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Introduction: Some patients with CLL will require treatment at diagnosis, but many other will remain untreated under observation for several years. In 2008, the International Workshop on Chronic Lymphocytic Leukemia(IWCLL) defined the criteria for treatment indications, which have been widely used in daily practice and in clinical trials. We have observed that many patients tolerate several of these clinical manifestations without treatment need, especially in public hospitals were resources and treatment options are scarce. We identified 5 reference centers for CLL that share the same profile of being more conservative in indicating treatment for CLL patients. We decided to analyze if more conservative local criteria for treatment indication impacts on patients' outcomes. Objective: To describe the outcomes of a series of CLL patients treated according to locally defined more conservative criteria for initiating treatment. Methods: The Brazilian Registry of CLL was started in 2004 as a prospective observational data collection tool. Inclusion criteria for enrollment followed the IWCLL guidelines. We retrospectively evaluated all patients with CLL in the Brazilian Registry of CLL who were followed between January 2013 and April 2020 at the 5 reference centers (3 public and 2 private). The following local criteria were used for treatment indications to all patients included: 1) persistent and progressive symptomatic cytopenias (no predefined minimum levels), 2) Massive or symptomatic lymphadenopathy, 3) Massive or symptomatic splenomegaly, 4) Disease-related symptoms, only if persistent and if other causes were excluded, and 5) Autoimmune complications including anemia or thrombocytopenia non-responsive to steroids. Progressive lymphocytosis and extranodal manifestations were not considered for treatment indication. Results: A total of 581 patients were followed during the observation period of 7 years (median follow-up was 40 months (range: 3-86). Median age was 65 years (range: 32-98) and most patients were male (57%). Binet stage was A in 67%, B in 14% and C in 19% of cases. FISH, performed in only 199 patients (34%), was normal in 47%, and showed del13q in 22%, trisomy 12 in 17%, del11q in 8% and del17p in 7%. According to IWCLL criteria, 257 (44%) presented indication for treatment over the time: 140 (55%) at diagnosis and 117 (45%) during follow-up. Based on the local criteria, 148 (25%) patients met criteria for indication of treatment. Therefore, 109 patients with IWCLL indication were not treated to date according to the local criteria. The IWCLL indications for these untreated patients were: cytopenias in 50 patients (48%), constitutional symptoms in 37 patients (35%), progressive lymphocytosis in 9 (9%), and lymphadenopathy or splenomegaly in 8 (8%). The median observation time for these untreated patients from the time of indication of treatment by IWCLL until the analysis was closed was 39 months, ranging from 3 to 86 months. Of the 109 untreated patients, 12 (13%) died during follow-up: 4 from infections probably unrelated to CLL (all patients were elderly, Binet A, non-neutropenic and non-hypoglobulinemic), 2 from cardiac causes, 1 from a car accident and 5 of unknown causes (lost follow-up after at least 2 years). No deaths were attributable to LLC. Overall survival at 4 years was 90% for the patients who were treated versus 89% for the patients who were not treated (P=0.85). Conclusion: Our data suggest that it is feasible and safe to adopt more conservative criteria to indicate treatment in a CLL patient. A more restrict approach may not only reflect in a significant financial impact to the health care system but also avoid premature exposition to prolonged and/or potentially toxic treatments. This finding might be of special interest to low-income countries. Disclosures No relevant conflicts of interest to declare.

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with thebcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

The Bcr-Abl tyrosine kinase found in Philadelphia chromosome (Ph1)-positive leukaemia is the result of a reciprocal translocation between chromosomes 9 and 22. Depending on the breakpoint of the bcr gene, different isoforms of Bcr-Abl appear and are associated with specific types of leukemias; in chronic myelogenous leukaemia (CML), a 210kDa protein is responsible for a deregulated proliferation and consequently accumulation of myeloid cells and their precursors, whereas in acute lymphocytic leukaemia (ALL) a 185kDa Bcr-Abl protein is the oncogenic counterpart. This chimeric protein not only induces transformation of cell lines, but also regulates cell proliferation and is one of the most potent anti-apoptotic molecules responsible for resistance to a variety of apoptosis-inducing agents. The signalling pathway of apoptosis which may be regulated by Bcr-Abl is still unknown, but our latest findings suggested that Bcr-Abl act at the mitochondrial level, preserving the mitochondria integrity during several apoptogenic stimuli thereby preventing release of cytochrome c and the consequent activation of effector caspases. To investigate the mechanisms whereby Bcr-Abl interferes with the cell apoptotic machinery, we asked where in Fas signalling cascade that Bcr-Abl blocks the apoptogenic signal. For this purpose, HL-60.neo, HL-60.Bcr-Abl, HL-60.Bcl-2 and HL-60.Bcl-xL cells were stimulated with anti-Fas antibodies (CH11), in the presence or not of cycloheximide, and the levels of apoptosis and activation of caspases where compared between wild-type and transfected cells with anti-apoptotic molecules. Our results demonstrate that Bcr-Abl blocks both mitochondria dependent and independent Fas pathway, suggesting that Bcr-Abl has a bigger spectrum of action than Bcl-2 and Bcl-xL. CNPq, FAPESP and IEP-HIAE

Leukemic cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) are very resistant to apoptosis induced by DNA-damaging agents and other chemotherapeutic drugs, due to the presence of Bcr-Abl, a chimeric cytoplasmic tyrosine-kinase that confers both malignancy and resistance to apoptosis. Efficient treatment of CML can be achieved with a normal bone marrow transplant, which induces a graft-versus-leukemia response, and more recently by the use of the specific inhibitor imatinib mesylate (glivec. Novartis). Glivec blocks Bcr-Abl kinase activity and, as a consequence, the malignant cell dies by apoptosis. However most glivec-treated patients, mainly in the acute and blast phases, develop resistant forms of the disease. Since resistance to apoptosis in Bcr-Abl+ cells is probably related to the inhibition of mitochondrial release of cytochrome c, an obligatory step in most apoptotic pathways, we sought to investigate expression of Bcl-2 family genes in Bcr-Abl+, glivec-treated cells. By semi-quantitative RT-PCR we analyzed the gene expression of several pro- and anti-apoptotic molecules in the transduced cell line HL-60.Bcr-Abl and the wild-type HL-60, after a 1, 4 and 8h treatment with 10μM glivec. Bcr-Abl′s kinase activity is promptly inhibited by glivec (within 5 to 15min) and HL-60.Bcr-Abl cells begin to show mitochondrial depolarization 24h after treatment with the drug, dying 48h later, whereas no effects are observed in HL-60. Soon after glivec addition some genes are transcriptionally regulated in HL-60.Bcr-Abl cells. The major differences were observed for bcl-xL (2-fold reduction), c-flip (2-fold increase), bcl-w (30% increase) and mcl-1 (20% reduction). Some pro-apoptotic molecules such as noxa also displayed differential regulation in HL-60.Bcr-Abl cells. No differences were observed in HL-60 cells. In conclusion we describe a complex transcriptional regulation mechanism dependent on Bcr-Abl tyrosine-kinase activity, which has not been previously described by the use of microarrays, and could contribute to the understanding of the mechanisms involved in protection of apoptosis and drug resistance of Bcr-Abl+ cells.

The bcr-abl oncogene is generated by the Philadelphia chromosome (Ph) translocation, fusing the BCR gene to the ABL gene and occurs mainly in two different forms. In chronic myelogenous leukemia (CML) a 210kDa Bcr-Abl protein is associated with proliferation and accumulation of myeloid cells and their precursors, whereas a 185kDa form is responsible for the pathogenesis of acute lymphocytic leukemia (ALL). Bcr-Abl not only induces cellular transformation but also regulates cell proliferation and confers resistance to a variety of apoptosis-inducing agents. Much attention has been focused on the development of novel therapies based on mechanistic understanding of Bcr-abl signaling in CML cells. However, we still do not fully understand how this molecule interferes with apoptotic machinery. The aim of this study was to investigate which step of the apoptotic machinery was most influenced by ectopic expression of Bcr-Abl myeloblastic (HL-60) and B lymphoblastic (SKW6.4) cell lines. Ectopic expression of Bcr-Abl conferred resistance to apoptosis induced by staurosporine and anti-Fas in HL-60 cells, but not in SKW6.4 cells, although increased phosphotyrosine containing proteins could be easily observed in SKW6.4.Bcr-Abl cells. The protein levels of Bcl-xL, Mcl-1 and Flip detected by western-blot were greater in HL-60.Bcr.Abl, when compared to wild type HL-60 cells. In contrast, the proapoptotic protein Bid was considerably reduced in HL-60. Bcr-abl cells. Two independent lines of SKW6.4.Bcr-Abl cells had a small reduction of in Bid levels, but no difference was observed in the expression of the anti-apoptotic proteins Bcl-xL, Mcl-1 and Flip. At the mRNA level SKW6.4 and SKW.Bcr-Abl presented similar expression of Bcl-xL, Mcl-1 and Flip. In addition a greater expression of anti-apoptotitc proteins A1 (IDV A1/actin=0.6) and Bcl-w (IDV Bcl-w/actin=1.6) was found in SKW6.4.Bcr-Abl compare to SKW (0.2 and 0.01, respectively) wild type. Interestingly, the upregulated levels of these anti-apoptotic genes were not sufficient to prevent apoptosis in these cells. Taken together these results suggest that Bcr-abl is not capable of protecting SKW6.4 cells from apoptosis induced by staurosporine and anti-Fas, perhaps because it interacts differently with the apoptotic machinery in B lymphoblasts compared to myeloblast cell lines. Further stydies are underway in our laboratory in order to better understand the Bcr-abl-mediated anti-apoptotic signaling in B lymphoblast cells.