R. Versace

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.

BARD1: an independent predictor of survival in non-small cell lung cancer

BRCA1 mRNA overexpression is correlated with poor survival in NSCLC. However, BRCA1 functions depend on the interaction with BARD1 for its stability, nuclear localization and ubiquitin ligase activity. Expression of alternatively spliced BARD1 isoforms that lack the BRCA1‐interaction domain was found upregulated and correlated with poor prognosis in breast and ovarian cancer. These BARD1 isoforms are essential for proliferation of cancer cells in vitro. We investigated whether BARD1 isoforms are expressed in NSCLC. While in lung tissues from healthy controls BARD1 expression was undetectable on the mRNA level and protein level, we found two novel isoforms in addition to previously identified mRNAs expressed in all NSCLC samples tested. Furthermore, the pattern of BARD1 isoform expression was similar in tumor and morphologically normal peri‐tumor tissues, and only one novel isoform π was specifically upregulated in tumors. Immunohistochemistry revealed that all 100 NSCLC cases tested expressed isoform‐specific BARD1 epitopes, while BARD1 expression was undetectable in biopsies from healthy controls. Statistical analysis showed that the expression of epitopes PVC and WFS, present on isoform π, or epitope WFS alone, expressed on isoforms π, κ and β, were significantly correlated with decreased patient survival. These findings were corroborated in a mouse model of chemically induced lung cancer. Immunostaining of mouse tumors showed that BARD1 epitopes PVC and WFS were specifically upregulated in invasive, but not in confined lung tumors. Thus, BARD1 isoforms might be involved in tumor initiation and invasive progression and might represent a novel prognostic marker for NSCLC.

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.

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.