Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival

Abstract Background : Two previous studies have reported significant but inconsistent associations between the presence of JAK2V617F in primary myelofibrosis (PMF) and older age at diagnosis, risk of thrombosis, higher leukocyte count, and inferior survival (Tefferi, et al. BJH2005;131:320, Campbell, et al. Blood2006;107:2098). The clinical relevance of V617F allele burden in PMF has not been previously studied. Methods : Diagnosis of PMF was based on the World Health Organization criteria and study eligibility included the availability of bone marrow-derived DNA that was collected either at time of diagnosis or within one year of diagnosis. Quantitative allele specific PCR was utilized to meaure V617F allele burden. Results I. V617F-positive vs. V617F-negative comparisons: A total of 199 patients (60% males; median age 61 years) were suitable for analysis of comparisons between mutation-positive and mutation-negative disease. The Dupriez prognostic scoring system (PSS) risk distributions were 61% low-risk, 31% intermediate-risk, and 8% high-risk. Hypercatabolic symptoms were present in 27% of the patients and ≥1% peripheral blood (PB) blasts in 37%. At a median follow-up of 23 months (range 0–266), 57 patients (29%) had died, 17 (9%) developed leukemic transformation (LT) and 10 (5%) experienced major thrombosis. V617F mutational frequency was 58%. Univariate analysis identified older age (p=0.0007), platelet count ≥ 100 x 109/L (p=0.05), and PB blast percentage < 3% (p=0.001) as being associated with a positive mutational status; all three variables sustained their significance during multivariable analysis. The presence of the mutation did not affect the incidence of thrombosis (p=0.78), overall survival (p=0.22) or leukemia-free survival (p=0.5). Results II. Clinical correlates of V617F allele burden: Quantitative measurement of V617F allele burden was performed in 129 patients that were divided into four groups: V617F-negative (n=53) and V617F-positive with mutant allele burden in the lower quartile (n=19), middle quartiles (n=38), or upper quartile (n=19) range (median and range of V617F allele burden ratio was 29% and 1% to 74%). Kaplan-Meier plots revealed significantly shortened overall (Figure; p = 0.0008) and leukemia-free (p = 0.01) survival for the lower quartile allele burden group; survival significance was sustained in a multivariable analysis that included the Dupriez PSS. Lower quartile allele burden was also associated with lower leukocyte count (p = 0.003) and presence of hypercatabolic symptoms (p=0.05). Thrombosis incidence was not affected by allele burden. Conclusions: In PMF, patients with a low V617F allele burden, compared to those with either undetectable (i.e. wild-type) or higher allele burden, display significantly shorter overall and leukemia-free survival. In contrast, the presence or absence of the mutation, by itself, does not result in distinct groups that differ significantly in terms of survival, LT, or incidence of thrombosis. These data suggest that a low V617F allele burden in PMF is a surrogate for the development of dominant V617F-negative subclones that are more likely to undergo LT. Figure Figure

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Thrombotic Events in Primary Myelofibrosis: Prevalence and Prognostic Factors in 207 Consecutive Patients.

Blood ◽

10.1182/blood.v114.22.3915.3915 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3915-3915

Author(s):

Michelle Elliott ◽

Terra Lasho ◽

Christy Finke ◽

Animesh D. Pardanani ◽

Ayalew Tefferi

Keyword(s):

Platelet Count ◽

Prognostic Factors ◽

Mutant Allele ◽

Primary Myelofibrosis ◽

Hemoglobin Level ◽

Snp Genotyping ◽

Taqman Assay ◽

Allele Burden ◽

Vein Thrombosis ◽

Jak2v617f Allele Burden

Abstract Abstract 3915 Poster Board III-851 Background Thromboembolic disease (TE) is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). There is limited information on the prevalence of TE in primary myelofibrosis (PMF) and associated prognostic factors. Methods Study patients were recruited form the Mayo Clinic database for PMF. Diagnosis was according to the 2001 WHO criteria. Clinical and laboratory information was collected at time of diagnosis and correlated with the occurrence of TE in general and arterial (AT) or venous (VT) thrombosis in particular. Leukocytosis was defined as a leukocyte count of > 10 × 109/L. Qualitative JAK2V617F analysis was done according to previously published PCR-based methods. In addition, a subset of the study patients underwent quantitative PCR for JAK2V617F and rs12343867 SNP genotyping (performed by a commercially available Taqman assay). All molecular studies were performed on stored bone marrow. Standard statistical methods were used to test significance of associations between thrombosis and other variables. Results 207 patients with PMF were studied (median age 62, range 28-87; 132 males). At presentation, the median (range) of hemoglobin (Hb), leukocytes (WBC) and platelets (plt) were 11 g/dl (6.4-15.4), 8.6 × 109/L (0.8-156.7) and 273 × 109/L (13-1916), respectively. Dupriez prognostic scores at diagnosis were low, intermediate and high in 57%, 33% and 10% of the patients, respectively. 130 (63%) patients were JAK2V617F positive. Among 73 patients in whom quantitative JAK2V617F analysis was performed, the mutation was not detected in 30 (41%) patients and the median mutant allele burden in the 43 mutation-positive patients was 28% (range 1-85). The same 73 patients had rs12343867 SNP genotyping performed that revealed C/C genotype in 14 (19%) patients, C/T in 34 (47%) and T/T in 25 (34%). At or before diagnosis, a total of 29 patients (14 %) had TE: 23 (11%) AT and 9 (4%) VT. The AT included acute coronary syndromes (ACS; n=19), transient ischemic attack/ cerebrovascular accidents (TIA/CVA; n=6) and peripheral arterial thrombosis (PAT; n=1). The VT included abdominal vein thrombosis (AVT; n=4), deep vein thrombosis/pulmonary embolism (DVT/PE; n=6) and dural sinus thrombosis (n=1). Several patients had more than one event and some both AT and VT. During follow-up, 22 (11%) patients experienced TE: 17 (8%) VT and 8 (4%) AT. The post-diagnosis VT included AVT (n=8), DVT/PE (n=13) and both DVT/PE and AVT (n=4). Post-diagnosis AT included CVA (n=4), TIA (n=1), ACS (n=2) and PAT (n=1). AT at or before diagnosis was associated with advanced age (p=0.003) but not with sex, leukocytosis, hemoglobin level, platelet count, JAK2 mutational status, JAK2 mutant allele burden or rs12343867 SNP genotype. VT at or before diagnosis also did not correlate with all these variables or age. These variables were also evaluated regarding their relationship with post-diagnosis AT, which showed significant associations with hemoglobin level (p=0.006), platelet count (p=0.04) and high JAK2V617F allele burden (0.03). The corresponding significant variables for post-diagnosis VT were leukocytosis (p=0.02) and high JAK2V617F allele burden (p=0.01). Of note, AT or VT at or before diagnosis did not predict post-diagnosis events. During multivariable analysis, only higher hemoglobin level predicted post-diagnosis AT. Conclusion The current study provides an accurate description of both arterial and venous thrombotic events in PMF. The association between higher hemoglobin and leukocyte counts at diagnosis and post-diagnosis occurrence, respectively, of arterial and venous thrombosis is similar to observations in patients with PV or ET and suggests a potential benefit from early institution of cytoreductive therapy for the appropriate patient groups. Disclosures: No relevant conflicts of interest to declare.

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Correlation of Novel Molecular Markers in Patients with Myeloproliferative Neoplasms.

Blood ◽

10.1182/blood.v114.22.4968.4968 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4968-4968

Author(s):

Kai Kaufmann ◽

Sabina Swierczek ◽

Shulian Shang ◽

Albert Gruender ◽

Rona Singer Weinberg ◽

...

Keyword(s):

Molecular Markers ◽

Mrna Expression ◽

Myeloproliferative Neoplasms ◽

Hematological Parameters ◽

Hemoglobin Concentration ◽

Primary Myelofibrosis ◽

Hematopoietic Stem ◽

Allele Burden ◽

The Relationship ◽

Jak2v617f Allele Burden

Abstract Abstract 4968 Elucidation of the molecular aberrations underlying the development of myeloproliferative neoplasms (MPNs) has progressed rapidly during the previous years. In addition to the JAK2V617F mutation, which is found in over 90% of patients with polycythemia vera (PV) and in around 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), several novel markers have recently been described. These include mutations in the Ten-Eleven-Translocation-2 (TET-2) gene, as well as overexpression of the hematopoietic transcription factor nuclear factor erythroid-2 (NF-E2). While the individual frequency of these abnormalities is well known, so far, studies that evaluate the relationship between these markers and their correlation with hematological parameters have not been conducted. The International Myeloproliferative Research Consortium (MPD-RC) has instituted a Tissue Bank (MPD-RC Trial 106) allowing the collection of MPN patient samples from member institutions in the US and Europe. Using this resource, we have investigated the relationship among the following molecular markers and hematological parameters in a series of 66 MPN patients: TET-2 mRNA expression, JAK2V617F allele burden, NF-E2 mRNA expression, granulocyte clonality (female patients) as well as hematocrit, hemoglobin concentration, platelet numbers and leukocyte counts. Our crossectional cohort included 37 PV patients, 14 ET, 4 PMF and one post PV MF patient. Sixtyeight percent of the patients were treated, medication including hydroxyurea, anagrelide, ASA, interferon and one patient treated with imatinib mesylate. We acknowledge that treatment may affect the molecular markers being investigated. In addition, the desired effect of treatment on hematological parameters may not be paralleled by a similar effect on molecular markers. Therefore, investigation of treated patients may not reveal biological relationships present in untreated diseases states. We thus tested the effect of cytoreductive treatment on the expression of molecular markers by comparing treated and untreated patients in our cohort. The JAK2V617F allele burden was significantly lower in treated patients than in untreated patients (p = 0.008). The same difference was noted when PV patients were analyzed alone (p = 0.006) In contrast, neither TET-2 nor NF-E2 mRNA expression were affected by treatment. In the 66 MPN patients evaluated, a significant inverse Spearman correlation of -0.25 was noted between NF-E2 mRNA expression and hemoglobin concentration (p = 0.05). This relationship was more pronounced when PV patients were analyzed alone (Spearman's r = -0.41, p = 0.01). In addition, a correlation of 0.51 between JAK2V617F allele burden and NF-E2 mRNA expression was noted (p = 0.0007). Occurrence of the recently discovered TET-2 gene mutations, which are present in around 15% of MPN patients, was measured indirectly by determining TET-2 mRNA expression. None of the other markers and parameters assessed was significantly correlated with the amount of TET-2 mRNA expressed. We report a previously unrecognized inverse relationship between NF-E2 mRNA expression and hemoglobin concentration. These data complement several recent findings in MPN patients. While NF-E2 is overexpressed in granulocytes of all three MPN subtypes, PV, ET and PMF, its overexpression in CD34+ hematopoietic stem cells has so far only been observed in PMF. We have recently shown that ex vivo overexpression of NF-E2 in CD34+ hematopoietic stem cells drastically reduces erythroid colony formation. It was previously noted that mean JAK2V617F allele burdens in PMF patients are higher than in PV or ET. Here, we confirm the previously noted positive correlation between JAK2V617F allele burden and NF-E2 mRNA expression. Our data therefore suggest that high levels of JAK2V617F in PMF patients directly or indirectly augment NF-E2 overexpression, which may contribute to the anemia of Primary Myelofibrosis. Disclosures No relevant conflicts of interest to declare.

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PB2206 HEMOSTATIC MARKERS OF HYPERCOAGULABILITY IN PRIMARY MYELOFIBROSIS PATIENTS, RELATION WITH JAK2V617F ALLELE BURDEN

HemaSphere ◽

10.1097/01.hs9.0000567304.01393.ea ◽

2019 ◽

Vol 3 (S1) ◽

pp. 988-989

Author(s):

N. Korsakova ◽

N. Silina ◽

E. Efremova ◽

M. Fominykh ◽

V. Kobilyanskaya ◽

...

Keyword(s):

Primary Myelofibrosis ◽

Allele Burden ◽

Hemostatic Markers ◽

Jak2v617f Allele Burden

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A JAK2 Germline Genetic Variation Is Associated with Shortened Survival in Primary Myelofibrosis: Clinical and JAK2V617F Allele Burden Correlates of rs12343867 SNP Genotype in 132 Cases.

Blood ◽

10.1182/blood.v114.22.2895.2895 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2895-2895 ◽

Cited By ~ 2

Author(s):

Ayalew Tefferi ◽

Terra Lasho ◽

Christy Finke ◽

Kebede Hussein ◽

Michelle Elliott ◽

...

Keyword(s):

Mutant Allele ◽

Cox Regression ◽

Conflicts Of Interest ◽

Multivariable Analysis ◽

Primary Myelofibrosis ◽

Taqman Assay ◽

Snp Analysis ◽

Allele Burden ◽

Mutational Status ◽

Jak2v617f Allele Burden

Abstract Abstract 2895 Poster Board II-871 Background: Recent studies have reported a significant association between JAK2V617F acquisition and a specific JAK2 germline haplotype; rs12343867 (located at intron 14 of the JAK2 gene) is one of several SNPs that tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with primary myelofibrosis (PMF) in order to study the clinical and laboratory correlates of the particular JAK2 haplotype and its effect on survival. Methods: Study patients were recruited form the Mayo Clinic database for PMF. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results: 132 patients with PMF were studied (median age 62, range 28-82; 82 males). Risk distribution according to the International Prognostic Scoring System (IPSS) for PMF were low in 39 (29%) patients, intermediate-1 in 41 (31%), intermediate-2 in 25 (20%) and high in 27 (20%). 77 (58%) patients were JAK2V617F positive; median mutant allele burden was 26% (range 1-85) and 21 patients displayed > 50% mutant allele burden. The rs12343867 genotype distributions were C/C 22%, C/T 44% and T/T 34% . The corresponding figures in JAK2V617F positive/negative cases were 31%/9%, 38%/53%, 31%/38% (p=0.01). Among the 21 patients with > 50% JAK2V617F allele burden, 14 (67%) displayed the C/C allele, 2 C/T and 5 T/T. The specific SNP genotype was not significantly affected by age (p=0.33), sex (p=0.46), leukocyte count (p=0.39), platelet count (p=0.09), or IPSS risk category (p=0.63). Patients were followed for a median of 54 months and during this period 73 (55%) deaths were documented. The T/T SNP genotype was significantly associated with shortened survival, compared to either C/C or C/T (p=0.001; Figure). Multivariable analysis showed this association to be independent of IPSS, JAK2V617F mutational status, age or sex. The adverse prognostic effect of the T/T genotype was also apparent when JAK2V617F negative (p=0.01) or positive (p=0.06) cases were analyzed separately. Conversely, JAK2 mutational status or allele burden did not affect survival in patients with T/T or C/C allele. Conclusion: The current study illustrates the non-random haplotype distribution of JAK2V617F in patients with PMF. More importantly, a non-JAK2 haplotype, tagged by the rs12343867 T allele, was associated with inferior survival that is not accounted for by IPSS or JAK2V617F mutational status. These findings, together with previous observations regarding shortened survival associated with low JAK2V617F allele burden, suggest the presence of molecular events in PMF that are more aggressive than JAK2V617F and not necessarily linked to the JAK2 haplotype. Disclosures: No relevant conflicts of interest to declare.

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The A3669G Polymorphism of GR Is a Host Genetic Modifier Associated with Polycythemia Vera and Primary Myelofibrosis

Blood ◽

10.1182/blood.v116.21.3067.3067 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3067-3067

Author(s):

Lilian Varricchio ◽

Elena Masselli ◽

Giovanni Migliaccio ◽

Carolyn Whitsett ◽

Ronald Hoffman ◽

...

Keyword(s):

Polycythemia Vera ◽

Mononuclear Cells ◽

Ex Vivo ◽

Genetic Modifier ◽

Primary Myelofibrosis ◽

Jak2v617f Mutation ◽

Single Mutation ◽

Allele Burden ◽

Host Genetic ◽

Jak2v617f Allele Burden

Abstract Abstract 3067 The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) which are all associated with a gain-of-function mutation (JAK2V617F) of the JAK2 gene. There is ongoing discussion on the mechanism by which this single mutation may cause diseases with three different phenotypes. The consensus is that while the JAK2V617F may increase proliferation of hematopoietic cells in general, the lineage affected is determined by host genetic modifiers still to be identified. The glucocorticoid receptor (GR) is an important regulator of erythropoiesis in vivo and in vitro. It exerts its activity by synergistic transcriptional and membrane-associated mechanisms. Upon activation, GR forms a transcriptional complex with phosphorylated STAT-5 (P-STAT-5) which binds and alters the expression of a subset of the erythropoietin (EPO)- and stem cell factor (SCF)-target genes. Physical interaction between GR and the EPO receptor (EPO-R) on the cell membrane suppresses the ability of both receptors to phosphorylate STAT-5 when activated together. Insufficient GR/P-STAT-5 transcriptional activity has been suggested to result in delayed erythroid maturation and to induce eythrocytosis in patients exposed to excessive glucocorticoids. We had previously determined that erythroid cells (EBs) generated ex vivo from peripheral blood mononuclear cells of patients with PV and those obtained from normal donors (ND) stimulated with dexamethasone have similar biological (increased proliferation with delayed maturation) and biochemical (great levels of NFE-2, WT-1 and GATA2 and low levels of GATA1 and β-globin expression) properties (Varricchio et al,Blood 2009;114:3899a). This similarity is counterintuitive because STAT-5 phosphorylation was reduced in normal EBs exposed to dexamethasone and EPO while in EBs from PV patients it was constitutive due to the presence of the JAK2V617F mutation. In addition to the canonical GRα signalling isoform, cells may express the dominant negative GRβ isoform which binds poorly to glucocorticoids because its unique structure impairs ligand binding and determines nuclear retention. Expression of GRβ is regulated by the presence of the A3669G SNP in exon 9 which specifically stabilizes GRβ mRNA. The presence of this polymorphism has been associated with glucocorticoid resistance but its effects on erythropoiesis are unknown. To clarify the similarities between the biological properties of EBs expanded ex vivo from ND in the presence of dexamethasone and those obtained from PV patients, we determined the levels of expression (at the mRNA and protein level) of GRα and GRβ in EBs expanded ex vivo from 16 ND and 16 PV patients. While EBs from ND expressed GRα only, those expanded from PV patients expressed both GRα and GRβ. Therefore, in spite of constitutive STAT-5 phosphorylation, in EBs from PV patients formation of transcriptionally active GR/P-STAT-5 complexes is reduced by expression of GRβ. To clarify the mechanism underlying expression of GRβ in EBs obtained from PV patients, the frequency of the A3669G polymorphism was determined by specific PCR followed by sequencing of the amplified products using DNA obtained from the mononuclear cells of ND (7) and patients with PV (10). DNA from patients with PMF (10) and ET (14) was analysed for comparison. The polymorphism was detected in 60% of patients with PV but not in ND, indicating that GRβ mRNA is particularly stable in EBs from PV patients. In addition, the polymorphism was observed in 50% of patients with PMF but was not detected in patients with ET. A trend toward higher JAK2V617F allele burden was observed in A3669G positive PV patients (69.8±23.0) compared to A3669G negative PV patients (37.3±22.6) and ET patients (28.13±27.9) while the JAK2V617F allele burden in patients with PMF with or without the A3669G SNP was the same (51.2±37.5 vs 42.8±17.9). These data suggest that in addition to mutations of EPO-R and Von Hippel-Lindau gene, erythrocytosis may be induced by excess GR activation by glucocorticoids or, in association with the JAK2V617F mutation, by the presence of the A3669G SNP which favours GRβ expression. Moreover, the A3669G SNP may represent a host genetic modifier and a potential diagnostic tool to predict development of erythrocytosis in patients with MPN and GRβ may represent a potential therapeutic target for PV. Disclosures: No relevant conflicts of interest to declare.

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A randomized phase II, open-label trial of orally administered SAR302503 in patients with polycythemia vera (PV) or essential thrombocythemia (ET) who are resistant or intolerant to hydroxyurea.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.tps6641 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. TPS6641-TPS6641 ◽

Cited By ~ 1

Author(s):

Ayalew Tefferi ◽

Jorge E. Cortes ◽

Andreas Hochhaus ◽

Jean-Jacques Kiladjian ◽

Ruben A. Mesa ◽

...

Keyword(s):

Phase 1 ◽

Symptom Burden ◽

Primary Myelofibrosis ◽

Open Label ◽

Eligibility Criteria ◽

Allele Burden ◽

Phase 2 Trial ◽

Stat3 Phosphorylation ◽

Ecog Ps ◽

Jak2v617f Allele Burden

TPS6641 Background: Hydroxyurea is widely used to treat patients with high-risk PV and ET. However, some patients are either clinically resistant to hydroxyurea or have unacceptable side effects, supporting the need for active therapeutic options in this setting. SAR302503 is an orally administered selective JAK-2 inhibitor that reduced symptom burden and provided a durable clinical benefit, including a reduction in the JAK2V617F allele burden, with an acceptable safety profile in a Phase 1/2 trial of patients with primary myelofibrosis (MF), post-PV MF, or post-ET MF (J Clin Oncol 2011;29:789; Pardanani, ASH 2011; Abs 3838). Based on the results in MF, in October 2011 we initiated a Phase 2 trial to evaluate SAR302503 for the treatment of patients with PV and ET who are resistant or intolerant to hydroxyurea (NCT01420783; ARD12042). Methods: Eligibility criteria include age of at least 18 years, ECOG PS 0–2, and hydroxyurea resistant PV or ET as defined by European LeukemiaNet Consensus Criteria (Blood 2009;113:4829). Patients are being randomized (1:1:1) to receive 100, 200, or 400 mg of SAR302503 orally once daily in consecutive 28-day cycles for at least 8 cycles in the absence of progression or unacceptable toxicity. The primary end points will be assessed after the completion of 8 cycles and are: for PV, the proportion of patients maintaining a hematocrit below 45% without phlebotomy for at least 3 months; for ET, the proportion of patients with a platelet count lower than 400 × 109/L for at least 3 months. Secondary objectives include safety, response rate, pharmacokinetics, pharmacodynamics as measured by JAK2V617F allele burden and STAT3 phosphorylation inhibition, assessment of disease-related symptoms, and quality of life. Enrollment of the planned total of 90 patients is ongoing at approximately 45 sites globally. Twelve patients have been enrolled as of January 2012.

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