Impact of temozolomide on immune response during malignant glioma chemotherapy.

Malignant glioma, or glioblastoma, is the most common and lethal form of brain tumor with a median survival time of 15 months. The established therapeutic regimen includes a tripartite therapy of surgical resection followed by radiation and temozolomide (TMZ) chemotherapy, concurrently with radiation and then as an adjuvant. TMZ, a DNA alkylating agent, is the most successful antiglioma drug and has added several months to the life expectancy of malignant glioma patients. However, TMZ is also responsible for inducing lymphopenia and myelosuppression in malignant glioma patients undergoing chemotherapy. Although TMZ-induced lymphopenia has been attributed to facilitate antitumor vaccination studies by inducing passive immune response, in general lymphopenic conditions have been associated with poor immune surveillance leading to opportunistic infections in glioma patients, as well as disrupting active antiglioma immune response by depleting both T and NK cells. Deletion of O6-methylguanine-DNA-methyltransferase (MGMT) activity, a DNA repair enzyme, by temozolomide has been determined to be the cause of lymphopenia. Drug-resistant mutation of the MGMT protein has been shown to render chemoprotection against TMZ. The immune modulating role of TMZ during glioma chemotherapy and possible mechanisms to establish a strong TMZ-resistant immune response have been discussed.

Celecoxib desensitization: continued temozolomide/celecoxib chemotherapy after a celecoxib-induced hypersensitivity reaction.

Cox-2 inhibitors have been identified as promising candidates for cancer therapy. Several studies have recently proposed the use of celecoxib in long-term low-intensity chemotherapy protocols for recurrent tumors. However, drug-induced hypersensitivity reactions may force discontinuation of the medication and, thus, significantly complicate successful care. Here, we report on celecoxib desensitization after a celecoxib-induced skin reaction, thereby allowing the continuation of temozolomide/celecoxib chemotherapy in a young patient with recurrent astrocytoma.

Temozolomide associated with PEG-interferon in patients with metastatic melanoma: a multicenter prospective phase I/II study.

Metastatic melanoma treatment remains disappointing, and a combined approach by chemotherapy and immunotherapy might increase the response rates through a synergistic action. Accordingly, a clinical trial using oral temozolomide (TMZ) and subcutaneous PEG-interferon alpha-2b (PEG) in patients with metastatic melanoma was designed to determine the maximal tolerated dosage of both drugs and the antitumoral response. A multicenter, prospective, phase I/II study was conducted in 31 metastatic melanoma patients, without cerebral metastasis. Dose escalation was performed according to the modified continual reassessment method scale and resulted in four cohorts of patients: TMZ 150 mg/m2 5 days/week each 4 weeks and PEG 0.5 microg/kg/week - TMZ 150 mg/m2 5 days/week and PEG 1.0 microg/kg/week - TMZ 200 mg/m2 5 days/week and PEG 0.5 microg/kg/week - TMZ 200 mg/m2 5 days/week and PEG 1.0 microg/kg/week. Patients received a maximum of six cycles. Thirty-three patients were enrolled in this study: one in the first dose level, one in the second one, 18 in the third one and 11 in the fourth one. At level 4, four of 11 patients experienced dose-limiting toxicity and four nondose-limiting toxicity; toxicity was mainly hematologic (grade IV thrombocytopenia). An objective response was observed in five patients (two complete response and three partial response) receiving level 3 or 4 of treatment. The disease remained stable in three patients, and six of 31 patients were alive 24 months after enrollment. The association of oral TMZ with subcutaneous PEG in metastatic melanoma displayed an unacceptable hematological toxicity with the dosages of 200 mg/m2 5 days/week and 1 microg/week, respectively. At a lower level, this treatment was effective and deserves further investigations to define its indications in metastatic melanoma patients.

Ifosfamide and cyclophosphamide: effects on immunosurveillance.

Ifosfamide (IF) and cyclophosphamide (CTX) are chemotherapeutic agents frequently used in the treatment of human malignancies. These drugs can exhibit a bimodal mechanism of antitumor action with cytotoxic and immunomodulatory effects when associated with adoptive immunotherapy. In human peripheral blood lymphocytes, IF irreversibly inhibits the proliferative response to interleukin-2 in a dose-dependent manner and may also induce the phosphorylation of HSP27 by depleting glutathione. CTX promotes discrete cytokine profiles upregulating the expansion of Th1 cells, and this may be important to increase cellular immune response. The data presented in this report indicate that treatment regimens of CTX and IF may be used according to the tumor immunogenicity.

Effects of cyclophosphamide and soluble tumor-associated antigens on lymphoid infiltration, proliferative activity and rate of apoptosis in chemically-induced rat mammary tumors.

The aim of this study was to analyze the roles of proliferative activity, lymphoid infiltration and apoptotic rate in the cellular mechanism underlying the promotion effects of soluble tumor-associated antigens (sTAA) in mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Studies were performed on the following groups of rats: i) control tumor-bearing rats, ii) tumor-bearing rats treated with sTAA, iii) tumor-bearing rats treated with CPA, iv) tumor-bearing rats treated with CPA and sTAA. Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and the rate of lymphoid infiltration, T cell content (CD4+ and CD8+ cells), and mitotic and apoptotic indices in tumors were evaluated. In control tumor-bearing rats, high lymphoid proliferation, as well as a high number of CD8+ cells, was found in tumors. Treatment with sTAA further significantly increased the total number of lymphoid cells and the number of CD8+ lymphocytes. CPA sharply decreased the production of all lymphoid cells studied, especially of CD4+ lymphocytes. The combined treatment of CPA and sTAA increased the number of lymphoid cells, although they did not reach control levels. The mitotic index significantly decreased as a result of the treatment with CPA alone and especially after the combined treatment with CPA and sTAA. The results of our experiments demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of tumor cells, stimulated production of T lymphocytes and increased rate of apoptosis among tumor cells.

T cell kinetics and apoptosis in immune organs and mammary tumors of rats treated with cyclophosphamide and soluble tumor-associated antigens.

The aim of this review was to analyze the role of cell kinetics and apoptosis in the cellular mechanism underlying the effects of soluble tumor-associated antigens (sTAA) on chemically-induced mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and tumors, the spleen, thymus, bone marrow and lymph nodes were studied by morphometric and immunohistochemical methods. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of CPA, and that the tumor-suppressive antitoxic effects of sTAA result from their activation of T-lymphocyte production in this system, particularly in the spleen and lymph nodes. The results of our experiments have shown that vaccination with sTAA actively promotes generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of the tumor cells, stimulated production of T lymphocytes and an increased rate of apoptosis among tumor cells.

Response of the immune system of mammary tumor-bearing rats to cyclophosphamide and soluble low-molecular-mass tumor-associated antigens: rate of lymphoid infiltration and distribution of T lymphocytes in tumors.

We have shown previously that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this study, we analyzed the possible mechanism underlying this phenomenon. Studies were performed on tumors obtained from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, iv) rats treated with CPA and sTAA. All analyzed tumors represented different types of invasive duct carcinomas. The rate of lymphoid infiltration and T cell content (CD4+ and CD8+ cells) of tumors were analyzed immunohistochemically. In parallel, mitotic index was evaluated in tumor cells. In tumor-bearing rats, high lymphoid proliferation was found at the periphery of tumors, and to a lesser extent deep inside the tumors. In control tumors, CD4+ T cell content was very low whereas CD8+ cells were highly abundant, especially at the tumor periphery. Treatment with sTAA significantly increased the total number of lymph cells and the number of CD8+ lymphocytes inside the tumors. Cytoplasmic vacuolization, decreased mitotic index and various degrees of fibrosis were the most distinct changes in tumors treated with CPA alone. CPA also sharply decreased the activity of all lymph cells studied, especially of CD4+ lymphocytes which could no longer be observed following this treatment. The combined treatment of CPA and sTAA increased the number of lymph cells, although they did not reach control levels. Inhibition of mainly CD4+ lymphocyte synthesis of CPA was confirmed by the low CD4/CD8 ratio, which increased slightly after the combined treatment with CPA and sTAA. Findings in the present study demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response.

Response of the immune system of mammary tumor-bearing rats to cyclophosphamide and soluble low-molecular mass tumor-associated antigens: the spleen and lymph nodes.

In a previous study, we showed that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the underlaying mechanisms. Studies were performed on the spleen and lymph nodes from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, i.v.) rats treated with CPA and sTAA. Different zones of the spleen and lymph nodes were measured and their T cell content (CD4(+) and CD8(+) cells) was analyzed immunohistochemically. CPA decreased the size and cell content of follicles, splenic areas related to the production of B cells, of the marginal zone and to a lesser extent of the periarterial lymph sheath, and decreased the number of CD4(+) and, at a lower rate, of CD8(+ )T cells in the spleen. Addition of sTAA restored activity in the splenic zones producing these cells. Similar effects of CPA and sTAA were found in lymph nodes with accumulation of B lymphocytes in the primary and secondary follicles and of T lymphocytes, including both CD4(+) and CD8(+) cells, in the paracortical zone. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of chemotherapeutic drugs such as CPA and that the tumor-suppressive antitoxic effects of sTAA result from their activation of B- and T-lymphocyte production in this system, particularly in the spleen and lymph nodes.

Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material.

An antibody-directed enzyme prodrug therapy (ADEPT) system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4-[N,N-bis(2-iodoethyl) amino] phenoxycarbonyl L -glutamic acid (ZD2767P) and a conjugate of the F(ab')(2) anti-CEA antibody A5B7 and the bacterial enzyme carboxypeptidase G2 (CPG2). ZD2767P is converted by antibody-targeted CPG2 into an active bifunctional alkylating drug (ZD2767) at the tumour site. The IC(50) value of the prodrug against the human colorectal tumour LS174T cell line was 55 +/- 9 microM following a 1 h exposure. In contrast, co-incubation of ZD2767P with CPG2 resulted in 229-fold increase in activity. Using a modified comet assay, DNA interstrand cross links (ISC) were detected within 1 h of ZD2767P + CPG2 treatment and were repaired by 24 h. A clear dose-response was seen between the level of ISC, growth inhibition and ZD2767 concentration. Administration of a therapeutic dose of ZD2767P 72 h after the F(ab')(2) A5B7 conjugate to mice bearing LS147T xenografts resulted in extensive ISC in the tumour after 1 h; repair was seen at 24 h. Tumour biopsies and peripheral lymphocytes were studied in 5 patients on the ADEPT phase I clinical trial. In 4 patients no ISC were detected. These patients also demonstrated poor localization of conjugate and no tumour response was seen. However a significant level of ISC was detected in one tumour biopsy, which also showed evidence of conjugate localization and clinical response. These studies demonstrate the application of the comet assay in the measurement of ISC in vitro and in clinical material and confirm that activation of ZD2767P results in the formation of DNA crosslinks.

Antibody recognition of melphalan adducts characterized using immobilized DNA: enhanced alkylation of G-Rich regions in cells compared to in vitro.

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

Treatment of severe systemic lupus erythematosus with high-dose chemotherapy and haemopoietic stem-cell transplantation: a phase I study.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) who experience persistent multiorgan dysfunction, despite standard doses of intravenous cyclophosphamide, represent a subset of patients at high risk of early death. We investigated the safety and efficacy of immune suppression and autologous haemopoietic stem-cell infusion to treat such patients. METHODS: From 1996, we selected patients with persistent SLE despite use of cyclophosphamide. Patients underwent dose-intense immune suppression and autologous haemopoietic stem-cell (CD34) infusion. Peripheral blood lymphocytes were analysed by flow cytometry, ELISA, and T-cell-receptor spectratyping before and after transplantation. We mobilised autologous haemopoietic stem cells with 2.0 g/m2 cyclophosphamide and 10 microg/kg granulocyte colony stimulating factor daily, enriched with CD34-positive selection, and reinfused after immunosuppression with 200 mg/kg cyclophosphamide, 1 g methylprednisolone, and 90 mg/kg equine antithymocyte globulin. RESULTS: Nine patients underwent stem-cell mobilisation but two were excluded before transplantation because of infection. The remaining seven received high-dose chemotherapy and stem-cell infusion. Median time to an absolute neutrophil count higher than 0.5x10(9)/L and nontransfused platelet count higher than 20x10(9)/L was 9 days (range 8-11) and 11 days (10-13), respectively. At a median follow-up of 25 months (12-40), all patients were free from signs of active lupus. Renal, cardiac, pulmonary, and serological markers, and T cell phenotype and repertoire had normalised. INTERPRETATION: Patients remained free from active lupus and improved continuously after transplantation, with no immunosuppressive medication or small residual doses of prednisone. T-cell repertoire diversity and responsiveness was restored. Durability of remission remains to be established.