B-cell lymphocytic leukaemia (B-CLL) is an indolent non-Hodgkin’s lymphoma and the most frequent leukaemia. Even though the capacity of B-CLL leukemic cells to proliferate has been underestimated until recently, the accumulation of tumor cells mostly results from a defect in the apoptotic program. The ubiquitin/proteasome system is important in the turnover of regulatory proteins and as regulator of cell proliferation and apoptosis. This critical link between the apoptotic machinery and the ubiquitin/proteasome system led to an increased interest in designing inhibitory agents that target these pathways. A promising new drug, bortezomib (BZ), a dipeptidyl boronic acid proteasome inhibitor, was approved for treatment of relapsed multiple myeloma. Extensive preclinical data are being developed to study the potential application of this new drug in other cancers. Our preliminary results show that BZ induces a marked decrease in LLC-B cell viability by increasing the percentage of apoptosis. The aim of this study is to evaluate the potential therapeutic value of BZ in B-CLL patients studying the cytotoxicity mechanisms induced by this proteasome inhibitor. For this purpose, mononuclear cells isolated from 22 patients with B-CLL (14 without and 8 with conventional therapy) were cultured in absence and presence of bortezomib (BZ) (ranging concentration from 0.01 μM to 10 μM), as single agent, or plus 75 or 150 μM fludarabine (FDN), during 24 hours. Directly conjugated monoclonal antibodies to CD5 and CD19 was used to identify LLC-B cells. Cell death was evaluated by annexin V incorporation and detected by flow cytometry. The expression of the proteins involved in apoptosis regulation, namely the proapoptotic proteins, Bax and p53, and the antiapoptotic protein Bcl-2 was determined by flow cytometry using monoclonal antibodies. Our results show that BZ induces a marked decrease in B-CLL cell viability by increasing the percentage of apoptosis in a dose dependent manner (20 to 80% apoptosis). However, in concentrations higher than 1 mM a saturable effect is observed. On the other hand, a lesser sensitivity to BZ was observed in normal mononuclear cells (≤ 30% apoptosis). BZ apoptotic effect seems to be independent of previous therapy. We observed higher levels of apoptosis in B-CLL cells treated with BZ compared with cells not treated or in response to treatment with FDN, which may relate with the increase in Bax expression (average difference of 37.3 ± 18%). However, we observe identical apoptosis levels in B-CLL cells, as opposed to others (Duechler, M. et al., 2005), and an increase in apoptotic T cells levels in the presence of treatment with BZ plus FDN compared with those observed in cells treated with BZ alone. On the other hand, when compared with the results obtained with FDN alone (in 150 μM), an increase in apoptosis levels was detected (average difference of 69.5 ± 28% increase in apoptosis, for B-CLL cells and 33.6 ± 11.6%, for T cells). Our results support that bortezomib induces apoptosis as a single agent in a Bax-dependent way. The apoptotic effect show some selectivity for the transformed cells (B-CLL). These results suggests that this proteasome inhibitor may be useful as a therapeutic approach in B-CLL patients.
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Background:γδT cells are a minor circulant effector/cytotoxic population involved in immune surveillance, with evidence supporting their anti‐multiple myeloma (MM) role. Although lymphocyte recover after autologous hematopoietic stem cell transplantation (aHSCT) has been extensively studied, there are limited data about γδT cells, with no information on the impact of previous therapeutic in its recovery.Aims:To evaluate γδT cells recovery in MM patients(pts), its impact in posttransplant recovery and the effect of previous treatment/response in γδT subsets reconstitution.Methods:We prospectively analysed 44 MM pts submitted to aHSCT (2016‐2018), previously to conditioning with melphalan 200 mg/m2 (Dc) and at 30 (D30), 60 (D60), and 100 (D100) days after aHSCT. The proportion (%) of γδT cells, performed by flow cytometry based on the expression of CD3/CD45/Vδ1/Vδ2/Vγ9/CD27, indicated the proportion of these cells among all T cells. Pts were divided into 3 groups: those who receive just 1‐line therapy previous aHSCT (NEW) and those who receive more than 1 line due to primary refractory disease (REFAC) or relapse after response (RECID), according to IMWG criteria. Toxicities were graded according to CTCAEv4.03.Results:Median age was 63 years (40‐69), with 64.0% of males, 27.3% ISS III. Median of previous therapeutic lines was 1(1‐4) – all pts had received bortezomib, 27.3% lenalidomide, 18.8% thalidomide, 9.1% daratumumab, and 4.6% pomalidomide; 36.4% received ≥1 novel anti‐MM agent.Median time diagnosis‐aHSCT was 11.1 months(5.3‐112.1) – 68.2%(n = 30) NEW, 16.6%(n = 6) REFAC and 18.2% (n = 8) RECID. The median number of previous lines was higher in REFAC vs RECID (2vs2; p = 0.015). There were no other characteristics differences at diagnosis between the 3 groups. There was no significant difference between NEW vs REFAC vs RECID in achieving at least very good partial response (VGPR) or higher previous to aHSCT (p = 0.224) or at D100 pos‐aHSCT (p = 0.905).RECID group presented a higher % of Vδ1 + γ9 + T cells (vs REFAC)(p = 0.007) and a lower of Vδ2 + γ9 + T(vs REFAC and NEW)(p = 0.060 and p = 0.027, respectively) at D100. There were no other differences between the 3 groups.Time to neutrophils >0.5 × 109/L (TTN) was higher in RECID group (vs NEW and REFAC) (p = 0.003 and p = 0.015, respectively). A higher TTN was positively correlated with a higher %Vγ9 + T (Coef. 21,4;p = 0.012), Vδ1 + γ9 + T(Coef 17.7;p = 0.007) and Vδ1 + γ9 + CD27 + T(Coef 35.9;p = 0.008)(D60), even after adjusting for the study group. Time to platelets >20 × 109/L (TTP) was higher in RECID vs NEW (p = 0.005). REFAC group needed more platelet transfusion vs NEW (p = 0.011). A higher % Vγ9 + T(D60) was associated with higher platelet transfusion requirement (Coef 7.4;p = 0.016), even after group adjusting. Despite a positive association between TTP and Vγ9 + T (D60)(Coef 20.8; p = 0.019), there was a loss significance after adjusting for group (p = NS).For a median follow up of 17.6 months, progression free survival (PFS) at 1‐year post‐aHSCT was lower in REFAC, although not significant(p = 0.276). None subset of Vγδ + T cells presented impact in PFS.Summary/Conclusion:Our results, although conditioned by a small sample size and a short follow‐up, show a higher % of Vδ1 + Vγ9 + T and lower Vδ2 + Vγ9 + T cells in patients’ group RECID at D100. Furthermore, our data suggests that γδT subsets (particularly Vδ1+, Vδ1 + Vγ9+ and Vγ9+) seem to have a role in delaying haematological recovery, mainly in megakaryocytic line. Further studies are needed to evaluate the relationship between these T cells subpopulations and the response to therapeutics/aHSCT, as well as the benefit of its modulation.
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