P. Lai

Medroxyprogesterone acetate reduces the in vitro production of cytokines and serotonin involved in anorexia/cachexia and emesis by peripheral blood mononuclear cells of cancer patients.

Medroxyprogesterone acetate (MPA) is widely used in oncology both in the treatment of hormone-related cancers and as supportive therapy in anorexia/cachexia syndrome (ACS), but conclusive data are not yet available to explain its anticachectic effect. ACS is characterised by weight loss, changes in metabolism, reduction of appetite, nausea and vomiting. Several cytokines, mainly interleukin (IL)-1, IL-2, IL-6 and tumour necrosis factor alpha (TNF alpha), are involved in the pathogenesis of ACS. Additionally, nausea and vomiting can be mediated by factors inducing serotonin (5-HT) production and/or release by pleiotropic cells including activated T lymphocytes. In the present study, we report the effect of MPA on peripheral blood mononuclear cells (PBMC) from 10 cancer patients in advanced stage of disease (6 head and neck, 2 colon, 1 lung and 1 ovary). The proliferative response of PBMC to PHA, anti-CD3 monoclonal antibody (MAb) or recombinant IL-2 (rIL-2), the production of IL-1 beta, IL-2, IL-6, TNF alpha and 5-HT by PHA-stimulated PBMC and the expression of lymphocyte membrane-bound IL-2 receptor (IL-2R) subunities (CD25 and CD122) were studied. The addition of MPA significantly reduced the PBMC proliferative response to PHA and anti-CD3 MAb but not to rIL-2. MPA 0.2 microgram/ml was also capable of reducing the levels of IL-1 beta, IL-6, TNF alpha and 5-HT produced in culture by PHA-stimulated PBMC, whereas it did not induce any change in the percentage of PBMC expressing either CD25 or CD122 or both molecules after stimulation with PHA or anti-CD3 mAb.

Tumor‐associated lympho‐monocytes from neoplastic effusions are immunologically defective in comparison with patient autologous PBMCs but are capable of releasing high amounts of various cytokines

We studied several in vitro activities of tumor‐associated lympho‐monocytes (TALMs) and the concentrations of interleukin (IL)‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, tumor necrosis factor (TNF)α, interferon (IFN)γ and soluble IL‐2 receptor (sIL‐2R) in neoplastic effusions and in the serum of advanced stage cancer patients. Comparisons were made with autologous peripheral blood mononuclear cells (PBMCs). Autologous PBMCs were compared with PBMCs from normal subjects used as controls. TALMs were collected from 13 peritoneal and 18 pleural neoplastic effusions, secondary to primary tumors of different sites. After PHA stimulation, concentrations of IL‐1α, IL‐1β and TNFα in culture media of TALMs both from peritoneal and pleural effusions were lower than those of autologous PBMCs and, similarly, concentrations of IL‐4 and IL‐10 in culture media of TALMs from peritoneal effusions were lower than those of autologous PBMCs, whereas concentrations of IL‐4 and IL‐10 in culture media of TALMs from pleural effusions were in the same range as those of autologous PBMCs. On the contrary, IL‐2, IL‐6 and IFNγ amounts (only from pleural effusions) were significantly higher. IL‐1α, IL‐1β, IL‐2, IL‐6 and TNFα production from patient PBMCs was lower than that of control PBMCs, whereas production of IL‐4, IL‐10 and IFNγ was higher than that of control PBMCs. Both in peritoneal and in pleural effusions concentrations of IL‐1α, IL‐1β and IL‐4 were not different from those measured in autologous serum, whereas those of IL‐6, IL‐10, TNFα, IFNγ and sIL‐2R were significantly higher. The amounts of IL‐2 in pleural effusions were not different from those of autologous serum, but in peritoneal effusions they were higher than those of autologous serum. The amounts of IL‐1α, IL‐1β, IL‐2, IL‐6, TNFα and sIL‐2R were higher in patient than in control sera, whereas those of IL‐4, IL‐10 and IFNγ were in the same range in patient and in control sera. Cell cycle analysis of cultured TALMs and PBMCs (from 3 patients) showed a significant accumulation of TALMs in the non‐cycling G0/G1 cell population compared with autologous PBMCs.Int. J. Cancer 71:724‐731, 1997.

Neo-adjuvant chemo-(immuno-)therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin + 5-fluorouracil (5-FU) with cisplatin + 5-FU + recombinant interleukin 2

Abstract We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical response and toxicity of the combination chemotherapy cisplatin + 5-FU with the same combination plus s.c. recombinant interleukin-2 (rIL-2) in patients with advanced (stage III–IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical Al Sarraf treatment: 100 mg/m2 cisplatin i.v. on day 1 plus 1000 mg m−2 day−1 5-FU on days 1–5 as a continuous infusion. Regimen B was the same as regimen A plus 4.5 MIU/day rIL-2 s.c. on days 8–12 and 15–19. Treatment was repeated every 3 weeks for three cycles. A total of 33 patients were enrolled in the study; 30 were evaluable for toxicity and 28 for response. Seventeen patients were assigned to group A and 16 were assigned to group B. Three patients (20%) of group A and 4 (31%) of group B had a complete response, 9 patients (60%) of group A and 6 (46%) of group B had a partial response, with an overall response rate of 12 patients (80%) for group A and 10 patients (77%) for group B. Two patients (13%) of group A and 3 patients (23%) group B had stable disease; 1 patient (7%) of group A had progressive disease. Thus, there was not a statistically significant difference in response rate between the two groups and therefore there was no benefit from the addition of immunotherapy with rIL-2 to the standard chemotherapy. Both regimens were well tolerated. There were 2 toxic deaths (6.7%), 1 from hematological causes in group A and 1 from cardiac causes in group B. Myelosuppression and gastrointestinal toxicity, mainly nausea/vomiting and stomatitis, were the most frequent toxicities. The calculated number of patients for the sample has not yet been reached; however, the projection of our present results suggests that it is highly improbable that a clinically significant difference between the two treatment groups will be observed even if the calculated patient sample size is achieved.

Tumor-associated lymphocytes (TAL) are competent to produce higher levels of cytokines in neoplastic pleural and peritoneal effusions than those found in sera and are able to release into culture higher levels of IL-2 and IL-6 than those released by PBMC

This work was designed to study the proliferative response of tumor-associated lymphocytes (TAL) from neoplastic effusions against autologous tumor cells and the immunophenotype pattern of TAL from neoplastic effusions and that of PBMC of the same patients. We also compared the serum levels of the cytokines interleukin (IL) 1β, 2 and 6, tumor necrosis factor-α (TNFα) and soluble IL-2 receptor (sIL-2R) with those present in neoplastic effusions of the same patients. Moreover, we examined the ability of TAL and peripheral blood mononuclear cells (PBMC) to produce and release the cytokines and sIL-2R and to express membrane CD25 following their stimulation with phytohemagglutinin (PHA) in vitro. Finally, we compared the cytokines/sIL-2R production and membrane CD25 expression by PHA-stimulated PBMC of the patients with neoplastic effusions with a series of 90 cancer patients without neoplastic effusions and 20 normal healthy subjects. Thirteen neoplastic pleural and eight peritoneal effusions were collected from 11 patients with primary lung cancer, 7 with primary epithelial ovarian cancer, 1 with breast cancer, 1 with pleural mesothelioma, and 1 with pancreatic cancer. The proliferative response of TAL from neoplastic effusions against autologous tumor cells was lower than the response to PHA, IL-2, and anti-CD3, but significant. The percentage distribution of CD3+ and CD8+ lymphocyte subpopulations was higher in peritoneal than in pleural effusions, while the CD16+ subset was higher in pleural than in peritoneal effusions. The percentage distribution of CD16+ was significantly lower in pleural effusions than in PBMC of patients with pleural effusions. The CD39 antigen was higher on TAL from peritoneal effusions than on PBMC of the same patients. The levels of IL-1β and sIL-2R in peritoneal effusions did not differ from those measured in the sera of the same patients, while the levels of IL-2, IL-6, and TNFα were higher in the peritoneal effusions. The levels of IL-2, IL-6, TNFα, and sIL-2R, but not IL-1β, in pleural effusions were significantly higher than those found in the sera of the same patients. The amounts of IL-2 and IL-6 produced by TAL were generally higher than those released by PBMC. The secretion of cytokines IL-1α, IL-2, and sIL2R by PHA-stimulated PBMC was lower, but IL-1β and IL-6 secretion was higher in cancer patients with neoplastic effusions than in either cancer patients without neoplastic effusions or normal subjects. The CD25 expression on PHA-stimulated PBMC derived from cancer patients with neoplastic effusions was in the same range as that of cancer patients without neoplastic effusions and normal subjects. These findings suggest that TAL may be able to produce cytokines and may be amenable to immune manipulation.

Evidence that Cisplatin Induces Serotonin Release from Human Peripheral Blood Mononuclear Cells and that Methylprednisolone Inhibits this Effect

Corticosteroids counteract cisplatin (CDDP)-induced acute emesis but the mechanism involved is still unknown. Therefore, the aim of this study was to verify whether CDDP can induce serotonin (5HT) release from peripheral blood mononuclear cells (PBMC) and determine whether methylprednisolone (MP) can inhibit such release. Blood from 10 healthy volunteers was used. Our study showed that CDDP did induce 5HT release from PBMC dose-dependently (10 +/- 1 nM for controls, 18 +/- 4 nM for CDDP 0.01 microgram and 30 +/- 4 nM for CDDP 0.1 microgram, P < 0.001) and that the addition of MP to cultures of PBMC in the presence of CDDP induced a significant decrease of 5HT concentrations. Our results highlight a new mechanism through which CDDP could induce emesis and suggest a further mechanism by which corticosteroids mediate their anti-emetic effect.

Results of a dose-intense phase 1 study of a combination chemotherapy regimen with cisplatin and epidoxorubicin including medroxyprogesterone acetate and recombinant interleukin-2 in patients with inoperable primary lung cancer

Based on the role of cytokines in the pathogenesis of cancer-related anorexia–cachexia and the ability of progestins, such as medroxyprogesterone acetate, to reduce cytokine production and relieve cancer-related anorexia–cachexia symptoms, the authors designed an open, dose-finding phase I study of a combined chemotherapy regimen (cisplatin [CDDP], epidoxorubicin [EPI]), including recombinant interleukin-2 (IL-2) and medroxyprogesterone acetate for patients with stage IIIB to IV inoperable primary lung cancer. The end points were clinical response and toxicity with definition of dose-limiting toxicity and maximal tolerable dose; relief of cancer-related anorexia–cachexia symptoms; the assessment of patient serum levels of IL-1&bgr;, IL-6, tumor-necrosing factor-&agr; (TNF-&agr;), and soluble IL-2 receptor (sIL-2R). From March to October 1997, 16 patients (M:F ratio, 14:2; mean age, 60.5 years; age range, 41 to 74 years) were enrolled. All patients were evaluable for toxicity and 14 of them for response. The patients were assigned to increasing dose levels of drugs according to a dose-escalation schedule. The weekly schedule consisted of a combination of CDDP given intravenously on day 1, EPI given intravenously on day 1, 1 g/day medroxyprogesterone acetate given orally on days 1 to 7, and recombinant IL-2 1.8 MIU administered subcutaneously on days 2 to 7 plus 300 &mgr;g granulocyte-colony stimulating factor support given subcutaneously on days 2 to 5. Administration of medroxyprogesterone acetate began 1 week before the first cycle. Dose escalation of the drugs was as follows: 30 mg·m2·week−1 CDDP and 25 mg·m2·week−1 EPI (first level, two patients); 30 mg·m2·week−1 CDDP and 33 mg·m2·week−1 EPI (second level, 2 patients); 40 mg·m2·week−1 CDDP and 33 mg·m2·week−1 EPI (third level, 6 patients); and 40 mg·m2·week−1 CDDP and 40 mg·m2·week−1 EPI (fourth level, 6 patients). Six cycles were planned for each patient. The actual dose intensity delivered was more than 80% of the projected dose intensity of all drugs. After six cycles, clinical response (according to World Health Organization criteria), toxicity (according to World Health Organization criteria), Eastern Cooperative Oncology Group (ECOG) performance status, body weight, appetite, and serum levels of cytokines were evaluated. After six cycles, 9 of 14 patients (64.3%) had partial response, 3 of 14 (21.4%) had stable disease, and 2 of 14 (14.3%) had progressive disease, and the objective response rate was 64.3%. ECOG performance status and body weight did not change significantly after treatment, whereas appetite showed an increase that was of borderline statistical significance. Toxicity was acceptable and only hematologic. Dose-limiting toxicity was established at the fourth dose level; consequently, maximal tolerable dose was assessed at the third dose level. Before treatment, the serum levels of IL-1&bgr;, IL-6, and TNF-&agr; were significantly greater in the patients than in healthy persons. The comparison between pretreatment and posttreatment serum values of IL-1&bgr;, IL-6, TNF-&agr;, and sIL-2R did not reveal significant differences in the patients. Similar results were obtained when the patients were considered as responders (partial response) or nonresponders (stable or progressive disease) to therapy. Only IL-6 serum levels were increased (p = 0.014) after treatment.

Membrane-bound and soluble IL-2 receptors (p55 and p75 chains) on peripheral blood mononuclear cells from patients with solid malignancies.

The aim of the investigation was to study directly the IL-2 receptor (IL-2 R) and its subunits, p55 and p75 chains, either membrane-bound or soluble, on PBMC of patients with solid malignancies and, indirectly, the same patients’ PBMC ability to produce IL-2. Fifty-eight cancer patients, 29 men and 29 women, were studied: their mean age was 57.3 yr, range 35–79. Twenty-two healthy age-sex-matched subjects served as controls. The tumors were the most common and the most representative among human cancers, i.e., breast, lung, head and neck, digestive tract and liver, prostate and gynecologic cancers: they were generally in advanced stages and in 23 cases metastatic. The PBMC proliferative response to PHA, PHA plus IL-2, and IL-2 was evaluated along with the response to PHA in the presence of anti-p55, anti-p75 monoclonal antibodies, or both. Moreover, membrane-bound IL-2 R (p55 and p75 chains) on PHA-stimulated PBMC was detected, along with soluble IL-2 R in the serum and in the culture supernatants. The conclusions suggest that in solid malignancies: the membrane-bound IL-2 Rs, both p55 and p75 chains, are expressed normally, there is an high serum level of soluble IL-2 R, there is a normal release of soluble IL-2 R in culture, and there is an indirect evidence of a lack of IL-2 production. Therefore, no primary impairment of IL-2 R was found in solid tumors. Moreover, in our study we have found no difference in any parameter studied between patients with and patients without metastases.

Membrane-bound/soluble IL-2 receptor (IL-2R) and levels of IL-1α, IL-2, and IL-6 in the serum and in the PBMC culture supernatants from 17 patients with hematological malignancies

The present study investigated the peripheral blood mononuclear cells (PBMC) blastic responses to PHA, PHA plus recombinant IL-2 (rIL-2) and rIL-2 alone; the expression of membrane-bound IL-2R on PHA-stimulated PBMC; and the levels of IL-1α, IL-2, IL-6, and sIL-2R in serum and in culture supernatants from PHA-stimulated PBMC in 17 patients with hematological malignancies (mean age 58.5 yr, range 22–82): 6 with non-Hodgkin’s lymphoma (NHL), 4 with Hodgkin’s lymphoma (HL), 5 with Hairy cell leukemia, 1 with chronic myelogenous leukemia, and 1 with chronic lymphocytic leukemia. The patients with HL and NHL with active disease (AD) were separated from those in clinical remission. The patients with AD were studied at diagnosis (obviously before therapy) and the patients in clinical remission were out of therapy since at least 6 mo. The lymphocyte blastogenic response to PHA was significantly lower in patients with HL and NHL with AD than in the control group. The response to rIL-2 alone was in the same range in the control group and in HL and NHL AD patients. By adding rIL-2 to PHA there was an increase of the blastogenic response of the same patients. The percentage of CD25 expressed on PHA-stimulated lymphocytes from patients with HL and NHL AD and from normal subjects is in the same range. Serum levels of IL-2, IL-6, and sIL-2R were significantly higher in HL and NHL AD patients than in controls as well as in all other hematological malignancies. Supernatants derived from PHA-stimulated PBMC were assessed for the presence of cytokines and sIL-2R by ELISA. The levels of IL-2, IL-6, and sIL-2R were significantly lower in HL and NHL AD patients than in controls as well as in all other hematological malignancies.

Study of peripheral blood lymphocyte subset distribution and IL-2 receptor (IL-2 R) p55–p75 subunit expression in patients with cancer of different sites

The aim of the study was to evaluate the subset distribution and the IL-2 R p55–p75 subunit expression on unstimulated and phytohemagglutinin (PHA)-stimulated (at 3-d) peripheral blood mononuclear cells (PBMC), of patients with solid cancers of different sites. Indeed the expression of the two subunits of IL-2R is an essential prerequisite for The action of the IL-2 on CD8+, CD16+ lymphocytes as effectors in antitumor activity (LAK-cells). The subset distribution (CD3, CD4, CD8, CD16, DR) was assessed by cytofluorometry with specific monoclonal antibodies (MAbs); the p55 (CD25) and p75 subunit expression was evaluated by specific MAb (OKT26a and anti-p75). Ninety patients with advanced cancer (mainly non-small cell lung cancer [NSCLC], head and neck cancer, and gynecological cancer; mean age 55 yr; range 27–80) were studied. Thirty-five age- and sex-matched healthy subjects were studied as controls. Our data show that there is no significant difference in the subset distribution between cancer patients and controls. Furthermore, no difference has been found in the expression of p55 subunits on unstimulated PBMC between cancer patients and controls. No difference has been found in the expression of both p55 and p75 subunits on PHA-stimulated PBMC between cancer patients and controls. Our results can support the rationale for further clinical trials with IL-2 in solid malignancies.