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The treatment of chronic myeloid leukemia (CML) achieved a great leap forward with the development of imatinib, a BCR-ABL kinase inhibitor. Alterations in the chemical structure of the inhibitor have produced agents that are more potent in vitro. In these studies, two new second-generation BCR-ABL kinase inhibitors, nilotinib and dasatinib, are compared with imatinib; these new drugs produce more complete responses and do so faster than imatinib. Both also appear to reduce the rate of progression to accelerated-phase and blast-phase disease. Chronic myeloid leukemia (CML) in the chronic phase, a clonal myeloproliferative disorder, is caused by the constitutively active BCR-ABL tyrosine kinase resulting from the translocation that produces the Philadelphia (Ph) chromosome. 1 , 2 Imatinib (Gleevec, Novartis Pharmaceuticals), an inhibitor of the BCR-ABL kinase, is the standard first-line therapy for patients with chronic-phase CML. 3 – 6 Dasatinib (Sprycel, Bristol-Myers Squibb), a second-generation BCR-ABL kinase inhibitor, has been approved as a second-line treatment for patients with CML if imatinib therapy fails. 4 – 7 Dasatinib therapy induces a complete cytogenetic response in approximately 50% of patients who do not have a response to imatinib or cannot . . .

Chronic myeloproliferative neoplasms are characterized by clonal myeloid expansion driven by activating mutations in the JAK2 pathway and chronic inflammation. The aim was to investigate the contribution of circulating inflammatory mediators to the abnormalities in the megakaryocytic lineage characteristic of MF and ET. Plasma samples from 30 MF and 28 ET patients were incubated with normal cord-blood CD34 + progenitors and megakaryo/thrombopoiesis was evaluated. MF plasma increased megakaryocyte output, which was attenuated in sequential samples from ruxolitinib-treated patients. JAK1/2, MAPK and NF-kB inhibitors reverted this effect, revealing the concomitant involvement of all three pathways. Elevated levels of circulating IL-1β and IL-6 correlated with megakaryocyte output, which was reverted by blocking antibodies, indicating this phenotype is partly driven by these inflammatory cytokines. Instead, ET plasma promoted enhanced proplatelet formation, which was coupled with increased NFE2 and Bcl-xL expression. Elevated levels of circulating RANTES correlated with ET plasma-induced proplatelet formation, which was partially reverted by RANTES receptor CCR5 antagonist Maraviroc, indicating RANTES is involved in this process. These findings indicate that, in addition to clonal mutations, extrinsic inflammatory mediators play a direct role in MF and ET megakaryocyte abnormalities. The distinct cytokine profile could potentially be useful for the development of targeted therapies.

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in 2, , and , which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.

Treatment-free remission (TFR) is a key therapeutic goal for chronic myeloid leukemia (CML) patients in deep molecular response (DMR). While predicting patient outcome remains challenging, different NK cell populations seem crucial. We conducted an immunological sub-study from the Argentina Stop Trial (AST), including 46 patients in 2019 (AST I) and 35 new patients between 2022 and 2023 (AST II). To characterize NK cell subsets in patients attempting TFR, peripheral blood mononuclear cell samples were collected before stopping treatment and phenotype and functional characteristics were assessed by flow cytometry. Non-relapsing patients from AST I exhibited NK cell subpopulations with cytomegalovirus-related memory features, high expression of cytotoxicity markers, and robust functionality. Remarkably, though clinical variables were very similar between cohorts, significant immune differences were observed. NK cell percentage and CD16 and CD57 receptor expression levels were significantly reduced in AST II (p = 0.0051; p = 0.0222; p = 0.0033, respectively), whereas NKp46, NKp44 and PD-1 expression levels were significantly increased (p = 0.0081; p < 0.0001; p < 0.0001, respectively). NK cells from AST II patients demonstrated higher overall functionality and more memory-like subpopulations, characterized mainly by the expression of CD57, NKG2C, NKp30 and NKp46 receptors among CD56dim NK cells, also with enhanced functional performance. However, in AST II, we were unable to report an association with clinical outcome. Given the enrollment time of both cohorts and that they appear to be clinically homogeneous, we consider that COVID could be impacting the immune landscape; accordingly, serum samples from AST II, but not AST I, confirmed the presence of anti-SARS-CoV-2 IgG. The influence of the COVID pandemic and the different vaccine platforms on NK cells cannot be underestimated when evaluating the role of the immune system in cancer.

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver ( JAK2 / CALR / MPL ) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1β and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34 + CD38 − CD26 + and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34 + CD38 − CD26 + and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26 + ( BCR-ABL1 + ) vs. CD26 − ( BCR-ABL1 − ) CD34 + CD38 − fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34 + CD38 − fractions that distinguishes between CD26 + ( BCR-ABL1 + ) and their CD26 − ( BCR-ABL1 - ) counterparts, providing valuable data for future studies.

The ENESTop study evaluated treatment-free remission (TFR) in patients with chronic myeloid leukemia (CML) in chronic phase who had received ≥3 years of tyrosine kinase inhibitor therapy and achieved sustained deep molecular response only after switching from imatinib to nilotinib. After 1-year nilotinib consolidation, 126 patients attempted TFR. At 48 weeks (primary analysis), 57.9% (73/126) were in TFR. In the present analysis at 5 years, 42.9% (54/126) were in TFR. Since the 48-week analysis, among patients who left the TFR phase, 58% (11/19) did not have a loss of molecular response and discontinued for other reasons. Of the 59 patients who reinitiated nilotinib upon loss of major molecular response (MMR) or confirmed loss of MR 4 , 98.3% regained MMR, 94.9% regained MR 4 , and 93.2% regained MR 4.5 . Overall adverse event rates decreased over the 5 years of TFR. In patients reinitiating nilotinib, there was a cumulative increase in cardiovascular events with longer nilotinib exposure. No disease progression or CML-related deaths were reported. Overall, these results confirm the durability and safety of TFR for patients receiving second-line nilotinib. Cardiovascular risk should be carefully managed, particularly when reinitiating treatment after TFR.

There are different BCR-ABL1 fusion genes that are translated into proteins that are different from each other, yet all leukemogenic, causing chronic myeloid leukemia (CML) or acute lymphoblastic leukemia. Their frequency has never been systematically investigated. In a series of 45503 newly diagnosed CML patients reported from 45 countries, it was found that the proportion of e13a2 (also known as b2a2) and of e14a2 (also known as b3a2), including the cases co-expressing e14a2 and e13a2, was 37.9% and 62.1%, respectively. The proportion of these two transcripts was correlated with gender, e13a2 being more frequent in males (39.2%) than in females (36.2%), was correlated with age, decreasing from 39.6% in children and adolescents down to 31.6% in patients ≥ 80 years old, and was not constant worldwide. Other, rare transcripts were reported in 666/34561 patients (1.93%). The proportion of rare transcripts was associated with gender (2.27% in females and 1.69% in males) and with age (from 1.79% in children and adolescents up to 3.84% in patients ≥ 80 years old). These data show that the differences in proportion are not by chance. This is important, as the transcript type is a variable that is suspected to be of prognostic importance for response to treatment, outcome of treatment, and rate of treatment-free remission.

The mechanisms underlying increased thrombotic risk in chronic myeloproliferative neoplasms (MPN) are incompletely understood. We assessed whether neutrophil extracellular traps (NETs), which promote thrombosis, contribute to the procoagulant state in essential thrombocythemia, polycythemia vera and myelofibrosis (MF) patients. Although MPN neutrophils showed increased basal reactive oxygen species (ROS), enhanced NETosis by unstimulated neutrophils was an infrequent finding, whereas PMA-triggered NETosis was impaired, particularly in MF, due to decreased PMA-triggered ROS production. Elevated circulating nucleosomes were a prominent finding and were higher in patients with advanced disease, which may have potential prognostic implication. Histone-MPO complexes, proposed as specific NET biomarker, were seldomly detected, suggesting NETs may not be the main source of nucleosomes in most patients, whereas their correlation with high LDH points to increased cell turn-over as a plausible origin. Lack of association of nucleosomes or NETs with thrombosis or activation markers does not support their use as predictors of thrombosis although prospective studies in a larger cohort may help define their potential contribution to MPN thrombosis. These results do not provide evidence for relevant in vivo NETosis in MPN patients under steady state conditions, although availability of standardized NET biomarkers may contribute to further research in this field.