JAK2V617F Mutational Status Research Articles

Thrombosis in myelofibrosis: prior thrombosis is the only predictive factor and most venous events are provoked

In a retrospective analysis of 205 patients (median age 62 years) with primary myelofibrosis and known JAK2V617F mutational status, 13.2% experienced a vaso-occlusive event at or prior to their diagnosis. After a median follow up of 31 months, post-diagnosis thrombosis occurred in 22 patients (10.7%), including 9 (4.4%) and 16 (7.8%) patients with a total of 9 arterial and 24 venous events, respectively. The majority (71%) of the venous events were temporally associated with other exogenous risk factors for thrombosis such as surgery, line placement or hormonal therapy. On multivariable analysis that included age, JAK2V617F mutation status and leukocyte count as covariates, history of thrombosis was the only predictive variable in general (P=0.04) or when arterial (P=0.007) and venous (P=0.02) thromboses were analyzed separately. The current study demonstrates a higher prevalence of venous, as opposed to arterial, events in PMF, post-diagnosis, and clarifies their nature as being mostly provoked.

Dynamics of telomere's length and telomerase activity in Philadelphia chromosome negative myeloproliferative neoplasms

Telomere exhaustion and increased telomerase activity are associated with the acquisition of aggressive molecular events in a variety of haematological malignancies. In Philadelphia chromosome negative myeloproliferative neoplasms (Ph negMPN's), telomere dynamics during clonal evolution of these diseases have not yet been fully elucidated. Herein we demonstrated that telomere shortening is a global phenomenon in Ph negMPN's, irrespective of disease phenotype, treatment administration and JAK2V617F mutational status but the presence of additional cytogenetic abnormalities further affects them. Consistent with the above finding, TA was upregulated in CD34+ haemopoietic progenitors from almost all Ph negMPN subgroups compared to healthy donors. Moreover, TL below the cut-off value of 27% could predict disease progression in Ph negMPN patients (PFS at 5 years 39% vs 81%). Thus, TL emerges as a new prognostic marker in Ph negMPN, reflecting probably the genetic instability of highly proliferating MPN clones.

Increased circulating procoagulant activity and thrombin generation in patients with myeloproliferative neoplasms

Microparticles (MPs) are membrane fragments ranging in size from 0.1 to 1 μm, and are considered as biomarkers reflecting prothrombotic state in many clinical diseases. The clinical course of myeloproliferative neoplasms (MPN) being frequently complicated by thrombotic events, we determined the MPs activity, i.e. circulating procoagulant activity (CPA), in polycythemia vera (PV) and essential thrombocythemia (ET) patients. To evaluate the influence of MPs on the coagulation, a thrombin generation test was realized in the absence and presence of thrombomodulin (TM). Compared with controls, patients had a higher CPA (24.0 ± 9.0 vs 10.6 ± 4.4 nM, p < 0.001), which was associated with a lower inhibition of the thrombin generation in the presence of TM (20.1 ± 9.5% vs 28.4 ± 11.8%, p < 0.001), compatible with a low sensitivity to TM. This sensitivity was influenced by the JAK2V617F mutational status, homozygous patients presenting the lowest inhibition rate of the thrombin generation. Filtration through a 0.22 μm membrane increased the sensitivity to TM in plasma, suggesting an influence of MPs in the “TM-resistance” observed in patients. Moreover, MPN patients receiving antiplatelet and/or cytoreductive therapies, our study suggests that CPA might be influenced by cytoreductive therapy. In conclusion, our data evidence in MPN patients the occurrence of an acquired “TM-resistance” partly determined by circulating microparticles. This TM-resistance might contribute to the hypercoagulable state observed in MPN patients, but the predictive value of the “TM-resistance” for thrombosis had not been evaluated.

Plasma levels of angiogenic factors and circulating endothelial cells in essential thrombocythemia: correlation with cytoreductive therapy and JAK2–V617F mutational status

Recent studies have shown that angiogenesis plays an important role in the biology of hematological malignancies including essential thrombocythemia (ET). Using cytofluorimetric analysis, the levels of angiogenic factors, as well as the number of circulating endothelial cells (CECs), were determined in 65 patients with ET, including 33 previously untreated and 32 receiving cytoreductive therapy. Correlations between markers of angiogenesis and JAK2–V617F mutational status were also assessed. We found significantly higher levels of vascular endothelial growth factor (VEGF) and markedly decreased levels of placental growth factor in untreated patients with ET with respect to control subjects. VEGF levels were significantly increased in patients with white blood count >8.7 (× 109/L) vs. <8.7 (× 109/L). Furthermore, the levels of VEGF in patients on hydroxyurea (HU) therapy were markedly lower than in untreated patients. It was also demonstrated that the number of all CEC subpopulations (resting, activated, apoptotic, and circulating precursor endothelial cells) was increased in patients with ET in relation to controls, regardless of the JAK2–V617F status, and was not affected by cytoreductive treatment. In conclusion, our study highlights the possible role of angiogenesis in the pathophysiology of ET. It provides evidence that the number of CECs is elevated independently of JAK2–V617F status and is not down-regulated by HU or anagrelide therapy. Our data suggest that VEGF levels are particularly elevated in patients with high leukocytosis. Further investigation should be undertaken to determine the possible role of antiangiogenic therapy in ET.

Chromosome 9p24 abnormalities: prevalence, description of novel JAK2 translocations, JAK2V617F mutation analysis and clinicopathologic correlates

Current information is insufficient regarding the prevalence, cytogenetic details, JAK2 involvement, JAK2V617F mutational status, and clinicopathologic correlates of chromosome 9p24 abnormalities. The current study was designed to provide additional data in this regard. The Mayo Clinic cytogenetics database spanning the years 1989-2008 was screened for chromosome 9p24 abnormalities. Bone marrow (BM) morphology was re-reviewed and relevant clinical information retrospectively obtained. Structural abnormalities of JAK2 were examined by FISH and JAK2V617F mutation analysis. Among 24 262 unique patient cytogenetic studies, chromosome 9p24 abnormalities were identified in 25 patients ( approximately 0.06%): 12 with myeloid and 13 with lymphoid neoplasms. The associated karyotype was complex in 12 of the 13 lymphoid but in only four of the 12 myeloid cases. Archived BM was available in 23 cases that allowed additional FISH studies and JAK2V617F mutation analysis. FISH analysis disclosed JAK2 involvement in 10 patients including five myeloid and five lymphoid cases; the associated karyotype was complex in all the lymphoid but in none of the myeloid cases. Five patients displayed previously undescribed JAK2 translocations: t(8;9)(q22;p24), t(9;17)(p24;q23), t(4;9)(q25;p24), t(2;9)(p21;p24) and t(8;9)(q13;p24). JAK2-associated chromosomal translocations, in a non-complex karyotype setting, were documented in four patients, all of whom carried a diagnosis of myeloproliferative neoplasms (MPN) and harbored JAK2V617F. Two patients with diffuse large B cell lymphoma displayed complex karyotype and JAK2 amplification by FISH. Chromosome 9p24 abnormalities are rare and do not always involve JAK2. The subset with JAK2 translocations are usually associated with MPN and harbor JAK2V617F, suggesting a cause-effect relationship. The current study also describes five novel translocations involving JAK2.

Circulating endothelial cells in essential thrombocythemia and polycythemia vera: correlation with JAK2-V617F mutational status, angiogenic factors and coagulation activation markers

Angiogenesis plays an important role in the biology of hematological malignancies, including essential thrombocythemia (ET) and polycythemia vera (PV). Some data suggests that it has a role in the pathogenesis of thrombosis, the major clinical problem in ET and PV. The number of different subpopulations of circulating endothelial cells (CECs), plasma levels of vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 1 and 2 (sVEGFR-1,2) and placenta growth factor (PlGF) were determined in 30 patients with ET and 16 patients with PV. Correlations between angiogenesis and JAK2-V617F mutational status, risk factors for thrombosis and coagulation activation markers were also assessed. The number of CEC subpopulations, were markedly higher in ET and PV patients, irrespective of JAK2-V617F status, when compared to the control group. The median values of activated CECs were markedly higher in PV patients with WBC >8.7 (x10(9)/l). Significantly higher VEGF plasma levels were found in ET patients and a similar trend was seen in PV patients in relation to healthy volunteers. The plasma levels of sVEGFR-1 were significantly higher, and PlGF levels markedly lower, in the ET and PV group than in controls. Our study also demonstrated markedly increased levels of D-dimer and TAT complexes in the patient groups. In conclusion, we found that angiogenesis, as measured by CEC numbers, is increased in ET and PV patients regardless of JAK2-V617F mutational status. Our results demonstrated that angiogenic cytokines interact with known thrombotic risk factors. We confirmed the coagulation activation in ET and PV patients but found no differences in levels of coagulation activation markers in relation to JAK2-V617F mutational status.

The G allele of the JAK2 rs10974944 SNP, part of JAK2 46/1 haplotype, is strongly associated with JAK2 V617F-positive myeloproliferative neoplasms

Polycythemia vera, essential thrombocythemia, and primary myelofibrosis are myeloproliferative neoplasms, characterized in a majority of cases by a unique somatic point mutation, JAK2 V617F. Recently, it was shown that JAK2 V617F occurs more frequently on a specific JAK2 haplotype, named JAK2 46/1. We genotyped 149 myeloproliferative neoplasms patients (69 had polycythemia vera, 65 had essential thrombocythemia, and 15 had primary myelofibrosis) with a known JAK2 V617F mutational status and 150 controls for the JAK2 rs10974944 (C/G) single nucleotide polymorphism, in which the G allele tags the 46/1 haplotype. We found that the rs10974944 GG/CG genotypes were significantly enriched in patients compared to controls (p < 0.0001). After stratifying for the JAK2 V617F mutational status and for the mutant allele burden, we demonstrated that GG/CG genotypes were significantly more frequent in V617F positive compared to V617F negative patients (p = 0.001), but not in V617F negative patients compared to controls (p = 0.29). Similarly, the GG/CG genotypes were significantly enriched in V617F positive patients with a mutant allele burden >50% compared to those with a mutant allele burden <50% (p = 0.0006). Our results indicate that the G allele, part of the JAK2 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive myeloproliferative neoplasms. Moreover, JAK2 46/1 seems to be associated with mutant allele burden >50% in JAK2 V617F-positive myeloproliferative neoplasms patients.

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.

Abstract 2895Poster 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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

JAK2 Haplotype SNP Analysis in Essential Thrombocythemia: Clinical and JAK2V617F Allele Burden Correlates in 226 Patients.

Abstract 2893Poster Board II-869 Background:A specific JAK2 germline haplotype has been associated with the occurrence of JAK2V617F in myeloproliferative neoplasms (MPN). Several SNPs, including rs12343867, tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with essential thrombocythemia (ET) in order to study the clinical and JAK2V617F allele burden correlates of the particular SNP genotype. Methods:Study patients were recruited form the Mayo Clinic database for ET. 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:226 patients with ET were studied (median age 57, range 14-91; 145 females). Median hemoglobin, leukocyte and platelet counts at initial diagnosis were 13.8 g/dL, 9.7 × 109/L and 1000 × 109/L, respectively. 118 patients (52%) were JAK2V617F positive and among them, 90 (76%) displayed JAK2V617F allele burden of < 10% (low allele burden group) and the median (range) mutant allele burden in the remaining 28 patients (higher allele burden group) was 12% (12-68). As expected, JAK2V617F-positive cases, compared to their mutation-negative counterparts, were older (p=0.006) and displayed higher hemoglobin (p=0.0001) and leukocyte (p<0.0001) counts and lower platelet (0.03) count.The rs12343867 genotype distributions were C/C 15%, C/T 53% and T/T 33% (expected frequency in the HapMap-CEU listed control population is ∼ 6%, 46% and 48%, respectively). The corresponding figures in JAK2V617F positive/negative cases were 17%%/11%, 53%/53%, 30%/36% (p=0.29). Unlike the case with JAK2V617F mutational status, rs12343867 SNP genotype did not correlate with age (p=0.69), hemoglobin level (p=0.47), leukocyte count (p=0.25) or platelet count (p=0.33).Patients were followed for a median of 7.4 years and during this period 56 (25%) deaths were documented. rs12343867 SNP genotype did not correlate with survival (Figure). Similarly, survival did not correlate with the presence or absence of JAK2V617F. However, patients with higher JAK2V617F allele burden displayed a shorter survival with borderline significance, during multivariable analysis that included age as a covariate. Conclusion:The current study shows the JAK2 haplotype enrichment in a large series of ET patients. However, the particular phenomenon was not significantly different between JAK2V617F positive and negative cases, which suggests specific haplotype association with the disease rather than the mutation. Consistent with this contention, rs12343867 genotype did not correlate with clinical or laboratory variables that typically associate with JAK2V617F mutational status. Furthermore, unlike the case with primary myelofibrosis, rs12343867 SNP genotype did not correlate with survival, an observation that is not surprising considering the lower level of complexity in molecular pathogenesis in ET compared to PMF. [Display omitted] Disclosures:Pardanani:TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Tefferi:TargeGen: Research Funding.

The Extent in Telomere Attrition and the Number of Affected Leukocyte Subsets Distinguishes Patients with Different Myeloproliferative Neoplasms (MPN).

Abstract 1913Poster Board I-936 Background:Myeloproliferative neoplasms (MPN) are clonal disorders with an origin of the disease in a hematopoietic stem or progenitor cell. Except for chronic myeloid leukemia (CML), the diagnosis for Philadelphia-chromosome negative (Ph-neg.) MPN is less straightforward. Although many patients with a Ph-neg. MPN can be identified by mutations in JAK2 and/or TET2, the categorization into primary or secondary cythosis or specifically into polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) can be difficult. Since telomeres can be used to estimate the mitotic history of cells, our aims were to evaluate 1) whether the telomere length can be used to distinguish clonal from polyclonal hematopoiesis, 2) whether telomere attrition correlates to the mutational status of JAK2 and 3) whether the extent of telomere shortening in different subsets of leukocytes can point to the originating level of the hematopoietic stem or progenitor cell. Patients and Methods:So far, 32 patients with Ph-neg. MPN diagnosed according to WHO criteria (range: 32 – 85 years; PV: n=19, ET n=5, PMF n=8; JAK2V617F positive n=22, JAK2V617F negative n=16) and 11 patients with secondary erythrocythosis (range 39 – 61 years) were included in this study. 400 healthy individuals (range 0-102 years) served as controls. After informed consent peripheral blood was taken from the patients to measure the telomere length in subsets of leukocytes by automated multicolor flow-FISH. In order to correct for the age-dependent decline in telomere length, telomere length differences to the 50th percentile of the healthy cohort (DeltaTel) were calculated. Telomere length values below the 10th percentile of those from healthy donors were considered as substantially affected by telomere attrition. The mutational status of the JAK2V617F was assessed by allele-specific real time quantitative PCR. Results:The mean telomere length in granulocytes from patients with MPN was considerably shorter compared to healthy controls (mean ± STD DeltaTel: 2.73kb ± 1.20kb), whereas there was no remarkable difference in lymphocytes (0.70kb ± 0.81kb). Furthermore, we found significant DeltaTel between granulocytes from patients with MPN and with secondary erythrocythosis (mean ± STD: 2.73kb ± 1.20kb vs. 1.66kb ± 0.894kb, p<0.0001). Regarding the JAK2V617F mutational status we found no difference for the average DeltaTel (mean ± STD: 2.35kb ± 1.25kb (JAKV617F+), 2.28kb ± 1.12kb (JAKV617F-), p=0.84). Most interestingly, there was a clear difference in the average DeltaTel in granulocytes from patients with different types of MPN (ANOVA p=0.017). The most striking DeltaTel was seen between patients with PMF and patients with PV and ET (PMF: 3.72kb ± 0.30kb, PV and ET: 2.40kb ± 1.18kb, p=0.005). In addition, in patients with PMF most subsets of leukocytes demonstrated substantial telomere length differences compared to healthy controls (Granulocytes: 2.06kb ± 0.55kb, p=0.0001, T-cells: 0.84kb ± 1.07kb, p=0.016, B-cells: 1.62kb ± 1.29kb, p=0.0001, NK-cells: 0.95kb ± 1.49kb, p=0.039), whereas in patients with ET, except for the granulocyte subset, no significant DeltaTel values were detected (Granulocytes: 1.01kb ± 0.856kb, p=0.014, T-cells: -0.435kb ± 0.723, p=0.34, B-cells: 0.26kb ± 1.00kb, p=0.65, NK-cells: 0.30kb ± 0.86kb, p=0.58). In patients with PV one to three leukocyte subsets showed substantial DeltaTel, but in varying combinations. Conclusion:In our ongoing study we were able to confirm shorter telomeres in granulocytes of MPN patients compared to telomeres in granulocytes of healthy controls and of patients with secondary erythrocythosis. Based on the telomere length attrition patients with a clonal cythosis can be distinguished from such patients with a secondary cythosis, which could help diagnostically in uncertain cases. No correlation was found between the extent of telomere attrition and the JAK2V617F mutational status. The extremely short telomeres found in most subsets of leukocytes from patients with PMF could point to a very early hematopoietic stem cell as the cell of origin, whereas for ET and PV with only one or a few subsets of leukocytes affected by telomere attrition the cell of origin could be a hematopoietic stem cell at a somewhat later stage. Disclosures:No relevant conflicts of interest to declare.

Acute Panmyelosis with Myelofibrosis: Clinicopathological and Molecular Features of a Rare Malignant Bone Marrow Disease.

Abstract 3101Poster Board III-38 BackgroundAcute panmyelosis with myelofibrosis (APMF) is listed among the category of “AML, not otherwise specified” in the actual WHO classification of hematopoietic tumors. Since specific genetic alterations have not yet been defined, this myeloid neoplasm (MN) remains clinicopathologically assigned. The WHO classification addresses the difficulties concerning the differential diagnosis between APMF, myelodysplastic syndromes (MDS) with excess of blasts and concomitant fibrosis (RAEB-F) and acute megakaryoblastic leukaemia (AMGL). MethodsOur files were searched for patients with rapid-onset MN without a prior history of a myeloproliferative neoplasm or a MDS fulfilling the 2008 WHO criteria of APMF. An age- and gender-matched control group of RAEB-F cases with multilineage dysplasia was retrieved for comparison. Our approach included:i) the careful review of morphological, clinical and laboratory data with special regard to the proportion of immature CD34 + precursors/blasts in bone marrow (BM) biopsies; ii) the grading of BM fibrosis according to the European consensus guidelines; iii) the analysis of the JAK2-V617F, the MPL W515L and the GATA1 mutational status, the cytoplasmic localization of nucleophosphamin1 (NPM1) in BM blasts, and the expression the inhibitor of differentiation protein-2 (Id2) in hematopoietic and stroma cells; iv) the evaluation of the prognostic relevance of these parameters. ResultsFifty-four patients (32 males, 22 females) with a median age of 66, 9 years from our institutions were diagnosed APMF. Sufficient DNA for allele specific PCR for JAK2 genotyping was obtained from paraffin-embedded EDTA-decalcified BMB specimens from 22 APMF and 14 RAEB-F patients. In addition, an analysis of MPL W515L and GATA1 mutations was performed on 15 APMF cases. The relevant clinicopathological and molecular data are summarized in the table.APMFRAEB-FP valueMedianSurvival, median (months)0,0006,5 (*CI: 5,6-7,49)36,3 (CI: 0,9-71,7)MCV, mean (fl)<0,00190,29 (CI: 88,3-92,3)98,3 (CI: 95,2-101,3)LDH, mean ( U/l)<0,001401,8 (CI: 341,6-461,9258,3 (CI: 226,2-290,4)BM fibrosis, (grade)<0,0012,5 (CI: 2,4- 2,7)1,8 (CI: 1,6-2)BM Blasts (%)<0,00131,4 (CI: 27,2-35,5)13,3 (CI: 11,3-14,9)Aberrant NPM1 (%)n.s.12,97%JAK2V617 F + (%)n.s.36%21%MPL W515L +(%)0n.d.GATA1 + (%)0n.d.*CI: 95% confidence intervalSemiquantitative evaluation revealed a decreased expression level of Id2 in erythropoiesis of the APMF samples, while Id2 was increased in the stromal compartment. Within the total APMF cohort, patients aged ≤60 years had a longer OS (p=0,013). A comparison between the Kaplan-Meier curves of the APMF patients who underwent hematopietic cell transplantation (HCT) with those treated by conventional chemotherapy clearly showed that HCT significantly improved OS (median survival 6,1 vs. 11,1 months, p=0,005). Within the non-transplanted group, a bone marrow blast count ≤25% was associated with a longer OS (p=0,006). ConclusionsOur results point to significant differences between APMF and RAEB-F with regard to OS, MCV, LDH, BM fibrosis and BM blast cells. We detected a higher frequency of mutated JAK2 V616F in the MB samples than generally reported in the literature for other categories of AML or MDS. However, the JAK2 V617F mutation status or an aberrant cytoplasmic NPM1 localization were both not predictive of outcome. In contrast to data published for AMGL, no association between a MPL W515L mutation and APMF was observed. Survival was significantly worse in APMF than in RAEB-F. A rapid diagnosis based on a multiparameter approach and a proper management including the option of HCT even in elderly patients seems mandatory for APMF patients. Targeting the fibrotic BM microenvironment which is characterized by an increased Id2 expression may be considered for future therapeutic strategies. DisclosuresSkoda:Genentech Inc.: Consultancy.

Pomalidomide Therapy in Myelofibrosis: 2-Year Follow-up of a Randomized Phase 2 Study.

Abstract 1904Poster Board I-927 BACKGROUND:Pomalidomide is an IMiDsó immunomodulatory drug that was created by chemical modification of thalidomide with the intent to reduce toxicity and enhance therapeutic activity. In a phase-2 randomized study of patients with myelofibrosis (MF), treatment with pomalidomide alone or in combination with a short course of prednisone was shown to be well tolerated and active in alleviating anemia ( JCO 2009). Herein, we report observations during the longer-term follow-up of those patients who had responded to a pomalidomide-containing treatment regimen. METHODS:Both primary and post polycythemia vera/essential thrombocythemia MF were included in the current study. Protocol eligibility criteria included absolute neutrophil count of × 109/L, platelet count of 50 × 109/L and hemoglobin level of < 10 g/dL. Patients with prior thalidomide or lenalidomide therapy were excluded from study participation. Response was assessed by the International Working Group for Myelofibrosis Research and Treatment criteria (IWG-MRT) (Blood. 2006). Pomalidomide was administered daily and continued indefinitely in responding patients. Prednisone was given for the first 3 months only, in a tapering dose schedule, starting at 30 mg/day. RESULTS:A total of 84 patients were enrolled from April 2007 through May 2008 and received treatment with pomalidomide alone 2 mg/day (n=22), pomalidomide 2 mg/day + prednisone (n=19), pomalidomide 0.5 mg/day + prednisone (n=22) or prednisone alone (n=21). Short-term toxicity details have previously been published (Tefferi et al. JCO 2009) and were remarkable for the lack of neuropathy and low prevalence of grade 3 or 4 myelosuppression (5–16%).Based on an intent-to-treat analysis, 16 (25%) of the 63 patients receiving pomalidomide alone or with a 3-month course of prednisone responded at a median of 1.8 months (range 0.9–6.9) from treatment initiation; all responses represented improvement in anemia with little or no effect on splenomegaly. Specifically, 12 of the 16 treatment responders became transfusion independent and the remaining 4 had a hemoglobin increase of > 2 g/dL (median best hemoglobin response = 11.9 g/dL; range 8.5–15.9). Responding patients usually displayed increases in platelet count but there was no consistent change in serum lactate dehydrogenase level.As of August 2009, ten (63%) of the 16 treatment responders were still in anemia remission and receiving pomalidomide therapy; median time on treatment was 19 months (range, 14–26) and median response duration was 17 months (range, 13-24). No new side effects emerged during this period. Six patients discontinued treatment because of either loss of response (n=5) or progressive splenomegaly/leukocytosis (n=1); 3 patients with continued anemia remission have had substantial increases in their spleen size, which qualifies as spleen progression per IWG-MRT. Among 9 patients with available cytogenetic information, all 3 with unfavorable karyotype relapsed while none of the 6 with favorable karyotype did.A small number of patients (n=7) had follow-up bone marrow examination at 1 to 2 years, at the discretion of their treating physician; none of these patients displayed any change in bone marrow fibrosis or JAK2V617F mutational status. Suppression of a monosomy 7 clone was documented in two patients. In one, the percentage of abnormal metaphases decreased from 90% to 15% after 11 months of treatment with pomalidomide but increased back to 100% four months later, when the patient relapsed while still on treatment. In the second patient, the percentage of abnormal metaphases was 65% at baseline, remained at 75% after 10 months of pomalidomide therapy, but decreased to 7% after one month of treatment discontinuation for relapse; a repeat examination in this patient after another 3 months (still off therapy) showed monosomy 7 and new additional abnormalities (including 4q21q25 deletion) in 65% of metaphases evaluated. CONCLUSION:Longer-term follow-up of pomalidomide-treated patients with myelofibrosis confirm the drug's favorable side effect profile. Also, the majority of treatment responders maintain their remission, which is exclusively anemia remission, for at least one year and several remain in remission after 2 years of therapy. The drug has limited effect on splenomegaly and progressive splenomegaly might occur in some patients despite continued remission in anemia. The presence of unfavorable cytogenetic abnormalities predicts relapse. Disclosures:Gale:Celgene: Employment.

Hydroxyurea Effect On Marked Splenomegaly Associated with Primary Myelofibrosis: Response Rates and Correlation with JAK2V617F Allele Burden.

Abstract 4971 BackgroundMarked splenomegaly severely compromises quality of life in primary myelofibrosis (PMF). Preliminary results from the use of small molecule JAK2 inhibitors suggest prominent activity in reducing spleen size. In order to put such salutary effect of investigational drug therapy in context, it is important to determine the value of conventional therapy in this regard. MethodsStudy population was selected from the Mayo Clinic PMF database and consisted of patients treated with hydroxyurea (HU) as first-line therapy for symptomatic, marked splenomegaly (palpable at >10 cm below the left costal margin) and in whom follow-up information was available to assess response. Palpable spleen size measurements were recorded before and after 3, 6 and 12 months of HU therapy. Spleen response was defined as minimal (< 25% reduction), minor (25-50%), or major (>50%). Standard statistical procedures were used to correlate spleen response with other clinical and laboratory parameters, including cytogenetic and JAK2V617F mutational status. Results46 consecutive patients (median age, 62 years; range 38-75; 30 males) met the above stipulated eligibility criteria for study inclusion. Pre-treatment IPSS risk categories were low, intermediate-1 (int-1), int-2 and high in 4, 13, 10 and 19 patients, respectively; 7 patients were transfusion-dependent. Quantitative JAK2V617F and pre-treatment cytogenetic information were available in 25 and 36 patients, respectively; 15 (60%) patients were positive for JAK2V617F and mutant allele burden was > 50% in 6 patients; 17 (47%) patients carried either favorable (n=5) or unfavorable (n=12) cytogenetic abnormalities.Overall response rate (major and minor) was 35% (16 of 46 patients) and major response rate 17%. Overall response rates were 10%, 67% and 33% in patients with undetectable JAK2V617F or mutant allele burden of < or ≥ 50%, respectively (p=0.04). Overall response rates in patients with normal, favorable and unfavorable karyotype were 32%, 40% and 33%, respectively (p=0.9). Response was not affected by age, sex, HU dose or pretreatment status regarding spleen size, IPSS score or transfusion need. ConclusionsThe current study provides background information on the value of conventional chemotherapy for controlling PMF-associated marked splenomegaly. The study also suggests a positive effect of JAK2V617F on treatment response to HU, a detail that needs to be considered when assessing the corresponding effect from a JAK2 inhibitor therapy. DisclosuresOff Label Use: Hydroxyurea use in myelofibrosis.

MiRNAs Expression Pattern in Essential Thrombocythemia and Polycythemia Vera Patients.

Abstract 1912Poster Board I-935 Background:Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are myeloproliferative neoplasms (MPN) arising from a multipotent hematopoietic stem cell characterized by an unregulated production of platelets, red cells and white cells alone or in combination, a tendency to clonal evolution and an increased risk of thrombohemorrhagic complications. MicroRNAs (miRNA) are negative regulators of genes involved in cellular proliferation, apoptosis and/or carcinogenesis. Aim:To analyze the expression pattern of miRNA between PV and ET patients and to find distinctive signatures in ET patients according to JAK2V617F and c-MPL mutational status. Material and Methods:Total RNA was extracted from peripheral blood granulocytes of 50 ET patients, 10 PV patients and 10 controls. Median age of patients was 57 years (range, 20-88); males 36%. The JAK2V617F mutation was present in 23 (46%) of 50 ET patients and in all PV patients. MPL mutations were present in 5 (18%) of 27 JAK2V617F negative cases (3 cases MPLW515L, 1 case MPLW515K and 1 case MPLS505N). miRNA expression was profiled in 384 miRNA via Taqman Low Density Array in ABI PRISM 7900. Expression data was normalized with RNU48 and relative quantification was calculated with the 2–σσCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by means of Significant Analysis of MicroArrays (SAM), Prediction Analysis of MicroArrays (PAM) and Class Comparison methods using BRB array tools version 3.7.0 and TIGR multiexperiment viewer version 4.3. Results:We found a general downregulation of miRNA in ET and PV patients respect to normal controls. A set of 29 miRNA allowed us to discriminate between ET and PV versus normal controls; three of these miRNA were up-regulated and 16 down-regulated in PV and ET vs. normal controls with a >2 fold change and p value <0.01. A distinctive signature of 79 miRNAs differentiated ET, PV and controls and a hierarchical clustering analysis defined miRNA expression profiles of the three particular groups. When we compared miRNA differentially expressed between PV and ET patients, we found nine miRNA, 4 up-regulated and 5 down-regulated in ET with respect to PV patients (p<0.01). Statistical comparisons between ET JAK2V617F-positive and ET JAK2V617F-negative cases showed a distinctive signature of 13 miRNA that allowed us to discriminate between the two groups. In addition, we also found in JAK2V617F cases 19 miRNA differentially expressed between MPL positive and MPL negative patients, with a >2 fold change and p<0.01. Finally, an increased expression of miR-142-5p correlated with JAK2V617F allele burden in ET patients Conclusions:Our study demonstrates that ET and PV can be defined by specific signatures of miRNA. In ET, some miRNA allow us to discriminate cases according to JAK2V617F mutational status and also between MPL-positive and MPL-negative JAK2V617F-negative patients. Research Funding:FIS EC07/90791 Disclosures:No relevant conflicts of interest to declare.

Pathway level analysis by augmenting activities of transcription factor target genes

Many approaches to discovering significant pathways in gene expression profiles have been developed to facilitate biological interpretation and hypothesis generation. In this work, the authors propose a pathway identification scheme integrating the activity of pathway member genes with that of target genes of transcription factors (TFs) in the same pathway by the weighted Z-method. The authors evaluated the integrative scoring scheme in gene expression profiles of essential thrombocythemia patients with JAK2V617F mutation status, primary breast tumour samples with the status of metastasis occurrence, two independent lung cancer expression profiles with their prognosis, and found that our approach identified cancer-type-specific pathways better than gene set enrichment analysis (GSEA) and Tian's method using the original pathways [pathways that have TFs from database] and the extended pathways (including target genes of TFs of the original pathways). The success of our scheme implicates that adding information of transcriptional regulation is better way of utilising mRNA measurements for estimating differential activities of pathways from gene expression profiles more exactly.

The JAK2 46/1 haplotype confers susceptibility to essential thrombocythemia regardless of JAK2V617F mutational status—clinical correlates in a study of 226 consecutive patients

The germline JAK2 haplotype 46/1, tagged by the 'C' allele of single-nucleotide polymorphism (SNP) rs12343867 (C/T), has been associated with JAK2V617F (VF)-positive myeloproliferative neoplasms. SNP rs12343867 was genotyped using bone marrow DNA in 226 consecutive patients with essential thrombocythemia (ET) with concomitant analysis of VF allele burden. The incidence of the 46/1-linked C allele was significantly higher in ET (genotype: CC 15%, CT 52%, TT 33%; C-allele frequency: 41%) than in population controls (P<0.01). Genotype distributions were similar among VF-positive/VF-negative patients (genotype: CC 18/11%, CT 52/53%, TT 30/36%; C-allele: 44/38%; P=0.29). Haplotype 46/1 frequency was remarkably similar when comparing VF-negative patients to those with <10% VF allele burden, but significantly higher in the presence of >10% VF allele burden (genotype: CC 11/13/38%, CT 53/56/38%, TT 36/31/24%; C-allele frequency: 38/41/57%; P<0.01). The clinical features of 46/1-positive and -negative ET were indistinguishable, including blood counts, rate of thrombosis/disease transformation and survival. We conclude that JAK2 haplotype 46/1 confers susceptibility to developing ET independent of VF mutational status and does not seem to further affect the clinical phenotype or prognosis.

Prodromal myeloproliferative neoplasms: The 2008 WHO classification

The concept of prodromal chronic myeloproliferative neoplasms has been endorsed by the WHO classification implicating a stepwise evolution of disease. Histology of the bone marrow (BM) and borderline to mildly expressed clinical features play a pivotal role for diagnosing prefibrotic-early primary myelofibrosis. By lowering the platelet count for essential thrombocythemia and regarding BM morphology, early manifestations are tackled. Pre-polycythemic stages of polycythemia vera with a low hemoglobin level at onset are diagnosed by positive JAK2V617F mutation status, a low erythropoietin value, and characteristic BM features. The revised WHO classification incorporates hematological, morphological, and molecular-genetic parameters to generate a consensus-based working diagnosis.

Polycythemia vera erythroid precursors exhibit increased proliferation and apoptosis resistance associated with abnormal RAS and PI3K pathway activation

Polycythemia vera (PV) is characterized by erythrocytosis associated with the presence of the activating JAK2(V617F) mutation in a variable proportion of hematopoietic cells. JAK2(V617F) is detected in other myeloproliferative neoplasms, does not appear to be the PV-initiating event, and its specific role in deregulated erythropoiesis in PV is incompletely understood. We investigated the pathogenesis of PV to characterize abnormal proliferation and apoptosis responses and aberrant oncogenic pathway activation in primary PV erythroid precursors. Peripheral blood CD34(+) cells isolated from PV patients and healthy controls were grown in liquid culture to expand a population of primary erythroblasts for experiments designed to analyze cellular proliferation, apoptosis, JAK2(V617F) mutation status, cytokine-dependent protein phosphorylation and gene expression profiling using Affymetrix microarrays. The survival and proliferation of PV erythroblasts were growth factor-dependent under strict serum-free conditions requiring both erythropoietin (EPO) and stem cell factor. PV erythroblasts exhibited EPO hypersensitivity and enhanced cellular proliferation associated with increased EPO-mediated extracellular signal-regulated kinases 1 and 2 phosphorylation. EPO-induced AKT phosphorylation was observed in PV but not normal erythroblasts, an effect associated with apoptosis resistance in PV erythroblasts. Analysis of gene expression and oncogenic pathway activation signatures revealed increased RAS (p<0.01) and phosphoinositide-3 kinase (p<0.05) pathway activation in PV erythroblasts. Deregulated erythropoiesis in PV involves EPO hypersensitivity and apoptosis resistance of erythroid precursor cells associated with abnormally increased activation of RAS-ERK and phosphoinositide-3 kinase-AKT pathways. These data suggest that investigation of the mechanisms of abnormal RAS and phosphoinositide-3 kinase pathway activation in erythroblasts may contribute to our understanding of the molecular pathogenesis of PV.

Cytogenetic abnormalities in essential thrombocythemia: prevalence and prognostic significance

In the current study we describe cytogenetic findings as well as clinical correlates and long-term prognostic relevance of abnormal cytogenetics at the time of diagnosis of essential thrombocythemia (ET). The study cohort consisted of a consecutive group of patients with ET who fulfilled the World Health Organization diagnostic criteria, and in whom cytogenetic analysis was performed at diagnosis. A total of 402 patients were studied (median age, 56 yrs; median follow-up 70 months). The prevalence of abnormal cytogenetics at diagnosis was 7% (28 of 402). The most common cytogenetic anomalies were trisomy 9 (four patients), abnormal chromosome 1 (three patients) and trisomy 8 (two patients). Parameters at diagnosis that were significantly associated with abnormal cytogenetics included palpable splenomegaly (P = 0.03), current tobacco use (P = 0.04); venous thrombosis (P = 0.02), and anemia with a hemoglobin of <10 g/dL (P = 0.02); but did not include JAK2V617F mutation status, or advanced age. During follow up, patients with abnormal cytogenetics did not have shorter survival, or increased transformation to acute leukemia or myelofibrosis. Cytogenetic anomalies at diagnosis are relatively uncommon in ET, and do not predict evolution into more aggressive myeloid disorders, or inferior survival.

Diagnosis of chronic myeloproliferative neoplasms with special emphasis on early stages

In the interdisciplinary approach to the diagnosis of chronic myeloproliferative neoplasms (MPN), bone marrow (BM) histopathology, clinical findings and molecular genetic abnormalities play a pivotal role. The recognition of the early stages of MPN requires long-term follow-up studies, including sequential BM examinations in combination with biological tests and the JAK2 V617F mutation status. Based on these rationales, the early stages of MPN, including polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), are described with corresponding haematological data. With reference to relevant complications (thromboembolic and haemorrhagic events and myelofibrotic transformation) and ensuing therapeutic options, the diagnostic value of a proper evaluation of the initial BM biopsies can not be overemphasized. Pre-polycythaemic stage PV, which often presents with a low but increasing haemoglobin level at onset, can be diagnosed from a positive JAK2 V617F mutation status, the characteristic BM features of panmyelosis (trilineage increase in erythro-, granulo- and megakary-opoiesis) and a serum erythropoietin level below the normal range. Early manifestations of ET can be recognized by lowering the platelet count (>600 x 10 9/litre) to 450 × 10 9/litre, BM morphology (predominance of large to giant megakaryocytes without abnormalities of maturation), as well as relevant complications (haemorrhage and vascular events). Histopathological characteristics and accompanying clinical features play a distinctive role in the establishment of pre-fibrotic early PMF. Due to their significant differences in progression to myelofibrosis and outcome, an important issue is the discrimination between pre-fibrotic PMF, which frequently presents with thrombocytosis, and ET. Up-to-date diagnostic criteria, originally established by the World Health Organization (WHO) and recently revised by a panel of experts, should be generally applied and validated in prospective clinical trials to achieve a consensus-based working diagnosis.