Long-Term Follow-Up in Patients With Chronic Myeloid Leukemia Treated With Ponatinib in a Real-World Cohort: Safety and Efficacy Analysis.

BACKGROUND: Ponatinib is a third-generation tyrosine-kinase inhibitor (TKI), indicated in patients with chronic phase (CP), accelerated phase (AP), or blast phase (BP) chronic myeloid leukemia (CML), who are resistant or intolerant to ≥2 prior TKIs, patients for whom subsequent treatment with imatinib is not appropriate, and patients who have a T315I mutation. PATIENTS AND METHODS: We aimed to evaluate outcomes of ponatinib treatment, including safety, with focus on cardiovascular toxicity, in real-world patients from Argentina. Data from patients with CP CML treated with ponatinib was retrospectively retrieved from 2013 to 2023 in 7 centers. RESULTS: Seventy-two patients were included (median age: 44 years; male: 55.5%; T315I mutation: 32%: median treatment duration: 36 months. At baseline, 57 patients (79%) had a breakpoint cluster region-Abelson (BCR::ABL1) transcript level >10% on the international reporting scale (BCR::ABL1 IS). A molecular response (MR, BCR::ABL1 (IS) <1%) was achieved at 12 months in 51.6% of evaluable patients; 57% maintained MR at last follow-up. Overall, 43% and 25% maintained major MR (MMR) or deep MR (DMR) (MR4.0-MR5.0), respectively at last follow-up. Twelve (16.6%) ponatinib-resistant patients were rescued with allogeneic hematopoietic stem cell transplantation. The estimated 2-year progression-free survival (PFS) was 84%. Ponatinib dose was reduced during treatment in 22 patients; nevertheless, MMR was maintained in 50% of these patients. Severe arterial occlusive events (AOE) were reported in 10.9% of patients after a median treatment of 5 months. CONCLUSION: CV toxicity was consistent with clinical trials and other real-world registries. Older age, hypercholesterolemia and a SCORE risk >2% were significantly associated with higher risk of AOEs. Controlling CV risk factors and reducing doses at optimal time points may help to optimize ponatinib use in daily practice.

High cell-free DNA is associated with disease progression, inflammasome activation and elevated levels of inflammasome-related cytokine IL-18 in patients with myelofibrosis.

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-1ß 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.

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Introduction: 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%. Methods: 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. Results: 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). Discussion: 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.

Elevated plasma levels of IL-6 and MCP-1 selectively identify CML patients who better sustain molecular remission after TKI withdrawal.

Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is safe under adequate molecular monitoring, but questions remain regarding which factors may be considered predictive for TFR. Argentina Stop Trial (AST) is a multicenter TFR trial showing that 65% of patients sustain molecular remission, and the prior time in deep molecular response (DMR) was associated with successful TFR. Luminex technology was used to characterize cytokines in plasma samples. Using machine learning algorithms, MCP-1 and IL-6 were identified as novel biomarkers and MCP-1low/IL-6low patients showed eightfold higher risk of relapse. These findings support the feasibility of TFR for patients in DMR and MCP-1/IL-6 plasma levels are strong predictive biomarkers.

Ponatinib dose-ranging study in chronic-phase chronic myeloid leukemia: a randomized, open-label phase 2 clinical trial.

In PACE (Ponatinib Ph+ ALL and CML Evaluation), a phase 2 trial of ponatinib that included patients with chronic-phase chronic myeloid leukemia (CP-CML) resistant to multiple prior tyrosine kinase inhibitors (TKIs), ponatinib showed deep and durable responses, but arterial occlusive events (AOEs) emerged as notable adverse events. Post hoc analyses indicated that AOEs are dose dependent. We assessed the benefit/risk ratio across 3 ponatinib starting doses in the first prospective study to evaluate a novel, response-based, dose-reduction strategy for TKI treatment. Adults with CP-CML resistant to or intolerant of at least 2 prior BCR-ABL1 TKIs or with a BCR-ABL1 T315I mutation were randomly assigned 1:1:1 to 3 cohorts receiving ponatinib 45, 30, or 15 mg once daily. In patients who received 45 or 30 mg daily the dose was reduced to 15 mg upon response (BCR-ABL1IS transcript levels ≤1%). The primary end point was response at 12 months. From August 2015 through May 2019, 283 patients were randomly assigned to the cohorts: 282 (94 per dose group) received treatment (data cutoff, 31 May 2020). The primary end point (98.3% confidence interval) was achieved in 44.1% (31.7-57.0) in the 45-mg cohort, 29.0% (18.4-41.6) in the 30-mg cohort, and 23.1% (13.4-35.3) in the 15-mg cohort. Independently confirmed grade 3 or above treatment-emergent AOEs occurred in 5, 5, and 3 patients in the 45-, 30-, and 15-mg cohorts, respectively. All cohorts showed benefit in this highly resistant CP-CML population. Optimal benefit/risk outcomes occurred with the 45-mg starting dose, which was decreased to 15 mg upon achievement of a response. This trial is registered on www.clinicaltrials.gov as NCT02467270.

Treatment-free remission in patients with chronic myeloid leukemia: recommendations of the LALNET expert panel.

Tyrosine kinase inhibitors (TKIs) have dramatically changed the survival of chronic myeloid leukemia (CML) patients, and treatment-free remission (TFR) has recently emerged as a new goal of CML treatment. The aim of this work was to develop recommendations for TKI discontinuation in Latin America (LA), outside of clinical trials. A working group of CML experts from LA discussed 22 questions regarding TFR and reached a consensus for TFR recommendations in the region. TFR is indicated in patients in first chronic phase, with typical BCR-ABL transcripts, under TKI treatment of a minimum of 5 years, in sustained deep molecular response (DMR; molecular response 4.5 [MR4.5]) for 2 years. Sustained DMR must be demonstrated on at least 4 international reporting scale quantitative polymerase chain reaction (PCR) tests, separated by at least 3 months, in the immediate prior 2 years. After second-line therapy, TFR is indicated in previously intolerant, not resistant, patients. Molecular monitoring is recommended monthly for the first 6 months, every 2 to 3 months from months 7 to 12, and every 3 months during the second year, indefinitely. Treatment should be reintroduced if major molecular response is lost. Monitoring of withdrawal syndrome, glucose levels, and lipid profile is recommended after discontinuation. After TKI reintroduction, molecular monitoring is indicated every 2 to 3 months until MR4.0 achievement; later, every 3 to 6 months. For the TFR attempt, having standardized and reliable BCR-ABL PCR tests is mandatory. These recommendations will be useful for safe discontinuation in daily practice and will benefit patients who wish to stop treatment in emergent regions, in particular, with TKI-related chronic adverse events.

COVID-19 in chronic myeloid leukemia patients in Latin America.

This observational, multicenter study aimed to report the clinical evolution of COVID-19 in patients with chronic myeloid leukemia in Latin America. A total of 92 patients presented with COVID-19 between March and December 2020, 26% of whom were severe or critical. The median age at COVID-19 diagnosis was 48 years (22-79 years), 32% were 60 years or older, and 61% were male. Thirty-nine patients presented with at least one comorbidity (42.3%). Eighty-one patients recovered (88%), and 11 (11.9%) died from COVID-19. There was one case of reinfection. Patients with a major molecular response presented superior overall survival compared to patients with no major molecular response (91 vs. 61%, respectively; p = 0.004). Patients in treatment-free remission and receiving tyrosine kinase inhibitors showed higher survival rates than patients who underwent hematopoietic stem cell transplantation and those who did not receive tyrosine kinase inhibitors (100, 89, 50, and 33%, respectively; p < 0.001).

Platelet Toll-Like Receptors Mediate Thromboinflammatory Responses in Patients With Essential Thrombocythemia.

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, 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.

Long-term efficacy and safety of ruxolitinib versus best available therapy in polycythaemia vera (RESPONSE): 5-year follow up of a phase 3 study.

BACKGROUND: Polycythaemia vera is a myeloproliferative neoplasm characterised by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow due to mutations in the Janus kinase 2 (JAK2) gene. Ruxolitinib, a JAK 1 and JAK 2 inhibitor, showed superiority over best available therapy in a phase 2 study in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. We aimed to compare the long-term safety and efficacy of ruxolitinib with best available therapy in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. METHODS: We report the 5-year results for a randomised, open-label, phase 3 study (RESPONSE) that enrolled patients at 109 sites across North America, South America, Europe, and the Asia-Pacific region. Patients (18 years or older) with polycythaemia vera who were resistant to or intolerant of hydroxyurea were randomly assigned 1:1 to receive either ruxolitinib or best available therapy. Patients randomly assigned to the ruxolitinib group received the drug orally at a starting dose of 10 mg twice a day. Single-agent best available therapy comprised hydroxyurea, interferon or pegylated interferon, pipobroman, anagrelide, approved immunomodulators, or observation without pharmacological treatment. The primary endpoint, composite response (patients who achieved both haematocrit control without phlebotomy and 35% or more reduction from baseline in spleen volume) at 32 weeeks was previously reported. Patients receiving best available therapy could cross over to ruxolitinib after week 32. We assessed the durability of primary composite response, complete haematological remission, overall clinicohaematological response, overall survival, patient-reported outcomes, and safety after 5-years of follow-up. This study is registered with ClinicalTrials.gov, NCT01243944. FINDINGS: We enrolled patients between Oct 27, 2010, and Feb 13, 2013, and the study concluded on Feb 9, 2018. Of 342 individuals screened for eligibility, 222 patients were randomly assigned to receive ruxolitinib (n=110, 50%) or best available therapy (n=112, 50%). The median time since polycythaemia vera diagnosis was 8·2 years (IQR 3·9-12·3) in the ruxolitinib group and 9·3 years (4·9-13·8) in the best available therapy group. 98 (88%) of 112 patients initially randomly assigned to best available therapy crossed over to receive ruxolitinib and no patient remained on best available therapy after 80 weeks of study. Among 25 primary responders in the ruxolitinib group, six had progressed at the time of final analysis. At 5 years, the probability of maintaining primary composite response was 74% (95% CI 51-88). The probability of maintaining complete haematological remission was 55% (95% CI 32-73) and the probability of maintaining overall clinicohaematological responses was 67% (54-77). In the intention-to-treat analysis not accounting for crossover, the probability of survival at 5 years was 91·9% (84·4-95·9) with ruxolitinib therapy and 91·0% (82·8-95·4) with best available therapy. Anaemia was the most common adverse event in patients receiving ruxolitinib (rates per 100 patient-years of exposure were 8·9 for ruxolitinib and 8·8 for the crossover population), though most anaemia events were mild to moderate in severity (grade 1 or 2 anaemia rates per 100 patient-years of exposure were 8·0 for ruxolitinib and 8·2 for the crossover population). Non-haematological adverse events were generally lower with long-term ruxolitinib treatment than with best available therapy. Thromboembolic events were lower in the ruxolitinib group than the best available therapy group. There were two on-treatment deaths in the ruxolitinib group. One of these deaths was due to gastric adenocarcinoma, which was assessed by the investigator as related to ruxolitinib treatment. INTERPRETATION: We showed that ruxolitinib is a safe and effective long-term treatment option for patients with polycythaemia vera who are resistant to or intolerant of hydroxyurea. Taken together, ruxolitinib treatment offers the first widely approved therapeutic alternative for this post-hydroxyurea patient population. FUNDING: Novartis Pharmaceuticals Corporation.

miRNome profiling of LSC-enriched CD34+CD38-CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool.

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.

Evaluation of the primitive fraction by functional in vitro assays at the RNA and DNA level represents a novel tool for complementing molecular monitoring in chronic myeloid leukemia.

Quantification of BCR-ABL1 mRNA levels in peripheral blood of chronic myeloid leukemia patients is a strong indicator of response to tyrosine-kinase inhibitors (TKI) treatment. However, additional prognostic markers are needed in order to better classify patients. The hypothesis of leukemic stem cells (LSCs) heterogeneity and persistence, suggests that their functional evaluation could be of clinical interest. In this work, we assessed the primitive and progenitor fractions in patients at diagnosis and during TKI treatment using functional in vitro assays, defining a "functional leukemic burden" (FLB). We observed that the FLB was reduced in vivo in both fractions upon treatment. However, different FLB levels were observed among patients according to their response to treatment, suggesting that quantification of the FLB could complement early molecular monitoring. Given that FLB assessment is limited by BCR-ABL1 mRNA expression levels, we developed a novel detection method of primitive cells at the DNA level, using patient-specific primers and direct nested PCR in colonies obtained from functional in vitro assays. We believe that this method could be useful in the context of discontinuation trials, given that it is unknown whether the persistent leukemic clone represents LSCs, able to resume the leukemia upon TKI removal.

Treatment-Free Remission After Second-Line Nilotinib Treatment in Patients With Chronic Myeloid Leukemia in Chronic Phase: Results From a Single-Group, Phase 2, Open-Label Study.

Background: Treatment-free remission (TFR)-that is, stopping tyrosine kinase inhibitor (TKI) therapy without loss of response-is an emerging treatment goal in chronic myeloid leukemia (CML). Objective: To evaluate TFR after discontinuation of second-line nilotinib therapy. Design: Single-group, phase 2, open-label study. (ClinicalTrials.gov: NCT01698905). Setting: 63 centers in 18 countries. Patients: Adults with CML in chronic phase who received TKI therapy for at least 3 years (>4 weeks with imatinib, then ≥2 years with nilotinib) and achieved MR4.5 (BCR-ABL1 ≤0.0032% on the International Scale [BCR-ABL1IS]) while receiving nilotinib entered a 1-year consolidation phase. Those with sustained MR4.5 during consolidation were eligible to enter TFR. Interventions: Patients received nilotinib during consolidation; those who entered TFR stopped treatment. Patients with loss of major molecular response (MMR) (BCR-ABL1IS ≤0.1%) or confirmed loss of MR4 (BCR-ABL1IS ≤0.01%) during TFR reinitiated nilotinib treatment. Measurements: Proportion of patients without loss of MMR, confirmed loss of MR4, or treatment reinitiation within 48 weeks of stopping treatment (primary end point). Results: 163 patients who had switched from imatinib to nilotinib (for reasons including resistance, intolerance, and physician preference) enrolled in the study and entered the consolidation phase. Of these patients, 126 met the criteria for entering the TFR phase, and 73 (58% [95% CI, 49% to 67%]) and 67 (53% [CI, 44% to 62%]) maintained TFR at 48 weeks (primary end point) and 96 weeks, respectively. Of the 56 patients who reinitiated nilotinib therapy, 55 regained MMR or better and 52 regained MR4.5. None had CML progression to accelerated phase or blast crisis. Musculoskeletal pain was more frequent during the first 48 weeks after nilotinib discontinuation. Limitation: The study included a heterogeneous patient population and was not designed to compare outcomes between patients continuing and those stopping treatment. Conclusion: TFR seems achievable in patients with sustained MR4.5 after switching to nilotinib. Primary Funding Source: Novartis Pharmaceuticals Corporation.

Neutrophil extracellular trap formation and circulating nucleosomes in patients with chronic myeloproliferative neoplasms.

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.

GSTM1 and GSTP1, but not GSTT1 genetic polymorphisms are associated with chronic myeloid leukemia risk and treatment response.

BACKGROUND: Chronic myeloid leukemia (CML) is associated to the BCR-ABL1 oncogene and can successfully be treated with tyrosine kinase inhibitors (TKIs). However, it remains still under investigation which molecular factors may influence CML risk or varying responses to TKIs. The aim of this study was to assess the role of Glutathione-S-transferases (GSTs) genetic polymorphisms in CML susceptibility and TKI clinical outcome. MATERIALS: Deletion polymorphisms in GSTM1 and GSTT1 genes and the single nucleotide polymorphism in GSTP1 c.319A>G (rs1695; p.105Ile>Val) were genotyped by PCR methods in 141 CML treated patients and 141 sex- and age-matched healthy individuals. RESULTS: Individual analysis of each GST gene showed no association with CML risk. A trend toward significance (p=0.07) for a recessive model was found for GSTP1 (OR: 2.04; CI: 0.94-4.4). However, the combined analysis showed that GSTM1-null/GSTP1-GG as well as GSTT1-null/GSTP1-GG were associated with CML development (p=0.03; OR: 3.54 CI: 1.2-14.57; p=0.05; OR: 12.65; CI: 1.17-21.5). The relationship with treatment outcome showed that the presence of GSTM1 gene was significantly linked with an inferior rate of major molecular response (p=0.048) and poor event free-survival (EFS) (p=0.02). Furthermore, a group of patients with GSTP1-GG genotype were significantly associated with reduced EFS comparing to those carrying other GSTP1 genotypes (p=0.049). GSTP1-GG genotypes had short time to treatment failure in a group of patients unresponsive to TKIs comparing to other GSTP1 genotypes (p=0.03). CONCLUSIONS: This study highlights the significance of GSTM1 and GSTP1 polymorphisms on CML susceptibility and response to TKIs in the Argentinean population.

TP53 codon 72 polymorphism predicts chronic myeloid leukemia susceptibility and treatment outcome.

BCR-ABL1 gene is a key molecular marker of chronic myeloid leukemia (CML), but it is still unclear which molecular factors may influence CML risk or lead to variable responses to tyrosine kinase inhibitors (TKIs). The aim of this study was to investigate the impact of TP53 c.213 G>C(Arg72Pro; rs1042522) polymorphism on CML risk and its correlation with clinical outcome. Peripheral blood samples from 141 treated CML patients and 141 sex- and age-matched healthy individuals were genotyped by PCR-RFLP. Standard genetic models for disease penetrance were evaluated by logistic regression analysis and Kaplan-Meier method was performed to estimate survival curves. Our study suggests that TP53 c.213 G>C polymorphism may be involved in CML development considering a recessive model (p=0.01; OR: 0.19; CI: 0.06-0.68). In addition, a non-homogenous distribution was found for this polymorphism in males and patients youngers than 50years (p=0.02). According to clinical response, TP53-GG genotype was associated with higher levels of BCR-ABL1 transcripts (p=0.04) and shorter event free survival (p=0.04). Moreover, a trend toward significance was found for failure free survival (p=0.06) and time to imatinib failure (p=0.08). In conclusion, our data suggest that a;TP53 c.213 G>C may be a potential biomarker of CML susceptibility and clinical outcome.

Ruxolitinib versus best available therapy in patients with polycythemia vera: 80-week follow-up from the RESPONSE trial.

RESPONSE is an open-label phase 3 study evaluating the Janus kinase 1/Janus kinase 2 inhibitor ruxolitinib versus best available therapy for efficacy/safety in hydroxyurea-resistant or intolerant patients with polycythemia vera. This preplanned analysis occurred when all patients completed the Week 80 visit or discontinued. Objectives included evaluating the durability of the primary response (Week 32 phlebotomy-independent hematocrit control plus ≥35% spleen volume reduction), its components, and that of complete hematologic remission; and long-term safety. Median exposure was 111 weeks; 91/110 (82.7%) patients randomized to ruxolitinib remained on treatment. No patients continued best available therapy (98/112 [87.5%] crossed over to ruxolitinib, most at/soon after Week 32). At Week 32, primary response was achieved by 22.7% vs. 0.9% of patients randomized to ruxolitinib and best available therapy, respectively (hematocrit control, 60.0% vs. 18.8%; spleen response, 40.0% vs. 0.9%). The probability of maintaining primary and hematocrit responses for ≥80 weeks was 92% and 89%, respectively; 43/44 spleen responses were maintained until Week 80. Complete hematologic remission at Week 32 was achieved in 23.6% of ruxolitinib-randomized patients; the probability of maintaining complete hematologic remission for ≥80 weeks was 69%. Among ruxolitinib crossover patients, 79.2% were not phlebotomized, and 18.8% achieved a ≥35% reduction from baseline in spleen volume after 32 weeks of treatment. New or worsening hematologic laboratory abnormalities in ruxolitinib-treated patients were primarily grade 1/2 decreases in hemoglobin, lymphocytes, and platelets. The thromboembolic event rate per 100 patient-years was 1.8 with randomized ruxolitinib treatment vs. 8.2 with best available therapy. These data support ruxolitinib as an effective long-term treatment option for hydroxyurea-resistant or intolerant patients with polycythemia vera. This trial was registered at clinicaltrials.gov identifier: 01243944.

MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the recommended treatment for patients with chronic myeloid leukemia (CML). The MDR1/ABCB1 gene plays a role in resistance to a wide spectrum of drugs, including TKIs. However, the association of MDR1/ABCB1 gene polymorphisms (SNPs) such as C1236T, G2677T/A, and C3435T with the clinical therapeutic evolution of CML has been poorly studied. We investigated these gene polymorphisms in CML-patients treated with imatinib, nilotinib and/or dasatinib. METHODS: ABCB1-SNPs were studied in 22 CML-patients in the chronic phase (CP) and 2 CML-patients in blast crisis (BC), all of whom were treated with TKIs, and compared with 25 healthy controls using nested-PCR and sequencing techniques. RESULTS: Seventeen different haplotypes were identified: 7 only in controls, 6 only in CML-patients, and the remaining 4 in both groups. The distribution ratios of homozygous TT-variants present on each exon between controls and CML-patients were 2.9 for exon 12, and 0.32 for the other 2 exons. Heterozygous T-variants were observed in all controls (100%) and 75% of CML-patients. Wt-haplotype (CC-GG-CC) was observed in 6 CML-patients (25%). In this wt-group, two were treated with nilotinib and reached a major molecular response. The remaining 4 cases had either a minimal or null molecular response, or developed bone marrow aplasia. CONCLUSION: Our results suggest that SNPs of the MDR1/ABCB1 gene could help to characterize the prognosis and the clinical-therapeutic evolution of CML-patients treated with TKIs. Wt-haplotype could be associated with a higher risk of developing CML, and a worse clinical-therapeutic evolution.

Molecular monitoring of imatinib in chronic myeloid leukemia patients in complete cytogenetic remission: does achievement of a stable major molecular response at any time point identify a privileged group of patients? A multicenter experience in Argentina and Uruguay.

BACKGROUND: Monitoring minimal residual disease (MRD) by real-time quantitative polymerase chain reaction (RT-PCR) in chronic myeloid leukemia (CML) patients is mandatory in the era of tyrosine kinase inhibitors. Achieving a major molecular response (MMR) at 12 and 18 months predicts a better progression and event-free survival. PATIENTS AND METHODS: The objective of this prospective, multicentric study was to evaluate MRD by standardized RT-PCR in 178 patients with chronic-phase CML who were treated with imatinib at different institutions in Argentina and Uruguay and to determine if achievement of a stable MMR (BCR-ABL transcript levels < 0.1%) identifies a low-risk cytogenetic relapse group. The median age of the patients was 50 years, and 55% of them had received imatinib as first-line therapy. BCR-ABL transcript levels were measured after achievement of complete cytogenetic remission (CCyR) and at 6-month intervals. RESULTS: MMR was detected in 44% patients at the start of the study. This value increased to 79% at month 36 of evaluation. Complete molecular response (CMR) also increased from 24% to 52% of patients. Not achieving a stable MMR determined a higher risk of cytogenetic relapse (9% of MMR patients not achieving an MMR vs. 1% of patients who achieved MMR). Patients with sustained MMR had a significantly better cytogenetic relapse-free survival at 48 months (97% vs. 87%; P = .008) but showed no differences in overall survival. Patients who did not remain in CCyR changed treatment. CONCLUSIONS: A stable MMR is a strong predictor for a durable CCyR. Standardized molecular monitoring could replace cytogenetic analysis once CCyR is obtained. These results emphasize the validity and feasibility of molecular monitoring in all standardized medical centers of the world.

Clinical outcome of chronic myeloid leukemia imatinib-resistant patients: do BCR-ABL kinase domain mutations affect patient survival? First multicenter Argentinean study.

In imatinib-treated patients with chronic myeloid leukemia (CML), BCR-ABL mutations are the most common mechanism of resistance. Here we report the first multicenter Argentinean study investigating mutations in those patients with CML who fail or lose response to imatinib, with or without previous interferon treatment. Point mutations were detected in 36 of 154 patients by direct sequencing. In our series, the single most common mutations were G250E, E255K/V, and M351T. The presence of mutations correlated significantly with accelerated phase, lack of molecular response, and lower cytogenetic and hematological responses. While overall survival did not differ between patients with or without mutations, the probability of progression was higher in patients with mutations. Cases with non-P-loop mutations showed a significantly better overall survival from diagnosis. Multivariate analysis showed that the most significant variables related to the development of mutations were accelerated phase, duration of imatinib treatment, and time delay to starting imatinib. Our results demonstrated that mutation frequency increased with the progression of disease, and suggest that imatinib treatment should be started early.

BAX/BCL-XL gene expression ratio inversely correlates with disease progression in chronic myeloid leukemia.

BCR-ABL fusion gene is implicated in the pathogenesis of chronic myeloid leukemia (CML), encoding the oncoprotein p210(BCR-ABL) with anti-apoptotic activity. The inability to undergo apoptosis is an important mechanism of drug resistance and neoplastic evolution in CML. The gene transcript expression of mitochondrial apoptotic related genes BAX and BCL-XL was evaluated by quantitative Real Time PCR (qPCR) in vitro in K562 cells and in vivo in peripheral blood of 66 CML patients in different stages of the disease: 13 cases at diagnosis, 34 in chronic phase (CP), 10 in accelerated phase (AP) and 9 in blast crisis (BC). Our results in K562 cells showed that all treatments with different tyrosine kinase inhibitors (TKIs) induced a decreased expression of the antiapoptotic oncogene BCL-XL, whereas the proapoptotic gene BAX remains constant with minor modifications. A significantly lower BAX/BCL-XL expression ratio (mean±SEM) than a group of healthy individuals (4.8±0.59) were observed in CML patients at diagnosis (1.28 ± 0.16), in AP (1.14±0.20), in BC (1.16±0.30) and in 18% of cases of patients in CP (2.71±0.40). Most CP cases (82%) showed a significantly increased ratio (10.03±1.30), indicating that the treatment with TKIs efficiently inhibited the expression of BCL-XL by blocking BCR-ABL oncoprotein. The BAX/BCL-XL ratio showed a significant inverse correlation (Spearman P<0.0001) with BCR-ABL/ABL relative expression indicating that low BAX/BCL-XL was associated with disease progression. Accordingly, the follow up of a cohort of eight cases during 6months from diagnosis showed that while the BAX/BCL-XL ratio rapidly increased after treatment in seven cases with good evolution, it decreased in the single case that showed rapid evolution and short survival. Our data suggest that BAX/BCL-XL expression ratio may be a sensitive monitor of disease progression and an early predictor of TKI therapy responsiveness in CML patients.