Relationship Between Granulocyte JAK2 (V617F) Mutant Allele Burden and Risk of Progression to Myelofibrosis in Polycythemia Vera: a Prospective Study of 338 Patients.

Abstract Abstract 751 An identical gain-of-function mutation of JAK2 is found in about 95% of patients with polycythemia vera (PV). According to a two-step model [N Engl J Med. 2005 Apr 28;352(17):1779-90], the occurrence of JAK2 (V617F) gives rise to a clone that is heterozygous and expands to replace hematopoietic cells without the JAK2 mutation. A mitotic recombination in a hematopoietic cell that is heterozygous for JAK2 (V617F) later generates uniparental disomy and 9pLOH. The daughter cell that is homozygous for JAK2 (V617F) gives rise to a new clone that expands and replaces the previous heterozygous clone. Therefore, variable proportions of JAK2 (V617F) mutant alleles are found in myeloid cell populations from PV patients. A mutant allele dosage effect on phenotype has been described, and PV patients with high mutant allele burden have been found to have a more severe disease. Patients with post-PV myelofibrosis have the highest mutant allele burdens [median value of about 90% - Blood. 2008 Apr 1;111(7):3383-7]. Interestingly, JAK2 (V617F) activates circulating granulocytes, and by this means likely plays a role in the constitutive mobilization of CD34-positive cells into peripheral blood that characterizes the transformation of PV into post-PV myelofibrosis [Blood. 2006 May 1;107(9):3676-82]. Since all these observations may suggest that the mutant allele burden contributes to determining the myelofibrotic transformation of PV, we examined PV patients enrolled in a prospective observational cohort study. As of August 10, 2009, 338 patients diagnosed with PV according to the 2008 WHO criteria have been enrolled in this study. Of these patients, 320 (94.7%) carried JAK2 (V617F), 14 (4.1%) had JAK2 exon 12 mutations, and 4 (1.2%) did not carry JAK2 (V617F) nor exon 12 mutations despite a typical PV phenotype. Of the 320 patients carrying JAK2 (V617F), 146 were enrolled at diagnosis and 174 at follow-up. Patients were routinely treated with phlebotomy and low dose aspirin, while those at high risk for thrombosis (history of previous thrombosis and/or age greater than 60 years) were given also cytoreductive therapy. Diagnosis of post-PV myelofibrosis was based on the IWG-MRT criteria, while diagnosis of myelodysplastic syndrome or acute myeloid leukemia (AML) was done according the 2008 WHO criteria. In order to accurately assess the granulocyte mutant allele burden, we refined a previously described quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay. This assay is now routinely calibrated using defined standards [Haematologica. 2009 Jan;94(1):38-45] and has a sensitivity equal to 0.2% mutant alleles. Within 320 JAK2 (V617F)-positive patients, the median mutant allele burden was 47% (range 1.1-100%); 167 (52%) patients had less than 50%, while 153 (48%) had more than 50% mutant alleles. PV patients at diagnosis had significantly lower mutant allele burdens than those enrolled in the study at follow-up (P = .002). During the study period, disease transformation occurred in 18 patients. Eight patients, all with more than 50% JAK2 (V617F) mutant alleles at study entry, progressed to post-PV myelofibrosis, while 10 patients developed AML. Since about half of the patients were enrolled at follow-up, survival analyses were carried out accounting for left censoring of the observation. Cox proportional hazard regression showed that the JAK2 mutant allele burden, analyzed as a continuous variable, was related to hematologic transformation-free survival (HR: 1.025, 95% CI 1.005-1.046; P = .015). By categorizing the mutant allele burden, patients with more than 50% mutant alleles had a significantly worse hematologic transformation-free survival (HR: 6.54, 95% CI 1.47-29.1; P = .013) compared with those with lower mutant allele burden. After adjusting for age in a multivariable analysis, the 50% cutoff retained statistical significance (P = .048). With respect to the risk of progression to post-PV myelofibrosis, the JAK2 mutant allele burden, considered as a continuous variable, was significantly related to myelofibrosis-free survival (HR: 1.04, 95% CI: 1.004-1.08; P = .029). In a multivariable analysis with allele burden and age as covariates, the mutant allele burden showed an independent effect on myelofibrosis-free survival (P= .038). By contrast, the risk of developing AML was not significantly related to the mutant allele burden. In conclusion, the findings of this study suggest that a high mutant allele burden represents a risk factor for progression to myelofibrosis in patients with PV. Disclosures: No relevant conflicts of interest to declare.

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Sequential Analysis By Next Generation Sequencing of 18 Genes in Polycythemia Vera and Essential Thrombocythemia Reveals a Association Between Mutational Status and Clinical Outcome after 3-Year Follow-up

Blood ◽

10.1182/blood.v126.23.2808.2808 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2808-2808

Author(s):

Damien Luque Paz ◽

Aurelie Chauveau ◽

Caroline Buors ◽

Jean-Christophe Ianotto ◽

Francoise Boyer ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Jak2 V617f Mutation ◽

Poor Prognostic Factor ◽

Disease Evolution ◽

Allele Burden ◽

V617f Mutation

Abstract Introduction Myeloproliferative neoplasms (MPN) are molecularly characterized by driver mutations of JAK2, MPL or CALR. Other somatic mutations may occur in epigenetic modifiers or oncogenes. Some of them have been shown to confer a poor prognosis in primary myelofibrosis, but their impact is less known in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). In this study, we investigated the mutational profile using NGS technology in 50 JAK2 V617F positive cases of MPN (27 PV and 23 ET) collected at the time of diagnosis and after a 3 year follow-up (3y). Patients and Methods All patients were JAK2 V617F positive and already included in the prospective cohort JAKSUIVI. All exons of JAK2, MPL, LNK, CBL, NRAS, NF1, TET2, ASXL1, IDH1 and 2, DNMT3A, SUZ12, EZH2, SF3B1, SRSF2, TP53, IKZF1 and SETBP1 were covered by an AmpliseqTM custom design and sequenced on a PGM instrument (Life Technologies). CALR exon 9 mutations were screened using fragment analysis. Hotspots that mutated recurrently in MPN with no sequencing NGS coverage were screened by Sanger sequencing and HRM. A somatic validation was performed for some mutations using DNA derived from the nails. The increase of a mutation between diagnosis and follow-up has been defined as a relative increase of twenty percent of the allele burden. An aggravation of the disease at 3y was defined by the presence of at least one of the following criteria: leukocytosis >12G/L or immature granulocytes >2% or erythroblasts >1%; anemia or thrombocytopenia not related to treatment toxicity; development or progressive splenomegaly; thrombocytosis on cytoreductive therapy; inadequate control of the patient's condition using the treatment (defined by at least one treatment change for reasons other than an adverse event). Results As expected, the JAK2 V617F mutation was found in all patients with the use of NGS. In addition, we found 27 other mutations in 10 genes out of the 18 genes studied by NGS (mean 0.54 mutations per patient). Overall, 29 of 50 patients had only the JAK2 V617F mutation and no other mutation in any of the genes analysed. No CALR mutation was detected. Nine mutations that were not previously described in myeloid malignancies were found. The genes involved in the epigenetic regulation were those most frequently mutated: TET2, ASXL1, IDH1, IDH2 and DNMT3A. In particular, TET2 mutations were the most frequent and occurred in 20% of cases. There was no difference in the number or in the presence of mutations between PV and ET. At 3y, 4 mutations appeared in 4 patients and 15 out of 50 patients (9 PV and 6 ET) were affected by an allele burden increase of at least one mutation. At 3y, 24/50 patients suffered an aggravation of the disease as defined by the primary outcome criterion (16 PV and 8 ET). The presence of a mutation (JAK2 V617Fomitted) at the time of the diagnosis was significantly associated with the aggravation of the disease (p=0.025). Retaining only mutations with an allele burden greater than 20%, the association with disease aggravation is more significant (p=0.011). Moreover, a mutation of ASXL1, IDH1/2 or SRSF2, which is a poor prognostic factor in primary myelofibrosis, was found in 8 patients, all having presented an aggravation of their disease (p=0.001). Only 4 patients had more than one somatic mutation other than JAK2 V617F and all of them also had an aggravation at 3y (p=0.046). In this cohort, appearance of a mutation at 3y was not associated with the course of the disease. Conversely, the increase of allele burden of at least one mutation was associated with an aggravation (p=0.019). Discussion and conclusion Despite the short follow-up and the limited number of patients, this study suggests that the presence of additional mutations at the time of the diagnosis in PV and TE is correlated to a poorer disease evolution. The increase of mutation allele burden, which reflects clonal evolution, also seems to be associated with the course of the disease. These results argue for a clinical interest in large mutation screening by NGS at the time of the diagnosis and during follow-up in ET and PV. Disclosures Ugo: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: ASH travel.

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Similar Rate of Thrombosis in Essential Thrombocythemia and Polycythemia Vera Patients after Stratification for JAK2 V617F Allele Burden.

Blood ◽

10.1182/blood.v112.11.1745.1745 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1745-1745

Author(s):

Alessandra Carobbio ◽

Guido Finazzi ◽

Elisabetta Antonioli ◽

Paola Guglielmelli ◽

Alessandro M. Vannucchi ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Vascular Complications ◽

Jak2 V617f ◽

Similar Rate ◽

Wild Type ◽

Allele Burden ◽

Real Time Quantitative Pcr ◽

Venous Thromboembolic

Abstract Patients with Essential Thrombocythemia (ET) can be categorized as either JAK2 V617F mutated (V617F+) or wild type (V617F−). Mutated patients display multiple features resembling Polycythemia Vera (PV), with significantly higher hemoglobin level and neutrophil counts, lower platelet count, more pronounced bone marrow erythropoiesis and granulopoiesis and higher tendency to transform in PV. Presence of the mutation and/or allele burden has been variably associated with the rate of vascular complications in ET and PV, but a direct comparison between the two disorders under this respect has not been performed. To tackle this issue, we compared the rate of major thrombosis in 867 ET patients (57% were JAK2 V617F+) with that in 415 PV patients (all V617F+). The median follow-up was 4.9 (0 – 39) and 3.8 (0 – 26) years in ET and PV, respectively. High risk ET patients (age ≥ 60 years and/or previous thrombosis) received Hydroxyurea whereas the vast majority of low-risk remained untreated. PV patients were treated according to the current risk-stratified recommendations. Thrombotic episodes were recorded over time and calculated as rates % per patient/year (pt/yr). After adjusting for age, the thrombosis-free survival curves of JAK2 V617F+ and V617F− ET patients were superimposable until 10 years after the diagnosis, then they diverged so that the actuarial probability of major thrombosis in mutated ET patients reached that of PV (48% vs 55%, test for trend p=0.05). We found that JAK2 V617F+ allele burden measured by real-time quantitative PCR influenced these rates in a comparable way in both ET and PV. Actually, in JAK2 wild type ET (n=376, 43%) the rate was 1.4% pt/yr. In ET patients with JAK2 V617F+ allele burden ranging from 1 to 25% (N=190; 49%) the rate was 1.9 % pt/yr compared to 1.2 in PV patients (N=64, 19%); in the group with 26–50% the rate was 2.0 % pt/yr in ET (N=177; 45%) and 3.0 in PV patients (N=118, 36%); in cases of V617F+ allele burden greater than 50% the rate was 3.8 % pt/yr in ET (N=23; 6%) and 2.9 in PV patients (N=147, 45%). In conclusion, from this retrospective analysis, we conclude that in patients with ET harboring JAK2 V617F mutation the rate of stroke, myocardial infarction and venous thromboembolic complications is similar to that of PV patients and increases in dependence of V617F allele burden, supporting the hypothesis that ET and PV may be viewed as a continuum also in terms of vascular complications

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JAK2 (V617F)-Positive Essential Thrombocythemia and Polycythemia Vera Are Different Expressions Of a Genotypic/Phenotypic Continuum

Blood ◽

10.1182/blood.v122.21.1592.1592 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1592-1592

Author(s):

Elisa Rumi ◽

Daniela Pietra ◽

Chiara Elena ◽

Ilaria Casetti ◽

Emanuela Sant 'Antonio ◽

...

Keyword(s):

Polycythemia Vera ◽

Median Time ◽

Essential Thrombocythemia ◽

Cumulative Incidence ◽

Mutant Allele ◽

Clinical Phenotype ◽

Jak2 V617f ◽

Wild Type ◽

Allele Burden ◽

Wbc Count

Abstract Background About 95% of patients with polycythemia vera (PV) and 60-70% of those with essential thrombocythemia (ET) carry the unique JAK2 (V617F) mutation. Previous observations suggest that JAK2 (V617F)-positive ET and PV form a biological continuum, in which the degree of erythrocytosis is determined by physiological and genetic factors. Aims In this work, we studied the natural history of JAK2 (V617F)-positive ET and PV with the aim of establishing whether the two disorders indeed represent different phenotypic expression of a genotypic/phenotypic continuum. Methods We identified 1269 patients diagnosed with ET or PV at our Division between 1980 and 2012, for whom at least one DNA sample was available. The JAK2 (V617F) mutation was assessed using allele-specific quantitative PCR. As patients carrying JAK2 exon 12 or MPL mutations were excluded, the final study population included 1214 patients, 719 of whom with ET (463 JAK2 mutated, 256 JAK2 wild-type) and 495 with PV. Results I: presenting features The clinical phenotype of ET patients at diagnosis differed according to JAK2 mutational status. JAK2 mutated ET presented with older age at diagnosis, higher hemoglobin (Hb) level and white blood cell (WBC) count, lower platelet (PLT) count and erythropoietin level compared to JAK2 wild-type ET (Wilcoxon rank-sum test: P<.001 in all comparisons). The median V617F allele burden was significantly lower in JAK2 mutated ET than in PV (18.4% vs 43.4%, P<.001). A mutant allele burden greater than 50% was observed in 2% of patients with JAK2 mutated ET and 41.5% of those with PV (Fisher exact test: P<.001). In both JAK2 mutated ET and PV, the mutant allele burden was directly related to WBC count and Hb level. Results II: PV evolution Evolution to PV was observed in 53 JAK2 mutated ET patients (incidence 95% CI: 1.4-2.4 per 100 p-years) vs none of the 256 JAK2 wild-type ET (incidence 95% CI: 0-0.2 per 100 p-years), resulting in a significantly different occurrence. The median time to PV evolution was 54 months (range 3.5-220). The cumulative incidence of PV evolution in JAK2 mutated ET patients at 15 years was 28.8% (95% CI: 20.7-37.3; Figure 1). PV evolution was significantly associated with higher JAK2 allele burden at diagnosis (Cox regression HR=1.04, P<.001). Based on the hypothesis that PV patients might have had a silent “pre-PV phase”, we did an ad hoc search for any complete blood count (CBC) collected before diagnosis. Among PV patients, 177 (36%) had a previous CBC, collected at a median time of 22 months (range 1-305) before PV diagnosis. A normal CBC was observed in 15% of patients; the remaining subjects showed thrombocytosis (≥450 x 109/L) and/or leukocytosis (≥10 x 109/L) and/or erythrocytosis. The median time to PV onset was significantly shorter in patients showing at least one CBC abnormality than in those with normal CBC (24 vs 48 months, P=.011). Results III: clinical course The median follow-up was 5.1 years (range, 0-32 years). JAK2-mutated ET and PV did not differ in terms of cumulative incidence of thrombosis (25.3% vs 33.7% at 15 years, P=.35; Figure 2A) and had similar overall survival (OS) (90.3% vs 82.6% at 15 years, P=.29; Figure 2B). Conversely, JAK2 wild-type ET showed a better OS in comparison with both JAK2 mutated ET (P=.028) and PV (P=.004) and a lower incidence of thrombosis (12.7% at 15 years) than JAK2 mutated ET (P=.002) and PV (P<.001). A similar cumulative incidence of disease progression (leukemia and myelofibrosis) was observed in JAK2 mutated (11.7% at 15 years) and JAK2 wild-type ET (12.1% at 15 years), whereas a higher cumulative incidence was observed in PV (26% at 15 years; P=.011 and P=.007 when compared with JAK2 mutated ET and JAK2 wild-type ET respectively). Conclusions This study supports the hypothesis that JAK2 mutated ET and PV are different expressions of a genotypic/phenotypic continuum, in which the mutant allele burden contributes to determine the clinical phenotype. The risk of progression from JAK2 mutated ET to PV is about 2% per year. This work was supported by grant #1005 from Associazione Italiana per la Ricerca sul Cancro (AIRC) “Special Program Molecular Clinical Oncology 5x1000” to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative - http:www-progettoagimm.it). Disclosures: No relevant conflicts of interest to declare.

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Mutational Profile of Patients with Polycythemia Vera Treated with Ruxolitinib in the Phase III Controlled Response Study

Blood ◽

10.1182/blood.v126.23.4087.4087 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4087-4087

Author(s):

Paola Guglielmelli ◽

Annalisa Pacilli ◽

Giada Rotunno ◽

Alessandro Pancrazzi ◽

Tiziana Fanelli ◽

...

Keyword(s):

Polycythemia Vera ◽

Board Of Directors ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Phase Iii ◽

Duration Of Treatment ◽

Advisory Committees ◽

Allele Burden ◽

Response Trial

Abstract Background. The JAK1/2 inhibitor ruxolitinib (RUX) demonstrated clinical benefit compared with best available therapy (BAT) in patients (pts) with polycythemia vera (PV) in a phase 3 study (RESPONSE TRIAL) (NEJM 2015; 372:426). A reduction of JAK2 V617F allele burden of 12.1% from baseline at week (w)32 was observed in pts receiving ruxolitinib compared to BAT (1.2%). Conversely, no information was available about other mutations that may occur in some PV pts. Aim. To analyze the molecular landscape of PV pts enrolled in the RESPONSE trial specifically as regarded subclonal mutations. Methods: In the RESPONSE study, PV pts with intolerance or refractoriness to hydroxyurea, showing an enlarged spleen volume (SV) >450 ml and phlebotomy requirement, were randomized (1:1) to receive RUX (n = 110) or BAT (n = 112). After institutional approval and informed written consent, samples for genotyping were available in 150 cases (67.5% of total, 75 each RUX and BAT). Mutations in 22 genes (JAK1, JAK2, JAK3, EZH2, ASXL1, TET2, IDH1, IDH2, CBL, SRSF2, DNMT3A, NFE2, SOCS1, SOCS2, SOCS3, SH2B3, STAT1, STAT3, STAT5A, STAT5B, SF3B1, U2AF1) were analyzed in blood DNA at baseline and at the latest available sample by deep sequencing with Ion Torrent-PGM. CALR mutations were analyzed by capillary electrophoresis. JAK2 V617F allele burden was confirmed by RT-qPCR assays. Results. 149/150 patients had informative sequencing results (RUX, n = 74 and BAT, n = 75). Mutation frequency at baseline in RUX pts was: JAK2 V617F 97.3% (mean allele burden=83.7±20.0%); JAK2Exon12 1.3%; other mutations in JAK2 5.4%; JAK3 1.3%; ASXL1 4.0%; TET2 20.3%; EZH2 4.0%; CBL 1.3%; SH2B3 2.0%; SOCS1 2.7%; NFE2 2.7%; STAT5A 1.3%. No somatic variants were detected in CALR, JAK1, SRSF2, IDH1, IDH2, SOCS2, SOCS3, DNMT3, STAT1, STAT5B, SF3B1, U2AF1. These frequencies were comparable in BAT arm. One patient was un-mutated in all assessed genes. 28.4% and 8.1% of RUX and 32.0% and 8.0% in BAT pts had 1 and >2 subclonal mutations, respectively. The proportion of PV pts harboring at least 1 mutation in either ASXL1, EZH2, SRSF2, IDH1/2, was significantly lower (8.1% in RUX and 10.6% in BAT) compared to reference series of primary myelofibrosis pts (31%) as it was for those having >2 HMR mutations (0.0 in RESPONSE vs 7.4% in PMF) (Leukemia 2014;28:1804) The median duration of treatment corresponding to the latest available sample for analysis was 82.8w; at that time, 43 pts (58.1%) randomized to RUX achieved a JAK2 V617F allele burden reduction ≥10%, of which 15 (20.3%) had >50% reduction. Among the latter pts, the median allele burden was 83.7% at baseline, 84.9%, 55.1% and 44.3% at 1, 2, and 3 years. Three patients attained an allele burden below 5% (from 65.1%, 17.3% and 83.7% at baseline to 3.2%, 0.5% and 1.4%, respectively, at latest follow up). Of the 27 pts harboring subclonal mutations at baseline, 12 (44.4%) presented a reduction of mutational allele burden ≥10%: 4 in JAK2 (other than JAK2 V617F/exon 12 mutations), 4 in TET2, 3 in ASXL1 and 1 each in JAK3, EZH2 and SH2B3. In 10 of the 12 pts, comparable decreases in JAK2 V617F allele burden were observed, suggesting reduction of a single clone expressing both mutations. Conversely, in 5 pts (18.5%) the allele burden of a baseline TET2 clone at increased by ≥10% (range: 10-37%); of these, one had concurrent reduction of JAK2 V617F burden from 17.3 to 0.5, thereby suggesting two independent clones. Eight pts (29.6%) acquired new mutations: 3 in TET2, 3 in U2AF1, 1 in DNMT3A and 1 in IDH1. Among these, 4 pts had achieved a reduction ≥10% of JAK2 V617F allele burden (18.4%, 25.4%, 30.5% and 39.4%, respectively). Three pts (4.0%) progressed during treatment (2 myelofibrosis,1 acute leukemia); no novel acquired mutation in the 22 genes was observed in these pts. All 3 pts were homozygous for V617F (92.3%, 72.7% and 59.0%) and did not show appreciable changes of allele burden during treatment. Conclusions. The current study identifies mutations and mutational combinations at baseline and during follow up in a representative cohort of pts enrolled in RESPONSE trial and treated with Ruxolitinib. We observed progressive reduction of JAK2 V617F allele burden that in some cases was associated with concurrent reduction of subclonal mutations. Conversely, emergency of novel clones as observed in some pts, whose significance might be clarified by ongoing analysis of hydroxyurea and phlebotomy treated patients that will be presented at the meeting. Disclosures Mahtab: Novartis Pharma AG: Employment. Rodriguez:Novartis Pharma: Employment. Vannucchi:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

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Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model

Blood ◽

10.1182/blood-2012-03-415646 ◽

2013 ◽

Vol 121 (7) ◽

pp. 1188-1199 ◽

Cited By ~ 30

Author(s):

Lucia Kubovcakova ◽

Pontus Lundberg ◽

Jean Grisouard ◽

Hui Hao-Shen ◽

Vincent Romanet ◽

...

Keyword(s):

Mouse Model ◽

Competitive Advantage ◽

Polycythemia Vera ◽

Mutant Allele ◽

Jak2 V617f ◽

Blood Parameters ◽

Therapeutic Agents ◽

Wild Type ◽

Allele Burden ◽

Key Points

Key Points JAK2-V617F cells show a competitive advantage over wild-type cells in BM transplantation assays. A preclinical mouse model allows the examination of the effects of therapeutic agents on blood parameters and JAK2-V617F mutant allele burden.

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Age and Response after Autologous Stem Cell Transplantation (ASCT) Define a Group of Long-Term Event-Free Survivors among Patients (Pts) with Low-Grade Follicular Lymphoma (FL): A Single Institution Experience.

Blood ◽

10.1182/blood.v104.11.1883.1883 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1883-1883

Author(s):

Charalambos Andreadis ◽

Elise A. Chong ◽

Edward A. Stadtmauer ◽

Selina M. Luger ◽

David L. Porter ◽

...

Keyword(s):

Multivariable Analysis ◽

Disease Free Survival ◽

Autologous Bone Marrow ◽

Large Cell ◽

High Dose ◽

Low Grade ◽

Free Survival ◽

Disease Free

Abstract Introduction: FL is generally responsive to conventional-dose chemotherapy but long term disease-free survival (DFS) is uncommon. High-dose chemo-radiotherapy followed by ASCT has the potential to induce remission in this disease but the long-term benefit of this modality remains to be determined. Methods: Between 1990 and 2003, we transplanted 52 pts originally diagnosed with low-grade FL (31 grade 1, 21 grade 2). Twenty-five (48%) had biopsy-proven large cell transformation (FL grade 3 or diffuse large cell lymphoma) before ASCT. The median number of prior therapies was 2 (range: 1 to 7). Prior to ASCT, 45 pts (87%) were responsive to salvage therapy with 20 pts (38%) in CR. Five pts (10%) had chemo-resistant disease at the time of ASCT. High-dose regimens included BCNU-cyclophosphamide-etoposide (31%), melphalan/TBI (27%), and cyclophosphamide/TBI (25%). Thirty-eight pts (73%) received peripheral stem cells (PSCT) and 14 pts (27%) received autologous bone marrow (BM) with 4-hydroxyperoxycyclophosphamide (4-hc) purging in 9 cases (17%). The median age was 49 yrs (range: 29–65). Results: There was 1 treatment-related death during the first 100 days. After ASCT, 36 pts (69%) achieved a CR, 2 (4%) had a PR, and 7 (13%) had stable disease. Among those in CR, 20 (56%) had a CR pre-ASCT, 14 (41%) had a lesser response, and 1 (3%) was chemo-resistant. Median follow-up (f/u) of survivors was 5.3 yrs (range: 1.7 months to 12.4 yrs). The median overall survival (OS) has not yet been reached. The median event-free survival (EFS) is 3.4 yrs (range: 1.7 months to 12.4 yrs). Among complete responders, more than 50% are disease free at last follow-up (range 1.7 months to 12.1 yrs). Variables favorably affecting EFS and OS are age < 60 yrs (p = 0.007, 0.015 respectively), achievement of a CR after ASCT (p = 0.002, 0.001), absence of transformation (p = 0.038, 0.017), BM vs. PSCT (p = 0.042, 0.086), and 4-hc BM purging (p = 0.044, 0.059). Number of prior regimens, response prior to ASCT, type of preparative regimen, and addition of TBI, were not significantly associated with EFS, DFS, or OS. In multivariable analysis, achievement of CR after ASCT and age < 60 yrs are the only significant predictors of EFS and OS. Adjusted for age, 53% of pts with a CR after ASCT are alive and event-free at last f/u (range: 2.4 months to 12.4 yrs) (Figure 1). In contrast, the median EFS among pts without a CR is 0.5 yrs (range: 1.7 months to 5.3 yrs). Conclusion: ASCT is a reasonable therapeutic approach to FL, resulting in long term EFS for some pts, even with relapsed, refractory and/or transformed disease. In our experience, significant predictors of EFS and OS after ASCT are complete response and age <60. The appropriate application and timing of ASCT in the management of pts with FL needs to be further evaluated in randomized, controlled clinical trials. Figure Figure

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Influence of the Jak2V617F Mutational Load at Diagnosis on Major Clinical Aspects in Patients with Polycythemia Vera.

Blood ◽

10.1182/blood.v108.11.5.5 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5-5 ◽

Cited By ~ 4

Author(s):

Alessandro M. Vannucchi ◽

Elisabetta Antoniolim ◽

Paola Guglielmelli ◽

Alessandro Pancrazzi ◽

Costanza Bogani ◽

...

Keyword(s):

Platelet Count ◽

Polycythemia Vera ◽

Mutant Allele ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Clinical Aspects ◽

Inverse Regression ◽

Vascular Events ◽

Allele Burden ◽

Tyrosine Kinase 2

Abstract Most patients with polycythemia vera (PV) carry a mutation in the Janus tyrosine kinase 2 gene (JAK2V617F); in 20–30% of them, mitotic recombination leads to homozygosity. It has been suggested that phenotype may be in part dependent on the mutant allele burden and the residual amount of wild-type (wt) allele, since wt JAK2 exherted a dominant negative effect during co-transfection experiments. However, simply differentiating heterozygotes from homozygotes may be misleading, since it does not distinguish between true heterozygosity or homozygosity in a mixed normal background, nor does it provide any measure of the relative contribution of the two alleles; consistently, homozygous progenitors have been found almost invariably in PV patients. The aim of this study was to assess the influence of JAK2V617F mutant allele burden on hematologic and clinical parameters in 116 PV patients diagnosed according to the WHO criteria, for whom a blood sample, collected at, or within six months from, the diagnosis was available. To measure the ratio between mutated and wt JAK2, we employed an ARMS-PCR procedure on RNA, purified from ≥95% pure preparations of PB granulocytes; mutation-specific amplicons were resolved and quantitated using capillary electrophoresis. Detectable amount of JAK2V617F RNA in the granulocytes was found in 96 patients (83%), with a median value of JAK2V617F/JAK2WT ratio of 38% (range, 1 to 100%). JAK2V617F patients were divided into four classes based on the relative amount of mutant allele: 32 patients (28%) had JAK2V617F/JAK2WT ratio of 1–25%, 24 (21%) of 26–50%, 17 (16%) of 51–75%, and 23 (20%) had 76–100% ratio. There was no difference among these classes as concerned the relative frequency of patients, to indicate that the extent of mutant allele at diagnosis is heterogeneous and that acquisition of the highest allele burden is not necessarily a time-dependent event. The hematocrit, leukocyte count, LDH and ALP values were directly related to the relative amount of JAK2V617F RNA ( P<0.0001 for all), while the MCV progressively decreased with increasing JAK2V617F/JAK2WT ratio (P<0.001). There was also a trend to a lower platelet count with the highest JAK2V617F/JAK2WT ratios (P=0.067). The frequency of patients who had PRV-1 over-expression was 8% among JAK2WT, and rose to 53%, 77%, 100% and 100% in the four ratio classes, respectively (P<0.0001); there was also a significant inverse regression between PRV-1 CT levels and the JAK2V617F/JAK2WT ratio (r=−0.596, P<0.0001). Furthermore, the highest JAK2V617F/JAK2WT ratios at diagnosis pointed to patients more likely to have splenomegaly (relative risk (RR) 5.0 to 7.82) or presenting pruritus (RR 1.25 to 5.21) (P<0.0001 for both), and predicted for an exceedingly higher RR of requiring chemotherapy for the control of disease in the follow-up, that ranged from 10.6 to 15.6 in the different ratio classes (P<0.0001). The RR of presenting major thromboses was progressively higher up to 4-times in the 76–100% ratio class compared to JAK2WT patients. A multivariate analysis, including age, leukocytosis, hematocrit, platelet count, and treatment options, indicated that the JAK2V617F/JAK2WT ratio behaved as an independent risk factor for major vascular events (P= 0.027). These data support a meaningful correlation between the proportion of mutant JAK2 allele and the propensity to a more symptomatic disease in PV patients, and foresee the quantitation of JAK2 mutant allele as an approach for patient risk stratification.

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JAK2 Mutations at Exon 12 and 14 in Polycythemia Vera and Idiopathic Erythrocytosis: Incidence and Correlation with Clinical Characteristics.

Blood ◽

10.1182/blood.v110.11.2532.2532 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2532-2532

Author(s):

Carlos Besses ◽

Luz Martínez-Avilés ◽

Alberto Alvarez-Larrán ◽

Francisco Cervantes ◽

Juan Carlos Hernández-Boluda ◽

...

Keyword(s):

Polycythemia Vera ◽

Cell Sorting ◽

Clinical Characteristics ◽

Clinical Phenotype ◽

Cell Lineage ◽

Jak2 V617f ◽

Free Survival ◽

Mutational Status ◽

Cell Subpopulations ◽

Idiopathic Erythrocytosis

Abstract Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome. Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data. Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells. Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation. Table 1: JAK2 exon 12 and exon 14 mutations in blood cell subpopulations. Mutation Granulocytes Platelets CD14+ CD3+ CD19+ CD56+ * exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined Patient 1* K539L (AAA →TTA) + + − − − − Patient 2* K539L (AAA →CTA) + + + − − − Patient 3* H538_K539delinsL + + ND − − ND Patient 4* N542_E543del + + + − − − Patient 5# V617F (GTC →TTT) + + − − − ND C618R (TGT →CGT) + + − − − ND Patient 6# V617F (GTC →TTC) + + + − − ND C616C (TGT →TGC) + + + − − ND

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Identification of Homozygous JAK2V617F Mutation in Unfractioned Blood Samples in a Substantial Proportion of Myeloproliferative Disorders: Long Term Follow-Up Study.

Blood ◽

10.1182/blood.v110.11.4644.4644 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4644-4644

Author(s):

Irina Panovska ◽

Nadica Matevska ◽

Martin Ivanovski ◽

Sanja Trajkova ◽

Aleksandar Stojanovik ◽

...

Keyword(s):

Jak2 V617f ◽

Jak2v617f Mutation ◽

Study Group ◽

Hematopoietic Progenitors ◽

Blood Samples ◽

Allele Burden ◽

Long Term Follow Up ◽

Thrombotic Complications

Abstract The discovery of the activating V617F mutation in the JAK2 tyrosine kinase gene in patients with myloproliferative disorders (MPD) provides a major breakthrough in the understanding of the pathogenesis, proving clonality and securing diagnosis of these diseases. The JAK2 V617F allele is an acquired somatic disease allele that arises in hematopoietic progenitors and confers a selective growth advantage. The mutation has been traced to a primitive stem cell that is capable of erythroid and myeloid differentiation. The data for the potential involvement of the B and T lymphocytes with the JAK2V617F mutation are still inconsistent. We present the results from the study designed the evaluate the prevalence of the JAK2V617F mutation in MPDs patients in our population and to investigate whether MPD patients that carry the JAK2V617F mutation differ in clinical course and outcome from JAK2V617F negative MPD patients. The study group consisted of 64 living MPD patients diagnosed according to standard WHO criteria for diagnosis of MPD (26 patients were diagnosed as polycythemia vera (PV), 34 as essential thrombocythemia (ET), 6 as idiopatic myelofibrosis (MF) and 8 were classified as atypical MPD) with the median follow-up of 7,4 years. DNA samples were obtained from unfractionated blood samples and the frequency of V617F JAK2 mutation was analyzed by allele-specific PCR assay. The mutant allele burden in mutation positive samples was analyzed by DNA sequencing. Our results showed that the JAK2 V617F mutation was present in 79% of patients with PV (36% were homozygous for the mutation), 58% with ET (11% homozygous), 69% with MF (28% homozygous) and in 33% of patients with atypical MPD. The mutant JAK2V617F allele burden was greater than 95% in two PV patients, which in the presence of 27% lymphocytes in the peripheral blood of the patients indicate lymphocytes involvement with the mutation. The high frequency of observed homozygosity for JAK2V617F in our study group was probably due to the long disease duration (median follow-up 11,4 years) and favors the theory of a time dependent increase in clonal dominance. Correlations of clinical and laboratory features at diagnosis and subsequent follow-up, including incidence of thrombo-hemorrhagic events, disease transformation and survival of JAK2V617F-positive and JAK2V617F-negative patients did not reveal significant differences except for the incidence of thrombotic complications. The JAK2V617F positive group had a higher incidence of thrombotic complications (30%) compared with the JAK2-V617F-negative group (14%, P< 0.05). Although we observed a disparity in the incidence of the JAK2V617F mutation in different MPD entities in our population with respect to the expected frequency of JAK2V617F from the literature, our results confirm the diagnostic significance of the JAK2V617F mutation in MPDs and support the notion that patients with this mutation should be classified in a new entity of MPDs. Identification of homozygous JAK2V617F mutation in unfractionated blood samples in a substantial proportion of long term follow-up MPD patients, together with the identification of the mutant JAK2V617F allele burden greater than 95% in two PV patients, suggests that acquisition of the JAK2V617F mutation arises in early multilineage hematopoietic progenitors and warrants further investigation.

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Polycythemia Vera as a Predisposing Factor for Aortic Stenosis: Prevalence and Correlation with Blood Cells Count and Mutational Status

Blood ◽

10.1182/blood.v112.11.5247.5247 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5247-5247

Author(s):

Ri ta Barone ◽

Clementina Caracciolo ◽

Rosario di Maggio ◽

Giovanni Fazio ◽

Luciana d’Angelo ◽

...

Keyword(s):

Relative Risk ◽

Aortic Stenosis ◽

Blood Cell ◽

Polycythemia Vera ◽

Blood Cells ◽

Jak2 V617f ◽

Laboratory Findings ◽

Mutational Status ◽

Adhesive Molecules

Abstract The association between Polycythemia Vera (PV) and thrombosis is multi-factorial involving the complex interaction between activated leukocytes, platelets and endothelium. Recent reports have postulated that PV patients may over express adhesive molecules on red cell surface, likely by JAK2 mutation (Wautier M et al. Blood.2007;110(3):894–901). This process activates endothelium with production of vascular growth factors and other mechanisms leading to atherosclerosis. Aortic Stenosis (AS) is the commonest valvular heart disease in western countries; its pathogenesis is mainly related to a degenerative process sharing many characteristics with atherosclerosis. At the present is not known whether patients with PV are at high risk of developing AS. Objective of the study. We perform a case-control study for evaluating rate of AS and its correlation with blood cells count and mutational status in patients with PV. Materials and methods. Prevalence of AS among PV patients have been compared with control patients matched for age, cardiovascular risk factors (hypertension, hyperlipidemia, diabetes, smoke and alcohol abuse) and coexisting cardiac diseases (i.e. heart failure). Diagnosis of PV has been posted accordingly to PVSG criteria. Diagnosis and severity of AS has been posted by echocardiography: stenosis with a valve area <1.0 cm2 has been considered severe. Results. Over a period of 18 months we recruited 43 PV patients (28 males and 15 females) and 74 controls. No differences were found in regard of the above cited characteristics; median age was 66.7 among PV patients and 68.2 among controls. The average duration of PV was 5.7 years with an average follow-up of 2.5 years. Most of the PV patients were on antiplatelet/anticoagulant therapy (27/43, 62.7%) and have been treated with cytoreductive therapy. Twelve (27.9%) had a thrombotic event before PV diagnosis; 4 (9.3%) developed thrombosis during the follow-up (median 1.3 years). A moderate/severe AS was found in 11 PV patients (25.6%) in comparison to 4 (5.4%) in control group (P= 0.023), thus giving a Relative Risk of 4.7. Among PV patients, the multivariate analysis did not show any correlation regarding JAK2 V617F mutational status, duration of disease, previous thrombosis, cytoreductive therapy and other common cardiovascular factors. Comparison of laboratory findings is reported in Table 1; a not significant trend was demonstrated in favor of patients with elevated hematocrit (>55%). Conclusions. Our study clearly shows that PV patients carry a fourfold risk of developing AS, without a clear association with blood cell alterations or previous thrombosis. Whether high prevalence of AS may be related to expression of adhesive molecules on red cells or altered share stress is currently under investigation. Table 1. Comparison of laboratory findings between PV patients with and without AS Laboratory parameter* PV patients with AS (11) PV patients without AS (32) Relative Risk P value *At diagnosis Legend: PV (Polycythemia Vera), AS (Aortic Stenosis) White blood cell×109/L (mean± SD) 9520 ± 1230 12.900 ± 2120 0.73 .078 Hemoglobin, g/dL (mean ± SD) 17.5 ± 1.3 17.1 ± 1.2 1.02 .088 Hematocrit, % (mean ± SD) 56.2 ± 0.6 51.1 ± 0.8 1.1 0.06 Platelets × 109/L(mean ± SD) 415.9 ± 43 353 ± 55 1.1 0.76 JAK2 V617F, n/N (%) 11/11 (100%) 31/32 (97%) - -

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