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Background: Cannabinoid receptors have been detected in human gliomas and cannabinoids have been proposed as novel drug candidates in the treatment of brain tumors. Aims: To test the in vitro antitumor activity of COR167, a novel cannabinoid CB2-selective agonist displaying a high binding affinity for human CB2 receptors, on tumor cells isolated from human glioblastoma multiforme and anaplastic astrocytoma. Methods: Glioma cell cultures were established from two glioblastoma multiforme and two anaplastic astrocytomas. Proliferation was measured in the presence or absence of COR167 with a bromodeoxyuridine (BrdU) cell proliferation ELISA assay. CB2 receptor expression was detected by western blotting. Apoptosis was assessed with phycoerythrin (PE) annexin V flow cytometry kit. TGF-beta 1 and 2 levels were analyzed in culture supernatants with commercial ELISAs. Results: COR167 was found to significantly reduce the proliferation of both glioblastoma and anaplastic astrocytoma in a dose-dependent manner at lower doses than other known, less specific CB2 agonists. This activity is independent of apoptosis and is associated with a significant reduction of TGF-beta 1 and 2 levels in supernatants of glioma cell cultures. Conclusion: These findings add to the role of cannabinoid CB2 receptor as a possible pharmacological target to counteract glial tumor growth and encourage further work to explore any other pharmacological effect of this novel CB2 agonist useful in the treatment of human gliomas.

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV(UM-HCL) and 52 M-IGHV(M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV(5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P< .001), leukocytosis (3 of 5 vs 3 of 53, P= .006), and bulky spleen (4 of 5 vs 4 of 53, P< .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.

We have previously shown that vaccination of chronic myelogenous leukaemia (CML) patients with CMLVAX100 (a mixture of 5 p210-b3a2 breakpoint derived peptides) plus GM-CSF was able to induce an evident and durable peptide-specific CD4+T cell response in the majority of patients. Peptide-specific CD4+T cell proliferation was measured by standard [3H] thymidine incorporation assay and this response was mainly mediated by a 25 mer long breakpoint peptide (b3a2-25) included in the vaccine. In this pilot clinical trial we showed that about 60% of 28 CML patients vaccinated while on imatinib, showed a reduction of their long lasting molecular residual disease after immunization (first 6 vaccinations) and about 25% of them achieved in addition a complete molecular response. To investigate the contribute of this peptide specific CD4+T cell response in antitumor activity, we further characterize peptide-induced proliferating CD4+cells. Briefly, in 10 CMLVAX100 vaccinated patients in which a strong b3a2-25 specific CD4+proliferation was previously observed, we freshly isolated peripheral blood CD4+T cells and we cultured them 4 days only in the presence or not of b3a2-25 vaccine peptide or control PR-25 peptide. Afterwards, each culture condition was analyzed both for the co-expression of CD25, perforin and FOXP3 molecules and for standard [3H] thymidine incorporation assay. As expected all patients showed a strong b3a2-25 specific CD4+proliferation (mean stimulation index 43 -range 19–81-). When flow cytometric analysis was performed, we observed a consistent increase of two main CD4+T cells subsets only in the b3a2-25 stimulated CD4+T cells, not in “no peptide” or “control peptide” stimulated CD4+T cells.