI, a pilot study was conducted to assess the tolerability and the effects on host immunity of adjuvant chemotherapy associated with IL-2 + interferon alpha (IFN) in breast cancer patients after surgery. Ten patients underwent alternating 28-day cycles of chemoimmunotherapy [cyclophosphamide + methotrexate + 5-fluorouracil (CMF, days 1, 8) + IL-2 (days 15–19) + IFN (day 22)] and chemotherapy alone (CMF). With this regimen each patient was considered to be a reasonable “control” of herself. Blood cell count and natural killer cell activity (NKA) were tested on days 1, 8, 15, 22, and 23. Preliminary in vitro studies indicated that IL-2 or IFN antagonized the severe inhibition of NKA induced by hydroxy-peroxy-cyclophosphamide (in vitro active derivative of cyclophosphamide), alone or associated with methotrexate + 5-fluorouracil. Nine patients completed all six alternating cycles, whereas one patient proved to have metastatic lesions after four cycles. The protocol was well tolerated, although leukopenia (CMF alone) and leukopenia with fever and moderate or minimal flu-like symptoms (CMF + IL-2 + IFN) were generally observed. Treatment with IL-2 facilitated complete recovery of white cell counts and NKA after the nadir on day 15. In conclusion, the present protocol appears to be well tolerated and amenable to administration on an outpatient basis. Therefore, further investigations should be performed to verify whether CMF + IL-2 + IFN would be superior to CMF alone for adjuvant treatment after surgery in breast cancer. Received: 9 April 1998 / Accepted: 16 July 1998     

Combining interferon with cytotoxic chemotherapy in patients with advanced breast cancer

Summary Laboratory studies have suggested therapeutic synergy between alpha-interferons and various cytotoxic agents. The present study set out to monitor the toxicity of giving a standard cytotoxic drug regimen with a well evaluated, low-dose, schedule of lymphoblastoid interferon. The combined treatment resulted in significantly greater leucopenia than was seen with cytotoxics alone and two patients developed severe infections. Although total numbers were small there was no evidence of increased therapeutic response in the combined treatment group. We would therefore suggest caution in future studies combining cytotoxic drugs and interferons as dose reductions of one or other modality is probably necessary.     

Diminished expression of interleukin-2 receptors in vivo after prior chemotherapy in advanced cancer patients receiving recombinant interleukin-2

In a phase I/II dose escalation study performed at our institution, a total of 14 advanced metastatic cancer patients received between 4 and 16 weeks of subcutaneous recombinant interleukin-2. Doses were escalated at weekly intervals, starting at 1.8 million IU/m2/day up to a maximum dose of 14.4 million U/m2 daily. When comparing patients with (n = 4) and without (n = 7) prior chemotherapy on day 0 (i.e., before rIL-2), both patient groups exhibited Tac IL-2 receptor (CD25) positive peripheral blood lymphocytes at equal levels of positivity (8%). In contrast, 4-week systemic treatment with subcutaneous rIL-2 at escalating dose levels revealed a significant difference in the up-regulation by interleukin-2 of CD25 cell surface receptor. Thus, after 4 consecutive weeks of treatment, patients without previous chemotherapy showed a mean CD25 positivity of peripheral blood lymphocytes at 38%, as compared with 22% in patients who did receive prior chemotherapy (p less than 0.05). These data suggest that chemotherapy pretreatment may have a significant effect on biological response to rIL-2 in vivo.     

Natural cytotoxicity and interferon production in human cancer: deficient natural killer activity and normal interferon production in patients with advanced disease

Natural cytotoxicity was measured in 51 adult patients with solid epithelial malignant tumors and in 27 normal subjects. Peripheral blood leukocytes (PBL) from 31% of the patients and 7% of the controls failed to kill target cells (K562) in a short-term chromium-release assay. When patients were classified according to clinical stage, PBL from 12% of patients with localized cancers, but 50% of patients with advanced disease, failed to exhibit cytotoxicity within the normal range. Pretreatment of PBL with interferon alpha (IFN alpha) or with Newcastle Disease Virus (NDV), a potent inducer of IFN alpha, enhanced cytotoxicity from all normal subjects. Of patients whose PBL lacked spontaneous cytotoxicity, half were able to kill normally after pretreatment of PBL with IFN alpha or NDV. Virtually all the patients whose PBL were unable to kill despite pretreatment with IFN alpha or virus had disseminated malignancies. IFN alpha production by PBL exposed to NDV and to K562 cells was normal in all the patients regardless of stage of disease or ability to kill K562 cells. The observed defect in natural cytotoxicity is thus unlikely to be due to a failure of PBL to produce IFN alpha.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

Immunological effects of a single evening subcutaneous injection of low-dose interleukin-2 in association with the pineal hormone melatonin in advanced cancer patients.

Recent experiments have shown that a great variety of neurohormones can interact with IL-2 in the modulation of host antitumor immune response. On the basis of these data, a study was started to evaluate the effect of the pineal hormone melatonin (MLT) on IL-2-induced immune changes in cancer. The study included 30 advanced cancer patients. They were randomized to be treated with IL-2 at a dose of 3 million IU subcutaneously twice/daily (8.00 a.m. and 8.00 p.m.) for 6 days/week for 4 weeks, with IL-2 once/daily in the evening, with IL-2 once/daily plus MLT at 10 or at 50 mg/day. MLT was given orally at 8.00 p.m. Both IL-2 given twice daily and IL-2 given once daily and IL-2 given once daily in association with MTL induced a significant increase in mean number of lymphocytes, T lymphocytes, NK cells, CD25-positive cells and eosinophils, whereas the single administration of IL-2 alone was unable to determine a significant rise in the mean number of immune cells. Soluble IL-2 receptor and neopterin increase was significantly higher during IL-2 given twice/daily than during IL-2 plus MLT, while no difference was seen in TNF rise. This study would suggest that a single daily injection of low-dose IL-2 is able to efficiently activate the lymphocyte proliferation in cancer patients when it is given in association with the pineal hormone MLT.     

Clinical trials of intrasplenic arterial infusion of interleukin-2 (IS-IL-2) to patients with advanced cancer

We tried a infusion of interleukin-2 (IL-2) of a relatively low dose via an intrasplenic arterial catheter connected to a chronometric infusion (IS-IL-2). Eighteen patients of colorectal cancer with metastases to the liver or lung or of unresectable hepatoma received a 24 hour continuous infusion with low dose recombinant of IL-2 (mainly 8 × 10⁵ JRU/day) for 25–40 days. All patients tolerated this protocol of the therapy and the main toxic effects were fever and general fatigue. Such serious toxicity as previously reported by high dose IL-2 therapy was not observed. Data of hepatic and renal functions were normal. IS-IL-2 therapy induced a high incidence of eosinophilia (12/18) and thrombocythemia (12/18). Peripheral natural killer (NK) and LAK activities were augmented in all patients and total white blood cell counts were increased during IS-IL-2 therapy. An increase in IL-2 receptor expression of peripheral blood mononuclear cells and significant rises in numbers of Leull (CD16)+, OKMl(CD11)+ and OKIal(HLA-DR)+ were observed. Of 18 patients 12 were evaluable for their response to therapy. Partial response (PR) was observed in one unresectable hepatoma and 11 demonstrated no change (NC) or progressive disease (PD). Six patients were not evaluable because of additional therapy (3 cases) or decreasing tumor cell markers having no measurable lesions (3 cases). Three patients of colorectal cancer from an unresectable group were presumed to have micrometastases to the liver as suggested by an elevated serum CEA level. After receiving IS-IL-2 therapy they demonstrated a decrease in the serum CEA level for more than 3 years after treatment. We conclude that continuous IS-IL-2 administration can result in an increase of their therapeutic efficacy of IL-2 administration and in a decrease its toxicity.     

Immunomodulatory effects of ultra-low-dose interleukin-2 in cancer patients: a phase-IB study

High-dose interleukin-(IL-2) has been broadly studied in tumour therapy, yet it may be inhibitory to T-cell-dependent immunity. Therefore immune and tumour responses mediated by low-dose IL-2 were studied systematically with respect to the feedback organisation of immune responses. IL-2 was administered once daily at three dose levels: 0.18, 0.9, 4.5 MIU/m² according to three different schedules requiring subcutaneous (s.c.) injection once weekly (four doses, stratum I), thrice weekly every other day (nine doses, stratum II), or five times weekly every other week (ten doses, stratum III). A total of 46 patients with advanced cancer were randomly assigned to one of the nine treatment groups. Systemic effects were induced at doses as low as 0.18 MIU/m² IL-2 s.c. as demonstrated from measurable IL-2 serum levels, induction of circulating IL-6, a transient lymphopenia, and stimulation of delayed-type hypersensitivity (DTH) responses of the skin. Analysis of the different IL-2 schedules demonstrated (a) prolonged effects of once-weekly injections on DTH responses, lymphocyte and eosinophil counts, and (b) maximum increase of eosinophil counts and preferential expansion of activated NK cells with repeated injections every 48 h or 72 h (stratum II), while sequential treatment according to stratum III was found to be more potent in increasing the number of activated T cells. A tumour response was observed in 1/15 patients with renal cell carcinoma who experienced more than 50% tumour regression for 8 months; 12 patients had stable disease for 4 months (median). These data demonstrate prolonged immunological effects of ultra-low doses of s. c. IL-2 despite its short half-life. Furthermore, scheduling of IL-2 was found to affect immune responsiveness specifically as demonstrated by the differential effects on natural killer and T cell populations.Supported by Bundesministerium für Forschung und Technologie, Förderkennzeichen 01GA8901/3 to R. M.     

Combination of tumor necrosis factor alpha and interferon alpha induces apoptotic cell death through a c-myc-dependent pathway in p53 mutant H226br non-small-cell lung cancer cell line.

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-alpha) and natural human interferon alpha (IFN-alpha). Studies were performed with two human non-small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-alpha and TNF-alpha significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-alpha or TNF-alpha alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-alpha/TNF-alpha induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-alpha/TNF-alpha. Antisense c-myc inhibited IFN-alpha/TNF-alpha cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-alpha/IFN-alpha induce apoptosis through a c-myc-dependent pathway rather than a p53-dependent pathway. (c)2001 Elsevier Science.     

Serum concentrations of interleukin-18 in early and advanced cancer patients: enhanced secretion in metastatic disease

The recent availability of adequate methods for cytokine measurement could contribute to better understanding the immunophysiopathology of neoplastic disease. Unfortunately, very little data is available about cytokine secretion in cancer patients. At present, IL-2, IL-12 and IL-15 represent the major antitumor cytokines in humans. Preliminary clinical studies have shown a progressive decline in IL-2 levels with cancer progression, whereas IL-12 seems to increase in the advanced disease. IL-18 is the latest cytokine discovered by potential anticancer and anti-angiogenetic activity, and it has effects similar to those of IL-12. This preliminary study was carried out to analyze IL-18 secretion in early or advanced cancer patients. The study included 40 cancer patients (lung cancer, 21; gastrointestinal tumors, 19), 17 of whom had metastatic disease, and 50 healthy controls. Serum levels of IL-18 were measured by ELISA. No significant difference in IL-18 mean levels was seen between controls and non-metastatic patients. In contrast, metastatic patients showed significantly higher IL-18 mean values with respect to both healthy controls and non-metastatic patients. This preliminary study seems to suggest that metastatic disease may be characterized by enhanced IL-18 secretion the biological and prognostic significance to be established by successive clinical investigation.     

Serum levels of interleukin-2 in cancer patients: preliminary considerations

In order to investigate the production of interleukin-2 (IL-2) in human neoplasms, we determined IL-2 and soluble IL-2 receptors (sIL-2R) in serum from 18 patients with lymphoma and 28 patients with solid tumors, with (15 cases) or without (13 cases) metastases. As controls, 58 healthy subjects were evaluated. Low levels of IL-2 were not observed in patients with lymphoma or limited solid tumor but abnormally low concentrations of IL-2 were seen in 4/15 metastatic solid tumors, associated with abnormally high values of sIL-2R. This preliminary study confirms in vivo the reduced IL-2 production in metastatic solid neoplasms, previously documented in vitro.     

Immunotherapy with intralesional and systemic interleukin-2 of patients with non-small-cell lung cancer

Eight patients affected by non-small-cell lung cancer were treated with intralesional and systemic recombinant IL-2(rIL-2) injection with the aim of activating both tumour-infiltrating lymphocytes and circulating cytotoxic or killer cells. The schedule of treatment was as follows: a daily fine-needle transparietal intralesional rIL-2 injection (1×10⁵ Cetus units) from day 1 to day 5 and systemic rIL-2 infusion (1×10⁵ Cetus units kg^–1 day^–1) from day 6 to day 10. One to four cycles of treatment were received by each patient. Clinical and immunological evaluations were performed (a) before treatment, (b) following the intralesional rIL-2 administration, (c) 1 h after the beginning of rIL-2 infusion and (d) at the end of the systemic rIL-2 infusion. No complete remission was achieved, two patients showed a partial remission, three resulted in stable disease and three patients progressed. Natural killer and lymphokine-activated killer cell activity dramatically decreased 1 h after the beginning of rIL-2 infusion and increased at the end of treatment. A progressive increase of circulating CD8⁺ and HLA class II⁺ T cells as well as of CD8⁺ T cell clones, most of which displayed NK activity, was recorded following rIL-2 infusion. Present data indicate that (a) the local administration of rIL-2 coupled with systemic rIL-2 infusion may be suggested as an alternative approach for the immunotherapy of lung cancer, (b) rIL-2 induces different immunological modifications according to the route and the time of its administration and (c) rIL-2 administration increases the amount of circulating immune cells with potential antitumour activity.     

Tumor necrosis factor alpha and beta and interferon gamma gene polymorphisms in Turkish breast cancer patients

Cytokine genes are important for researching cancer predisposition to cancers that elicit anti-tumor immune response. In this study, we investigated the association between breast cancer and tumor necrosis factor alpha (TNF-α) -308 (G>A), TNF-β +252 (A>G), and interferon gamma (IFN-γ) +874 (T>A) gene polymorphisms in a Turkish population. This study involved 204 female breast cancer patients and 204 healthy female controls. Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by polymerase chain reaction, allele-specific oligonucleotide polymerase chain reaction, and restriction fragment length polymorphism. TNF-α -308 genotype was found to have no effect on breast cancer susceptibility. However, there were statistically significant differences between the genotype frequencies of patients and controls for TNF-β polymorphism (p?=?0.016) and the allele and genotype frequencies for the IFN-γ polymorphism (p?=?0.0312 and p?=?0.001, respectively). In the composite genotype analysis, the TNF-α/β GAAG composite genotype (p?=?0.0424), the TNF-α/IFN-γ GGTT and GATT composite genotypes (p?=?0.0296 and p?=?0.0129, respectively), the TNF-β/IFN-γ AGTT composite genotype (p?=?0.0003), and the TNF-α/β/IFN-γ GGAGTT and GAAGTT composite genotypes (p?=?0.0437 and p?=?0.0038, respectively) were estimated to have a protective effect against breast cancer. However, the TNF-α/IFN-γ GGTA composite genotype is a risk factor for breast cancer (p?=?0.0156). In conclusion, TNF-β +252GG genotype was found more frequent in Turkish breast cancer patients than controls and IFN-γ TA+AA genotypes were estimated to increase breast cancer risk significantly in Turkish population.     

Intrapleural application of natural IFN alpha in breast cancer patients with pleural carcinomatosis. Monitoring of immunotherapy by assaying serum interferon levels.

For resistant local recurrence, e.g. in breast cancer, or metastatic spread, local infiltration of IFN may be an interesting new approach. The aim of this study was to find out if intrapleurally administered interferon, in breast cancer patients with pleural carcinomatosis, can cause measurable serum concentrations and how soon after administration. Serum IFN concentrations were compared with those in the pleural fluid, and correlated with the presence of malignant cells in the pleural fluid. To uncover possible rhythmicity of serum interferon levels and its relationship to the timing of therapy, natural leukocyte interferon was administered intrapleurally at 10 a.m. Data on pharmacokinetics were obtained from blood samples drawn at -2, 0, 2, 8, 14, 22 and 46 h during the course of treatment. In contrast to our previous observations in healthy volunteers, levels of serum IFN before therapy had no circadian rhythmicity. Daily pharmacokinetic profile of individual patients on interferon therapy has shown that serum IFN peaks 8 h after intrapleurally administered IFN alpha. The peak depended on frequency and number of applied doses. During treatment with IFN alpha, malignant cells degenerated and finally disappeared from pleural fluid. At the same time reactive cells appeared. This effect is rather uniformly observed, but varies in degree. The number of patients is too small, however, to permit conclusions in regard to correlation of this clinical effect and the levels of serum IFN alpha.     

Recombinant interleukin-2 treatment in patients with metastatic colorectal cancer: Effect on natural cytotoxicity

Summary Natural cytotoxicity (natural killer, NK, and lymphokine-activated killer, LAK, activity) was documented in 12 patients with metastatic colorectal cancer, both before and after a 5-day course of continuous therapy with intravenous recombinant interleukin-2 (rIL-2). Treatment induced a substantial increase in circulating CD56⁺ lymphocytes (pretreatment: 12.1±6.9%, mean ± SD; posttreatment: 39.2±6.9%. Maximal NK cell activity was induced by treatment with rIL-2 but only suboptimal augmentation of LAK cell cytotoxicity was obtained. This study indicates that although continuous infusion of rIL-2 does have a significant effect on natural cytotoxicity, this is suboptimal and further studies are necessary to define the most efficacious immunity-enhancing regimens of therapy, thereby hopefully improving clinical outcome of rIL-2 treatment.