A. Aquino

Cetuximab ± chemotherapy enhances dendritic cell-mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen-specific cytotoxic T-cell response in vitro

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune‐mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti‐tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen‐cross presentation to cytotoxic T‐lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC‐mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug‐treated cetuximab‐coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen‐A(*)02.01+ donors. T‐cell cultures were characterized for immune‐phenotype and tumor‐antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L‐folinate + 5‐flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC‐mediated cross‐priming of antigens derived from mAb‐covered/drug‐treated cancer cells elicited a robust CTL anti‐tumor response. On the basis of our data, we suggest a possible involvement of CTL‐dependent immunity in cetuximab anti‐cancer effects.

Hypercalcemia in breast cancer

Hypercalcemia is relatively frequent in malignancy with or without osteolytic bone metastases. It is thought that neoplastic cells may secrete substances which not only stimulate osteoclastic activity but are also capable of modifying the absorption, excretion, and resorption of calcium and phosphate ions. Since 1987, we have studied 24 breast cancer patients with hypercalcemia (22 with bone metastases and two without). The group of 22 patients with bone metastases were divided into two subgroups. The first consisted of 10 patients with high serum levels of humoral factors, such as parathyroid hormone-related protein (PTHrP), and/or prostaglandin E2 (PGE2) and/or interleukin 1 (IL-1), and high levels of bone markers, such as alkaline phosphatase, bone Gla protein and urinary hydroxyproline. The second subgroup consisted of 12 patients with high levels of bone markers alone. Bone histologic analysis showed an osteoclastic activation surrounding metastatic tumor tissue in six out of 10 patients of the first subgroup, while an evident osteolysis caused by the tumor cells was noted in seven out of 12 patients of the second subgroup. The two patients without bone metastases showed normal biochemistry and bone histologic examination. The authors, having tried to explain the pathogenesis of hypercalcemia, emphasize the importance of humoral factors secreted by tumor cells as a direct or indirect cause of hypercalcemia. The origin of hypercalcemia remains unclear in two patients without bone metastases.

Weekly chemotherapy in advanced prostatic cancer.

This randomised phase II study was performed in order to evaluate the effectiveness of a weekly chemotherapy regimen in advanced prostatic carcinoma patients (stage D2) refractory to hormonal therapy. Seventy-two cases were studied: they were randomised in a 2:1 ratio to receive either epirubicin (30 mg m-2 weekly) or doxorubicin (25 mg m-2 weekly); 48 patients received epirubicin and 24 received doxorubicin. After 12 courses of chemotherapy, the 45 evaluable patients in the epirubicin arm showed a response rate of 37.7% and the 21 evaluable patients in the doxorubicin arm showed a response rate of 33.3% (P = 0.51). Pain intensity, bone and prostatic tumour markers rapidly and significantly decreased in responders. An improvement in physical symptoms, functional conditions and in emotional well-being was observed in the majority of the treated patients. The histological analysis of bone metastases, performed before and after 12 courses of chemotherapy showed a significant reduction in neoplastic invasion and in new bone formation in responders. Cardiac performance worsened in five out of 45 patients and in ten out of 21 during the first 12 courses of epirubicin or doxorubicin respectively (P = 0.014). The median survival was 12.5 months in the epirubicin arm and 8.0 months in the doxorubicin arm (P = 0.042). Our data indicate that in advanced prostatic carcinoma, a weekly epirubicin regimen may give rapid palliative results, similar to that of doxorubicin, but with less side-effects.

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

Abstract 5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses In Vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1 + healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity In Vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

TREATMENT OF ADVANCED COLORECTAL CANCER WITH HIGH-DOSE INTENSITY FOLINIC ACID AND 5-FLUOROURACIL PLUS SUPPORTIVE CARE

This randomised clinical trial, involving patients with advanced colorectal cancer, was carried out to compare the effectiveness of accelerated folinic acid (FA) plus 5-fluorouracil (5-FU) with that of the conventional regimen of 5-FU alone. Both regimens were administered with simulataneous supportive care. 185 patients were eligible: 94 were randomly allocated to receive FA 200 mg/m2 i.v. plus 5-FU 400 mg/m2 i.v. on days 1-5 every 3 weeks; and 91 to receive 5-FU 400 mg/m2 i.v. on days 1-5 every 4 weeks. The response rate was 33.3% in the accelerated FA/5-FU and 18.6% in the 5-FU arm (P = 0.045). Median survival was 13.5 months in the FA/5-FU arm and 7.5 months in the 5-FU arm (P = 0.039). Toxicity was mild and slightly more pronounced in the FA/5-FU arm (P = 0.078). This study indicates that, in patients with advanced colorectal cancer, accelerated chemotherapy with FA and 5-FU and simultaneous supportive care is capable of achieving a higher response rate and longer survival than conventional 5-FU alone, without severe toxicity.

Advanced breast cancer treatment with folinic acid, 5-fluorouracil, and mitomycin C

A total of 44 women with advanced breast cancer who had failed first-and second-line chemotherapy were given combination chemotherapy consisting of folinic acid (FA), 5-fluorouracil (5-FU) and mitomycin C (MMC). The treatment schedule was: 200 mg/m2 FA and 400 mg/m2 5-FU given i.v. over 2 h for 5 days plus 5 mg/m2 MMC given i.v. on days 3–5; in 19 patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 3–4 and bone marrow depression, the MMC dose was 3 mg/m2 given i.v. on days 3–5. In all, 41 patients were evaluable for response; 15 had a partial remission (PR), 18 had stable disease (SD), and 8 showed progressive disease (PD). The median response duration was 6 months and the median survival was 10 months. Toxicity was mild and consisted mainly of stomatitis, diarrhea, and leukopenia. A rapid improvement in performance status was noted in responding patients. A striking result was the reduction of analgesics in most cases and their complete withdrawal in responding patients. This combination chemotherapy achieved satisfactory effectiveness and improved the quality of life of patients.

Production of parathyroid hormone and parathyroid-hormone-related protein by breast cancer cells in culture

Parathyroid-hormone-related protein (PTHrP) has been implicated in the origin of malignant hypercalcaemia. However, PTHrP production is not restricted to neoplastic cells, it is widespread among a variety of normal cell types and tissues. A physiological role for PTHrP has not been well defined. We describe a case of breast cancer with bone metastases and humoral hypercalcaemia of malignancy, with high levels of plasma C-terminal parathyroid hormone (PTH), mid-molecule PTH and PTHrP. Cells from breast cancer biopsies were cultured and medium samples assayed for the C-terminal and mid-molecule fragments, intact PTH and PTHrP. The data indicate a progressive increase in both PTH fragments and PTHrP levels, over a period of 30 days. No temporal parallelism exists between PTH fragments and PTHrP concentrations, the former being maximum at the 14th day, and the latter at the 30th day from the beginning of the culture. Our results indicate a coproduction of PTH and PTHrP by the breast cancer cells both in vivo and in vitro.

6140 POSTER Cetuximab-mediated Immune-enhancing Effects in Vitro and in Metastatic Colorectal Cancer (mCRC) Patients

6140 POSTER Cetuximab-mediated Immune-enhancing Effects in Vitro and in Metastatic Colorectal Cancer (mCRC) Patients P. Correale1, C. Botta1, M.G. Cusi2, E. Bestoso1, S. Apollinari1, M. Caraglia3, M.T. Del Vecchio4, A. Abbruzzese3, A. Aquino5, P. Tagliaferri6. 1“S. Maria alle Scotte” Siena University Hospital, Department of Oncology, Siena, Italy; 2“S. Maria alle Scotte” Siena University Hospital, Department of Molecular Biology Microbiology Section, Siena, Italy; 3II University of Naples, Department of Biochemistry and Biophysics, Naples, Italy; 4“S. Maria alle Scotte” University Hospital of Siena, Section of Pathological Anatomy Department of Human Pathology and Oncology, Siena, Italy; 5“Tor Vergata” University, Department of Neurosciences Pharmacology Section, Rome, Italy; 6Magna Graecia University and Tommaso Campanella Cancer Center Catanzaro, Medical Oncology, Catanzaro, Italy Introduction: Cetuximab is a chimeric human-murine monoclonal IgG1 to the Epidermal-growth-factor-Receptor, approved for colorectal cancer treatment with chemotherapy. Its human constant fragment (Fc), throughout receptor binding, trigger additional immune-mediated effects, which may offer significant contribute to the final therapeutic effect. We investigated cetuximab ability in vitro to promote colon cancer cell phagocytosis and antigen-cross-priming by dendritic cells (DCs) to tumour-specific cytotoxic- T-cell (CTL) precursors. We also carried-out an immunological study in 26 mCRC patients enrolled in an on-going phase 2 trial, receiving an experimental biochemotherapy (GILFICet) regimen combining gemcitabine, irinotecan, fluoruracil, levofolinate, cetuximab, and metronomic sc. aldesleukine. Material and Methods: Transmission-electron-microscopy (TEM) and Flow cytometry were used to evaluate the susceptibility of multiple colon cancer cell lines to DC-mediated phagocytosis. Human DCs loaded with cetuximab-coated colon cancer cells, exposed or not to a combination of anti-cancer drugs, were used to in vitro sensitize human PBMCs from normal donors and cancer patients collected at baseline and after 3 GILFICet courses. T-cell cultures were characterized for immunephenotype and tumour-antigen specific CTL activity by Flow cytometry, LDH release and IFNg-ELISPOT. Results: ILF (irinotecan+ folinate+5-flurouracil) and GILF (gemcitabine+ ILF) poly-chemotherapies confirmed their ability to induce antigen remodelling and danger signals in colon cancer cells. After exposure to poly-chemotherapy and cetuximab these cells became highly susceptible to Fc-receptor-mediated phagocytosis/trogocytosis by human DCs, promoted their activation, and increased their ability to elicit a highly efficient CTL response on human PBMCs in vitro. Our study on the PBMCs of colon cancer patients enrolled in the GILFICet trial, revealed a significant treatment-related increase in na¨ive and central-memory-Tcells, activated-CTLs, NK/NKTs, mature-activated DCs and IFNg-releasing cells. Substantial differences were observed in T-cell lines generated from patients’ PBMCs taken before and after biochemotherapy. In the latter group there was in fact, a remarkable increase in proliferating CD8+Ki67+CTLs and tumour-antigen specific CTLs’ precursors. Discussion: These results suggest that cetuximab may exert immuneenhancing effects with potential antitumour activity

Quality of life in advanced colorectal cancer

Colorectal carcinoma is a knowingly chemoresistant neoplasia. Fluorouracil (5-FU) is considered the standard drug for the treatment of advanced colorectal cancer and it is known that the action of 5-FU is potentiated by Folinic-acid (FA).