Beatriz Moiraghi

Clinical significance of V617F mutation of the JAK2 gene in patients with chronic myeloproliferative disorders.

Abstract Objective: To determine the prevalence of JAK2 V617F mutation and its clinical correlation in patients with chronic myeloproliferative disorders (CMD): polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Materials and methods: Detection of JAK2 V617F mutation by allele specific-PCR. Results: One hundred and three patients with CMD were included in the study. JAK2 V617F distribution was PV 40/45 (89%), ET 30/43 (69%), and IMF 7/15 (47%). In PV and ET patients only, 18 had thrombosis at diagnosis and 12 during follow-up (these were microvascular: 11, venous: 7 and arterial: 12); of these 28/70 (40%) were JAK2pos versus 2/18 (11%) JAK2neg; P=0·02. In a median of 4 years, two patients with PV JAK2pos evolved to myelofibrosis and one patient with PV presented in leukemic transformation (JAK2pos before and after transformation); six patients died: four patients with IMF and two patients with PV. Conclusions: We found an association between JAK2 V617F and thrombotic events in patients with PV and ET.

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.

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.

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.

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.