ABSTRACT
In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.
Subject(s)
Butyrate Response Factor 1/genetics , DNA Methylation , Enhancer Elements, Genetic/genetics , Epigenomics/methods , Primary Myelofibrosis/genetics , Apoptosis/drug effects , Butyrate Response Factor 1/metabolism , Butyrate Response Factor 1/pharmacology , Case-Control Studies , Cell Line , Cell Proliferation/drug effects , Epigenesis, Genetic , Genes, Tumor Suppressor , HumansABSTRACT
BACKGROUND: Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS: We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS: The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as "TET2-first patients"), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first ("JAK2-first patients") had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2-TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS: The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.).
Subject(s)
DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/physiology , Janus Kinase 2/genetics , Mutation , Myeloproliferative Disorders/genetics , Proto-Oncogene Proteins/genetics , Age of Onset , Cell Proliferation/genetics , DNA Mutational Analysis , Dioxygenases , Gene Expression , Homozygote , Humans , Polycythemia Vera/genetics , Thrombocythemia, Essential/genetics , Thrombosis/genetics , Transcription, Genetic , Up-RegulationABSTRACT
JAK2V617F monitoring and NGS of non-driver genes was performed in 100 patients with polycythemia vera (PV) or essential thrombocythemia (ET) with long molecular follow-up. Patients who did not progress to myelofibrosis (MF) or acute myeloid leukemia (AML) after more than 10 years (n = 50) showed a low frequency of mutations at first sample (18%) and an incidence rate of 1.7 new mutations × 100 person-years. Mutations were detected at first sample in 83% of PV/ET patients who later progressed to AML (n = 12) with these patients having a rate of 25.6 mutations × 100 person-years. Presence of mutations at diagnosis was the unique risk factor for acquiring a new genetic event (HR 2.7, 95% CI 1.1-6.8, p = 0.03) after correction for age, PV diagnosis, and total duration of hydroxyurea (HU) exposure. Patients with additional mutation at first sample showed a higher probability of developing cytopenia under HU therapy and a higher risk of AML (HR 12.2, 95% CI 2.6-57.1, p = 0.001) with mutations in ASXL1 (p < 0.0001), TP53 (p = 0.01), SRSF2 (p < 0.0001), IDH1/2 (p < 0.0001), and RUNX1 (p < 0.0001) being associated with a higher probability of AML. Myelofibrotic transformation was more frequent in patients with additional mutations, especially in SF3B1 (p = 0.02) and IDH1/2 (p < 0.0001) although a persistently high or a progressive increase of the JAK2V617F allele burden while receiving cytoreduction was the strongest predictor of MF transformation (HR 10.8, 95% CI 2.4-49.1, p = 0.002). In conclusion, NGS may be useful to identify a minority of PV and ET patients with high genetic instability and increased risk of AML transformation.
Subject(s)
Cell Transformation, Neoplastic/genetics , Janus Kinase 2/genetics , Mutation, Missense , Polycythemia Vera/genetics , Thrombocythemia, Essential/genetics , Adult , Aged , Aged, 80 and over , Alleles , Amino Acid Substitution , Cytogenetic Analysis , Disease Progression , Female , Follow-Up Studies , Gene Frequency , Humans , Male , Middle Aged , Phenylalanine/genetics , Polycythemia Vera/pathology , Thrombocythemia, Essential/pathology , Valine/geneticsABSTRACT
The clinical significance of resistance/intolerance to hydroxycarbamide (HC) was assessed in a series of 890 patients with polycythaemia vera (PV). Resistance/intolerance to HC was recorded in 137 patients (15·4%), consisting of: need for phlebotomies (3·3%), uncontrolled myeloproliferation (1·6%), failure to reduce massive splenomegaly (0·8%), development of cytopenia at the lowest dose of HC to achieve a response (1·7%) and extra-haematological toxicity (9%). With a median follow-up of 4·6 years, 99 patients died, resulting in a median survival of 19 years. Fulfilling any of the resistance/intolerance criteria had no impact on survival but when the different criteria were individually assessed, an increased risk of death was observed in patients developing cytopenia [Hazard ratio (HR): 3·5, 95% confidence interval (CI): 1·5-8·3, P = 0·003]. Resistance/intolerance had no impact in the rate of thrombosis or bleeding. Risk of myelofibrotic transformation was significantly higher in those patients developing cytopenia (HR: 5·1, 95% CI: 1·9-13·7, P = 0·001) and massive splenomegaly (HR: 9·1, 95% CI: 2·3-35·9, P = 0·002). Cytopenia at the lowest dose required to achieve a response was also an independent risk factor for transformation to acute leukaemia (HR: 20·3, 95% CI: 5·4-76·5, P < 0·001). In conclusion, the unified definition of resistance/intolerance to HC delineates a heterogeneous group of PV patients, with those developing cytopenia being associated with an adverse outcome.
Subject(s)
Hydroxyurea/therapeutic use , Nucleic Acid Synthesis Inhibitors/therapeutic use , Polycythemia Vera/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Drug Resistance , Drug Tolerance , Female , Humans , Hydroxyurea/adverse effects , Kaplan-Meier Estimate , Leukocyte Count , Leukopenia/chemically induced , Male , Middle Aged , Nucleic Acid Synthesis Inhibitors/adverse effects , Polycythemia Vera/blood , Prognosis , Registries , Retrospective Studies , Treatment Outcome , Young AdultABSTRACT
OBJECTIVES: Population-based studies have reported an increased incidence of skin cancer in patients with essential thrombocythemia (ET) and polycythemia vera (PV). We have examined the risk factors for non-melanoma skin cancer (NMSC) in patients diagnosed with ET or PV during 1973-2012. METHODS: A case-control study was performed to compare the clinical and treatment-related data of 51 ET/PV patients who had NMSC with that of 401 patients who did not. We also evaluated whether polymorphisms in 12 genes involved in DNA integrity predisposed to NMSC. RESULTS: By multivariate logistic regression analysis, risk factors for NMSC were older age (OR: 1.7, 95% CI: 1.3-2.1, P < 0.001), male sex (OR: 2.1, 95% CI: 1.1-3.8, P = 0.023), higher cumulated hydroxycarbamide dose (OR: 1.3, 95% CI: 1.1-1.7, P = 0.017), and busulphan exposure (OR: 3.2, 95% CI: 1.05-10.0, P = 0.041). On the time-to-event prognostic model, factors independently associated with increased cumulative incidence of NMSC were age (5% increased risk per year; P < 0.001), male sex (91% increased risk; P = 0.022), and hydroxycarbamide exposure (22% increased risk; P = 0.065). No susceptibility gene variant was identified. CONCLUSIONS: These findings suggest that the risk to develop NMSC in ET/PV patients results from the combined effect of common risk factors (age, male sex) together with cytoreductive treatment.
Subject(s)
Polycythemia Vera/complications , Skin Neoplasms/epidemiology , Skin Neoplasms/etiology , Thrombocythemia, Essential/complications , Adolescent , Adult , Case-Control Studies , Child , Child, Preschool , Female , Genetic Predisposition to Disease , Humans , Infant , Male , Odds Ratio , Polycythemia Vera/drug therapy , Polycythemia Vera/genetics , Polymorphism, Single Nucleotide , Population Surveillance , Risk Factors , Thrombocythemia, Essential/drug therapy , Thrombocythemia, Essential/genetics , Young AdultABSTRACT
It is unclear whether anticoagulation guidelines intended for the general population are applicable to patients with polycythemia vera (PV) and essential thrombocythemia (ET). In the present study, the risk of thrombotic recurrence was analyzed in 150 patients with PV and ET treated with vitamin K antagonists (VKA) because of an arterial or venous thrombosis. After an observation period of 963 patient-years, the incidence of re-thrombosis was 4.5 and 12 per 100 patient-years under VKA therapy and after stopping it, respectively (P < 0.0005). After a multivariate adjustment for other prognostic factors, VKA treatment was associated with a 2.8-fold reduction in the risk of thrombotic recurrence. Notably, VKA therapy offset the increased risk of re-thrombosis associated with a prior history of remote thrombosis. Both the protective effect of VKA therapy and the predisposing factors for recurrence were independent of the anatomical site involved in the index thrombosis. Treatment periods with VKA did not result in a higher incidence of major bleeding as compared with those without VKA. These findings support the use of long-term anticoagulation for the secondary prevention of thrombosis in patients with PV and ET, particularly in those with history of remote thrombosis.
Subject(s)
Anticoagulants/administration & dosage , Polycythemia Vera/drug therapy , Thrombocythemia, Essential/drug therapy , Thrombosis/prevention & control , Administration, Oral , Aged , Female , Follow-Up Studies , Humans , Male , Middle Aged , Polycythemia Vera/epidemiology , Recurrence , Thrombocythemia, Essential/epidemiology , Thrombosis/epidemiology , Vitamin K/antagonists & inhibitorsABSTRACT
OBJECTIVES: Clonal dominance is characteristic of patients with post-polycythemia vera myelofibrosis (post-PV MF), whereas patients in chronic phase usually display polyclonal hematopoiesis. The aim of this work was to study the mutational burden of JAK2V617F at the progenitor level in patients with PV and correlate it with the evolutive phase of the disease and the presence of mutations in genes different to JAK2V617F. METHODS: JAK2V617F was measured in stem cells, progenitor cells, and granulocytes of 45 patients with PV (early chronic phase n = 26, late chronic phase n = 10, post-PV MF n = 9). In addition, screening of TET2, DNMT3A, ASXL1, SF3B1, SRSF2, U2AF1, and TP53 was performed with quantification of the mutation in CD34+ cells in positive cases. Moreover, we assessed whether JAK2V617F allele burden in granulocytes (at a single time point or monitoring) could be used as a surrogate of clonal dominance. RESULTS: Ten patients presented clonal dominance at progenitor level (PV at diagnosis n = 2, late chronic phase n = 1, post-PV MF n = 7). Additional mutations were identified in four patients at diagnosis, three in TET2, and one in DNMT3A gene, with clonal dominance present in three of them. At PV diagnosis, clonal dominance was demonstrated only in patients with additional mutations. JAK2V617F monitoring showed better diagnostic accuracy than single time point measurement as a marker of clonal dominance. CONCLUSIONS: Clonal dominance may be present at diagnosis, especially in those cases carrying other mutations. JAK2V617F monitoring during follow-up could help in the identification of patients with clonal dominance.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/metabolism , Janus Kinase 2/genetics , Polycythemia Vera/genetics , Primary Myelofibrosis/genetics , Proto-Oncogene Proteins/genetics , Adult , Aged , Aged, 80 and over , Alleles , Clone Cells , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , DNA-Binding Proteins/metabolism , Dioxygenases , Disease Progression , Female , Gene Expression , Granulocytes/metabolism , Granulocytes/pathology , Hematopoiesis/genetics , Hematopoietic Stem Cells/pathology , Humans , Janus Kinase 2/metabolism , Male , Middle Aged , Mutation , Polycythemia Vera/complications , Polycythemia Vera/diagnosis , Polycythemia Vera/pathology , Primary Myelofibrosis/diagnosis , Primary Myelofibrosis/etiology , Primary Myelofibrosis/pathology , Proto-Oncogene Proteins/metabolismABSTRACT
Low serum erythropoietin (EPO) is a minor criterion of Polycythaemia Vera (PV) but its diagnostic usefulness relies on studies performed before the discovery of JAK2 V617F mutation. The objective of the present study was to evaluate the diagnostic accuracy of serum EPO and JAK2 V617F allele burden as markers of PV as well as the combination of different diagnostic criteria in 287 patients (99 with PV, 137 with Essential Thrombocythaemia and 51 with non-clonal erythrocytosis). Low EPO showed good diagnostic accuracy as a marker for PV, with the area under the curve (AUC) of the chemiluminescent-enhanced enzyme immunoassay (CEIA) being better than that of radioimmunoassay (RIA) (0·87 and 0·76 for CEIA and RIA, respectively). JAK2 V617F quantification displayed an excellent diagnostic accuracy, with an AUC of 0·95. A haematocrit >52% (males) or >48% (females) plus the presence of the JAK2 V617F mutation had a sensitivity and specificity of 79% and 97%, respectively. Adding low EPO or the JAK2 V617F allele burden did not improve the diagnostic accuracy for PV whereas the inclusion of both improved the sensitivity up to 83% and maintaining 96% specificity. Haematocrit and qualitative JAK2 V617F mutation allow a reliable diagnosis of PV. Incorporation of EPO and/or JAK2 V617F mutant load does not improve the diagnostic accuracy.
Subject(s)
Erythropoietin/blood , Janus Kinase 2/genetics , Mutation, Missense , Point Mutation , Polycythemia Vera/diagnosis , Alleles , Amino Acid Substitution , Area Under Curve , Biomarkers , Diagnosis, Differential , Female , Hematocrit , Hemoglobins/analysis , Humans , Leukocyte Count , Male , Platelet Count , Polycythemia/diagnosis , Polycythemia Vera/blood , Polycythemia Vera/genetics , ROC Curve , Reproducibility of Results , Sensitivity and Specificity , Thrombocythemia, Essential/diagnosisABSTRACT
Criteria of response and definition of resistance and intolerance to hydroxyurea (HU) in polycythemia vera (PV) were proposed by the European LeukemiaNet (ELN). Such criteria were evaluated in 261 PV patients (median follow-up, 7.2 years) treated with HU for a median of 4.4 years. Complete response, partial response, and no response were observed in 24%, 66%, and 10% of patients, respectively. Achieving ELN response (complete or partial) or hematocrit response did not result in better survival or less thrombosis and bleeding. On the contrary, having no response in leukocyte count was associated with higher risk of death (HR, 2.7; 95% confidence interval [CI], 1.3%-5.4%; P = .007), whereas lack of response in platelet count involved a higher risk of thrombosis and bleeding. Resistance and intolerance to HU was registered in 11% and 13% of patients, respectively. Resistance to HU was associated with higher risk of death (HR, 5.6; 95% CI, 2.7%-11.9%; P < .001) and transformation (HR, 6.8; 95% CI, 3.0%-15.4%; P < .001). In summary, fulfilling the ELN definition for response to HU was not associated with a benefit in the clinical outcome in PV, whereas response in platelet and white blood cell counts were predictive of less thrombohemorrhagic complications and better prognosis, respectively. Resistance to HU was an adverse prognostic factor.
Subject(s)
Hydroxyurea/therapeutic use , Outcome Assessment, Health Care/standards , Polycythemia Vera/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Cell Transformation, Neoplastic , Drug Resistance , Drug Tolerance , Female , Follow-Up Studies , Humans , Leukocyte Count , Male , Middle Aged , Multivariate Analysis , Nucleic Acid Synthesis Inhibitors/therapeutic use , Outcome Assessment, Health Care/methods , Platelet Count , Prognosis , Remission Induction , Risk Assessment , Risk Factors , Survival Analysis , Young AdultABSTRACT
Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have an increased incidence of acute myeloid leukemia and new nonhematologic malignancies compared with the general population. However, information on the factors determining the risk for such complications is limited. In the present study, we investigated whether constitutional genetic variations in DNA repair predispose to leukemic transformation and new nonmyeloid neoplasias in patients with ET and PV. Case-control studies for predisposition to both types of malignancies were nested in a cohort of 422 subjects diagnosed with ET or PV during the period 1973-2010 in several institutions in Spain. A total of 64 incidence cases of leukemia and 50 cases of primary nonmyeloid cancers were accrued. At conditional regression analysis, the Gln/Gln genotype in the XPD codon 751 showed the strongest association with both leukemic transformation (odds ratio [OR] = 4.9; 95% confidence interval [95% CI], 2.0-12) and development of nonmyeloid malignancies (OR = 4.2; 95% CI, 1.5-12). Additional predictive factors were exposure to cytoreductive agents for leukemic transformation (OR = 3.5; 95% CI, 2.0-6.2) and age for nonmyeloid malignancies (OR = 2.0; 95% CI, 1.4-2.8). These findings provide further evidence about the contribution of inherited genetic variations to the pathogenesis and clinical course of myeloproliferative neoplasms.
Subject(s)
Leukemia/genetics , Polycythemia Vera/genetics , Polymorphism, Genetic , Thrombocythemia, Essential/genetics , Xeroderma Pigmentosum Group D Protein/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Leukemia/epidemiology , Leukemia/metabolism , Male , Polycythemia Vera/epidemiology , Polycythemia Vera/metabolism , Retrospective Studies , Thrombocythemia, Essential/epidemiology , Thrombocythemia, Essential/metabolism , Xeroderma Pigmentosum Group D Protein/metabolismABSTRACT
Therapeutic options for patients with polycythemia vera (PV) and essential thrombocythemia (ET) resistant or intolerant to hydroxyurea are limited. Busulfan is effective as first-line therapy, but there is scarce information on this drug as second-line treatment. The efficacy of busulfan in patients with advanced PV or ET refractory or intolerant to hydroxyurea was assessed in 36 patients (PV n = 15, ET n = 21) treated for a median of 256 days. Complete hematological response (CHR) was achieved in 83 % of patients, after a median time of 203 days (range 92-313). The probability of sustained CHR at 1 and 2 years was 87 and 62 %, respectively. Time to CHR was shorter in patients treated with ≥14 mg of busulfan per week than with lower doses (141 versus 336 days, p = 0.01). Partial molecular response was achieved in three out of nine (33 %) patients. Busulfan was stopped in 27 patients (75 %) due to CHR achievement in 18 cases (67 %), hematological toxicity in 8 cases (30 %), and disease transformation in 1 case. With a median follow-up of 721 days, six patients have died, with the probability of survival at 2 years being 85 %. The probability of thrombosis at 2 years was 11 %. Transformation into acute leukemia or myelodysplastic syndrome was observed in three cases, all of them in a JAK2V617F-negative clone carrying additional mutations. Busulfan, at a dose of 2 mg/day, is an effective option for elderly patients with PV or ET who fail to hydroxyurea, but a significant rate of transformation was observed.
Subject(s)
Alkylating Agents/therapeutic use , Busulfan/therapeutic use , Polycythemia Vera/drug therapy , Thrombocythemia, Essential/drug therapy , Aged , Aged, 80 and over , Blood Cell Count , Comorbidity , Disease Progression , Drug Resistance , Drug Substitution , Female , Hematocrit , Hemorrhage/etiology , Humans , Hydroxyurea/adverse effects , Hydroxyurea/therapeutic use , Janus Kinase 2/genetics , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/epidemiology , Leukemia, Myeloid, Acute/etiology , Male , Middle Aged , Polycythemia Vera/complications , Polycythemia Vera/genetics , Remission Induction , Risk Factors , Thrombocythemia, Essential/complications , Thrombocythemia, Essential/genetics , Thrombosis/etiology , Treatment OutcomeABSTRACT
Although approximately 95% of patients with polycythemia vera (PV) harbor the V617F mutation in JAK2 exon 14, several mutations in exon 12 have been described in the remaining patients. We conducted a European collaborative study to define the molecular and clinical features of patients harboring these mutations. Overall, 106 PVs were recruited and 17 different mutations identified. Irrespective of the mutation, two-thirds of patients had isolated erythrocytosis, whereas the remaining subjects had erythrocytosis plus leukocytosis and/or thrombocytosis. Compared with JAK2 (V617F)-positive PV patients, those with exon 12 mutations had significantly higher hemoglobin level and lower platelet and leukocyte counts at diagnosis but similar incidences of thrombosis, myelofibrosis, leukemia, and death. In a multivariable analysis, age more than 60 years and prior thrombosis predicted thrombosis. These findings suggest that, despite the phenotypical difference, the outcome of JAK2 exon 12 mutations-positive PV is similar to that of JAK2 (V617F)-positive PV.
Subject(s)
Janus Kinase 2/genetics , Mutation , Polycythemia Vera/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Exons , Female , Humans , In Situ Hybridization , Leukocytosis/etiology , Leukocytosis/genetics , Male , Middle Aged , Polycythemia/etiology , Polycythemia/genetics , Polycythemia Vera/complications , Polycythemia Vera/mortality , Polymerase Chain Reaction , Thrombocytosis/etiology , Thrombocytosis/genetics , Young AdultABSTRACT
Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.
Subject(s)
DNA Methylation/genetics , Gene Expression Profiling/methods , Gene Regulatory Networks/genetics , Polycythemia Vera/genetics , Primary Myelofibrosis/genetics , Chronic Disease , Humans , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/genetics , Polycythemia Vera/diagnosis , Primary Myelofibrosis/diagnosisABSTRACT
This study investigates whether the response criteria proposed by the European LeukemiaNet (ELN) to evaluate cytoreductive therapies in essential thrombocythemia (ET) correlate with clinically relevant outcomes in patients receiving anagrelide. We evaluated 154 ET patients treated with anagrelide (upfront in 87) for a median of 2.9 years. Complete response (CR), partial response, and no response were observed in 56, 30.5, and 13.5 % patients, respectively. Only 38 patients (25 %) achieved a sustained CR. Overall, the aggregated time on CR and without CR was 200.1 and 333.6 person-years, respectively. The incidence rate of thrombosis and hemorrhage was independent of the CR status. The only factor associated with shorter survival after anagrelide start was the patient's age, whereas achieving a CR with anagrelide had no predictive value for subsequent survival. In conclusion, CR according to the ELN definition is not associated with any measurable clinical benefit in ET patients treated with anagrelide.
Subject(s)
International Agencies/standards , Leukocyte Count , Platelet Aggregation Inhibitors/therapeutic use , Platelet Count , Quinazolines/therapeutic use , Spleen/pathology , Thrombocythemia, Essential/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers , Disease-Free Survival , Female , Follow-Up Studies , Humans , Hydroxyurea/therapeutic use , Interferons/therapeutic use , Kaplan-Meier Estimate , Male , Middle Aged , Organ Size/drug effects , Prognosis , Remission Induction , Retrospective Studies , Spain/epidemiology , Survival Analysis , Thrombocythemia, Essential/blood , Thrombocythemia, Essential/mortality , Thrombocythemia, Essential/pathology , Treatment Outcome , Young AdultABSTRACT
JAK2V617F-negative essential thrombocythemia (ET) is a heterogeneous disease including clonal cases and others without evidence of clonality. However, it is unknown if the detection of myeloid clonality in JAK2V617F-negative ET patients confers a different clinical outcome than those in whom clonal hematopoiesis cannot be demonstrated. The objective of the present study was to evaluate the clinical significance of clonality assessment in patients with JAK2V617F-negative ET. Clonality investigation including mutational status of MPL, TET2, and ASXL1 genes and human androgen receptor (HUMARA) assay was performed in 73 JAK2V617F-negative cases out of 186 subjects consecutively diagnosed with ET in a single institution, at diagnosis or during follow-up. Mutations in MPL, TET2, and ASXL1 were observed in 7, 4, and 2 cases, respectively, whereas clonality by HUMARA assay was demonstrated in 21 out of 46 (46 %) female patients. With a median follow-up of 8 years, death, thrombosis, bleeding, and disease transformation were registered in 7, 10, 8, and 6 patients, respectively. No differences in thrombosis, bleeding or survival were observed according to clonality assessment. The probability of disease transformation at 10 years was higher in patients showing clonal hematopoiesis by presenting mutations in either MPL, TET2, or ASXL1 (64 versus 2 % in patients without mutations, p < 0.001) and in those with HUMARA clonality (35 versus 0 % in patients with polyclonal hematopoiesis, p < 0.004). In conclusion, disease transformation is associated with evidence of clonality in JAK2V617F-negative ET.
Subject(s)
Janus Kinase 2/genetics , Mutation/genetics , Thrombocythemia, Essential/diagnosis , Thrombocythemia, Essential/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Clone Cells , Cohort Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Phenylalanine/genetics , Thrombocythemia, Essential/genetics , Valine/genetics , Young AdultABSTRACT
Primary familial and congenital polycythemia is a rare disease characterized by an increase in red cell mass that may be due to pathogenic variants in the EPO receptor (EPOR) gene. To date, 33 genetic variants have been reported to be associated. We analyzed the presence of EPOR variants in two patients with polycythemia in whom JAK2 pathogenic variants had been previously discarded. Molecular analysis of the EPOR gene was performed by Sanger sequencing of the coding regions and exon/intron boundaries of exon 8. We performed in vitro culture of erythroid progenitor cells. Segregation studies were done whenever possible. The two patients studied showed hypersensitivity to EPO in in vitro cultures. Analysis of the EPOR gene unveiled two novel pathogenic variants. Genetic testing of asymptomatic relatives could guarantee surveillance and proper management.
Subject(s)
Polycythemia , Receptors, Erythropoietin , Humans , Receptors, Erythropoietin/genetics , Polycythemia/genetics , Polycythemia/congenital , Polycythemia/pathologyABSTRACT
Molecular and cytogenetic studies are essential for diagnosis and prognosis in patients with myelodysplastic syndromes (MDSs). Cell-free DNA (cfDNA) analysis has been reported to be a reliable noninvasive approach for detecting molecular abnormalities in MDS; however, there is limited information about cytogenetic alterations and monitoring in cfDNA. We assessed the molecular and cytogenetic profile of a cohort of 70 patients with MDS by next-generation sequencing (NGS) of cfDNA and compared the results to sequencing of paired bone marrow (BM) DNA. Sequencing of BM DNA and cfDNA showed a comparable mutational profile (92.1% concordance), and variant allele frequencies (VAFs) strongly correlated between both sample types. Of note, SF3B1 mutations were detected with significantly higher VAFs in cfDNA than in BM DNA. NGS and microarrays were highly concordant in detecting chromosomal alterations although with lower sensitivity than karyotype and fluorescence in situ hybridization. Nevertheless, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA. In addition, we monitored molecular and cytogenetic alterations and observed an excellent correlation between the VAFs of mutations in BM DNA and cfDNA across multiple matched time points. A decrease in the cfDNA VAFs was detected in patients responding to therapy, but not in nonresponding patients. Of note, cfDNA analysis also showed cytogenetic evolution in 2 nonresponsive cases. In summary, although further studies with larger cohorts are needed, our results support the analysis of cfDNA as a promising strategy for performing molecular characterization, detection of chromosomal aberrations and monitoring of patients with MDS.
Subject(s)
Cell-Free Nucleic Acids , Myelodysplastic Syndromes , Chromosome Aberrations , Humans , In Situ Hybridization, Fluorescence , Mutation , Myelodysplastic Syndromes/diagnosis , Myelodysplastic Syndromes/geneticsABSTRACT
Standardized criteria of response to treatment and a unified definition of resistance/intolerance to hydroxycarbamide (HC) in essential thrombocythaemia (ET) have been proposed by the European LeukaemiaNet (ELN). We have retrospectively evaluated such criteria in 166 ET patients treated with HC for a median of 4·5 years. Overall, 134 patients achieved a complete clinicohaematological response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n = 15) or intolerance (n = 21) to HC. Fifteen cases developed anaemia with thrombocytosis, which was associated with a high incidence of myelofibrosis and death from any cause. Other definitions of resistance were less useful. Factors determining the thrombotic risk were a history of prior thrombosis and a baseline leucocyte count >10 × 109/ l. Of note, patients achieving a CR, even if sustained during the entire follow-up, did not benefit from a lower incidence of thrombosis or an improved survival. In conclusion, most ET patients respond to HC, but the achievement of response, as defined by the ELN, does not correlate with the patients' outcome. The best discriminating ELN criterion of resistance to HC was the detection of anaemia, which also identified a subgroup of patients with poor prognosis.
Subject(s)
Hydroxyurea/therapeutic use , Nucleic Acid Synthesis Inhibitors/therapeutic use , Thrombocythemia, Essential/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Drug Evaluation/methods , Drug Evaluation/standards , Drug Resistance , Epidemiologic Methods , Female , Humans , Hydroxyurea/adverse effects , Male , Middle Aged , Nucleic Acid Synthesis Inhibitors/adverse effects , Platelet Count , Prognosis , Thrombocythemia, Essential/blood , Thrombocythemia, Essential/complications , Thrombosis/etiology , Thrombosis/prevention & control , Treatment Outcome , Young AdultABSTRACT
This study investigates the differential gene expression profile of JAK2(V617F)-positive myeloproliferative neoplasm (MPN) patients, with and without response to hydroxyurea (HU) treatment. Twenty-one polycythemia vera, 28 essential thrombocythemia, eight secondary erythrocytosis, and 30 controls were studied. Thirty-four genes were overexpressed in patients who did not respond to HU. Of these, some participate in proliferative pathways: MAPK, AKT, Src kinase (SFK), and JAK2 pathway. JAK2 allele burden was similar between groups of responders and nonresponder. A molecular fingerprint distinguishes JAK2(V617F)-positive MPN patients without response to HU treatment, with overexpression of JAK2, MAPK14, PIK3CA, and SFK genes.