V617F-positive Patients Research Articles

Lack of Association Between the 46/1 JAK2 Haplotype and the Presence of JAK2V617F Mutation In Splanchnic Vein Thrombosis Patients

AbstractAbstract 4120 Aim:The majority of myeloproliferative neoplasms (MPN), i.e. Polycythemia vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis, are characterized by the presence of the acquired JAK2V617F gene mutation. Recent studies revealed that the 46/1 or “GGCC” haplotype located in the JAK2 gene is strongly associated with the development of a JAK2V617F positive MPN. However, this particular haplotype was also detected in excess in JAK2V617F negative MPN carrying mutations across JAK2 exon 12 or the MPL gene, suggesting that this germline genetic variation increases the risk of developing a MPN regardless of the acquisition of one particular mutation. A MPN is found in approximately half of the patients presenting with Splanchnic Vein Thrombosis (SVT) and JAK2V617F mutation is present in virtually all of these MPN patients. In this study we sought to clarify the impact of the JAK2 46/1 haplotype on the susceptibility to SVT. Patients and Methods:Peripheral blood was obtained after informed consent; following DNA extraction we proceeded to genotyping using commercially available TaqMan SNP genotyping assays for one tag SNP (rs10974944) which is in complete linkage disequilibrium with the 46/1 haplotype. Results were confirmed using another tag SNP (rs1234867). The study was performed on 170 patients with SVT, 58 patients with peripheral vein thrombosis and 31 patients with JAK2V617F positive PV. Results:In the 170 patients with SVT the frequency of G-allele that stands for the 46/1 haplotype was 0.28 (CC n=89; C/G n=67; G/G n=14), not significantly different from that of the published population controls (n=1500) from the Welcome Trust Case Control Consortium WTCCC (0.24; p=0.11 by the Fisher exact test). The frequency of the 46/1 haplotype in 58 patients with peripheral vein thrombosis was also similar to that of the WTCCC (0.24). However, the frequency of the 46/1 haplotype in SVT patients was significantly different from its frequency in a group of 31 patients with JAK2V617F positive PV (0.52; p=0.0005). When we analysed SVT patients according to their JAK2 mutational status, we found no difference in the frequency of the 46/1 haplotype between the JAK2V617F positive patients (0.27; n=75) and the JAK2V617F negative patients (0.28; n=95) (p=0.90). Of note, a JAK2 allele burden greater than 50% was observed in 11% of patients with JAK2V617F positive SVT and 45% of patients with JAK2V617F positive PV. Conclusions:In this large cohort of 170 patients with SVT the frequency of the 46/1 haplotype was not different from the cohort of controls of the WTCCC. This result suggests that the 46/1 haplotype is not a susceptibility locus for the development of SVT. Moreover, this haplotype was not overrepresented in the group of SVT patients harbouring a JAK2V617F mutation compared to JAK2V617F negative patients. This result is in apparent contradiction with the hypothesis that the 46/1 haplotype predisposes to the acquisition of JAK2V617F mutation or of a MPN, in agreement with recent studies reporting almost identical 46/1 frequencies between V617F-negative patients and V617F-positive patients with low mutation burden (i.e. <50%), which is the case of SVT patients in this series. Disclosures:No relevant conflicts of interest to declare.

LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients

AbstractAbstract 5054 Introduction:The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients. Objectives:To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients. Methods:In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay. Results:LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1). Conclusions:In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation. [Display omitted] Disclosures:Ma:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

Pegylated Interferon Alpha-2a Therapy for Patients with High Risk Essential Thrombocytemia: a Retrospective Analysis on 59 Patients.On Behalf of the French Intergroup of Myeloproliferative Disorders (FIM)

AbstractAbstract 1974 Background:Conventional therapeutic guidelines in essential thrombocytemia (ET) are based on established criteria of higher incidence of vascular complications: age more than 60 years, history of thrombosis, platelets more than 1000–1500 ×109/L and cardio-vascular risk factors. Hydroxyurea or anagrelide are the most commonly used cytoreductive therapy for high risk ET. Despite a proven activity of Interferon-alpha (IFN-α) in Ph negative myeloproliferative disorders, generalized use and efficacy have been hampered by frequent side effects. Nonetheless it is still a nonleukemogenic therapeutic option for younger patients and during pregnancy. More recently, phase II trials using Pegylated (Peg-) forms of IFN-α in patients with polycythemia vera (PV) or high risk ET have provided interesting results: first the superior toxicity profile is generally associated with a rapid hematologic response Second the molecular response described for a subset of JAK2 V617F positive PV or ET patients treated with Peg-IFN α-2a have indicated a selective targeting of the malignant clone with that latter agent. Methods:We revised cases of high risk ET having being or currently treated with IFN-α and reported by centres of the French Intergroup of myeloproliferative disorders (FIM). The current retrospective analysis was performed on data collected for the first 67 patients (pts) that have been observed for a median of 72 months (range 11–273). Among them, 59 pts have received Peg-IFNα-2a therapy. Assessment of the hematologic responses was performed at months 1, 2, 3 and then every 6 months. Results:For the entire cohort, median age was 37 years (range 17–64) and sex ratio F/M of 2.33. Previous thrombosis was observed in 19 cases and micro vascular disturbance in 4 pts. At diagnosis, median platelets count was 926.0×109/L (range 428.0; 4000.0), haemoglobin 14g/dl (8.0-14.1), leucocytes count 9.7×109/L (2.7-28.0). Detection of the Jak2V617F mutation was positive in 33 cases and negative for 26 pts.Median time from diagnosis to IFN-α was 50 months (1-256 months). IFN-α was generally administered as second line after Hydroxyurea. No prior therapy has been used for 6 pts. Reasons for IFN-α initiation were absence of hematologic response (31%), toxicity to the prior treatment (14%), pregnancy (16%), or medical decision for younger pts (39%).Of 67 pts of the cohort, 59 pts have been treated with Peg-IFNα-2a with a median duration of Peg-IFNα-2a of 16 months (range 1 day to 45 months). Efficacy and safety of the Peg-IFNα-2a were assessed in that subgroup. Thus 58 pts were evaluable for the hematologic response with at least 1 month exposure to Peg-IFNα2a.Overall, absence of response was observed in 4/58 (7%) pts, partial hematologic responses in 9/58 (16%) and complete hematologic response (CHR) was achieved for 45/58 (76%) of patients according to the European LeukemiaNet criteria. Cumulative incidence of CHR at 1, 3, and 6 months was 29%, 52% and 65% respectively. At 18 months, estimated probability of CHR was 83% (95%CI 71–92). At the last follow up 47/58 (81%) of the pts were still treated with Peg-IFNα-2a and overall 36 pts are in CHR. Median time to achieve CHR was 3 months. Median dose of Peg-IFNα-2a was 88μg weekly (range 28–180μg weekly). Reasons for discontinuation of Peg-IFNα2a (11pts) were related non hematologic toxicity in 7/59 pts (12%), 1 failure, and 3 pregnancies.By analysing the response according to the Jak2 status (58 pts), the overall CHR rates were 31 and 14 for the 37 JAK2 V617F mutated and the 21 JAK2 negative pts respectively. The median time to achieve CHR was 6 and 3 months. At 18 months the estimated CHR rates were 87% (73-95) and 66% (45-86). The difference is not statistically significant. Median doses of Peg-IFNα2a were 90μg (12-180) and 75μg (45-144) weekly. Duration of exposure to Peg-IFNα2a was 16 months (1-36) and 15 months (1-45). Conclusion:This retrospective analysis is the first attempt to present results on efficacy and tolerance of Peg-IFN on a large cohort of high risk ET not included in clinical trials. These results support the use of Peg-IFN in HR ET pts. Increase patients number, molecular analysis and update of these preliminary data will be presented. Disclosures:Off Label Use: Interferon alpha and Pegylated-Interferon alpha for myeloproliferative disorders.

Gender Differences In the Prevalence of Thrombosis In the JAK2 V617F-Positive Myeloproliferative Disorders

AbstractAbstract 3080 Introduction:Thrombotic events are prevalent and morbid conditions in essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), but the exact mechanisms aside from hyperviscosity and dysfunctional platelet behavior are largely undefined. Since ET, PV and PMF are chronic, latent disorders of middle and older age, we examined the influence of traditional thrombotic risk factors on the tendency toward thrombotic events. To this end, we retrospectively analyzed a cohort of 270 JAK2 V617F-positive patients from the Johns Hopkins Center for Myeloproliferative Disorders. Methods:Thrombotic events within 5 years preceding or at any time following an official diagnosis of ET (n=59), PV (n=166), or MF (post-ETMF, post-PVMF, and PMF) (n=45) were captured by retrospective review of the medical record; the events were characterized as microvascular or macrovascular, arterial or venous. Traditional thrombotic risk factors were recorded, including hypertension, diabetes, dyslipidemia, and history of smoking) and since there were gender differences in diagnostic class (ET and PV were more common in women), age at diagnosis, smoking history, dyslipidemia, and leukocyte count, these variables were incorporated into a multivariable logistic regression model. Result:Of the entire MPD cohort, 67 patients (25%) had 79 microvascular or macrovascular thrombotic events, including 14 (24%) with ET (18 events), 45 (27%) with PV (51 events), and 8 (18%) with MF (10 events). The thrombosis prevalence did not differ statistically by disease class. However, thrombosis prevalence did differ by gender: women had a higher prevalence of thrombosis compared to men (29% vs. 18%; p=0.035) and the odds ratio remained significant after multivariable adjustment (OR 1.98, p=0.04). There were no gender differences with respect to treatment (aspirin or cytoreductive agents), nor were there differences due to age (median 56 years) or disease duration at the time of thrombosis. The frequency of JAK2 V617F homozygosity also did not differ by gender. In men, 9 of 13 (69%) thrombotic events occurred at diagnosis or in the preceding 5 years; however, in women, thrombotic events during follow-up were as likely since only 20 of 45 (44%) thrombotic events occurred at diagnosis or in the preceding 5 years. Nine patients experienced multiple thrombotic events, of these, 6 were women; in 8 cases, these thrombotic events involved different vascular beds, suggesting a general proclivity for thrombosis. Overall, a dependence between sex and the type of thrombosis could not be identified, possibly due to inadequate power (p=0.36). However, there was an indication of gender differences in some thrombotic subgroups: for example, of 20 abdominal venous thromboses (AVT, including hepatic vein, portal vein, mesenteric vein and splenic vein thrombosis), 16 (80%) occurred in women (p=0.07). AVT was the most common event in PV, occurring in 23% of cases, and of these, 11 of 12 were reported in women. Further, in 7 of these women, an AVT was the presenting manifestation of their MPD. AVT was also the most common type of thrombosis in MF, occurring in 50% of cases; of these, 4 of 5 also occurred in women. In contrast to PV, rather than manifesting at diagnosis, 3 of the 5 events (60%) complicated splenectomy. Neurologic events also appeared to be more frequent in women: 6 of 7 CVA's (86%) occurred in women (p=0.14), and of 11 TIA's, 8 (73%) occurred in women (p=0.4). Conclusion:In this retrospective analysis of 270 cases, thrombotic events were equally prevalent in ET, PV, and MF, though importantly more common in women in this patient cohort. There was also a trend suggesting differences in thrombosis type between women and men, since TIA, CVA, and especially, AVT appeared to be more common in women. Indeed, in women, AVT was often the presenting feature leading to a diagnosis of PV. This study indicates unique disease and gender specific interactions in the generation of thrombosis in the MPD, and suggests that women are paradoxically at higher risk for thrombosis in the MPD. Though a mechanism is not yet defined, this analysis contributes to a growing literature emphasizing gender differences in all three MPD, which already includes differences in the JAK2 V617F allele burden and disease class. Further, this analysis supports the important impact of individual and host variation on the clinical manifestations of the MPD. Disclosures:No relevant conflicts of interest to declare.

Disease burden at the progenitor level is a feature of primary myelofibrosis: a multivariable analysis of 164 JAK2 V617F-positive myeloproliferative neoplasm patients

Suppression of normal hematopoiesis by the neoplastic clone (clonal dominance) is a feature of the myeloproliferative neoplasms, but the determinants that predict clonal dominance are unknown. The objective of this study was to identify clinical and laboratory variables that associate with the JAK2 V617F CD34(+) progenitor allele burden and clonal dominance, which was defined by congruence of the JAK2 V617F CD34(+) progenitor and neutrophil allele burdens. A cross-sectional analysis was performed on 164 consecutive JAK2 V617F-positive patients: 30 with essential thrombocytosis (ET), 100 with polycythemia vera (PV), and 34 with myelofibrosis (MF), including 8 post-ET MF and 3 post-PV MF. The JAK2 V617F CD34(+) progenitor and neutrophil allele burdens were measured using an allele-specific, quantitative real-time polymerase chain reaction assay. After adjusting for genotype, sex, age at diagnosis, and disease duration, disease type was the strongest predictor of clonal dominance, with the odds ratio being nearly 61.9 times higher for MF patients when compared with ET patients (p < 0.001), and 9.7 times higher when compared with PV patients (p = 0.002). Additionally, clonal dominance was associated with a clinical phenotype of an increased spleen size (p = 0.006), increased white blood cell count (p = 0.009), and lower hemoglobin (p < 0.001), even after adjusting for disease type and duration. These data indicate that loss of wild-type clones at the progenitor level is a feature of MF (primary MF, post-ET MF, and post-PV MF), presumably due to expansion of the JAK2 V617F clone and that this characteristic is surprisingly independent of JAK2 V617F homozygosity, suggesting that additional genomic lesions may contribute to this unique molecular process that distinguishes MF from ET and PV.

Observation versus antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia

AbstractThe effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F–positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 × 109/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

Abstract B6: Identification of novel mutations in the inhibitory adaptor protein LNK in patients with JAK2 V617F-negative and -positive chronic myeloproliferative neoplasms

Abstract Dysregulated JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs), as evidenced by the identification of activating mutations in JAK2, and the thrombopoietin (TPO) receptor MPL in a subset of MPN patients. Clinical trials with highly specific inhibitors of JAK2 are currently ongoing, and clinical responses have been observed in the majority of MPN patients, validating JAK2 as an important therapeutic target in these patients. In addition, responses have been observed in patients lacking known mutations in JAK2 or MPL, suggesting that other regulatory elements in this pathway are altered. However, the molecular basis for this observation is not well understood. One regulator of JAK-STAT signaling is LNK (SH2B3), a member of a family of adaptor proteins that share several structural motifs, including a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain, an SH2 domain, and a conserved C-terminal tyrosine residue. LNK binds to MPL via its SH2 domain and co-localizes to the plasma membrane via its PH domain. Upon cytokine stimulation with TPO, LNK binds strongly to JAK2 and inhibits downstream STAT activation, thereby providing critical negative feedback regulation. LNK-/- mice exhibit an MPN phenotype, including an expanded hematopoietic stem cell compartment, megakaryocyte hyperplasia, splenomegaly, leukocytosis, and thrombocytosis. We sequenced LNK in a cohort of MPN patients, leading to the identification of novel mutations in 7/159 (4.4%) patients. One patient with JAK2 V617F-negative primary myelofibrosis (PMF) exhibited a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. Six additional patients were found to have point mutations affecting conserved residues in the PH domain. Interestingly, a point mutation leading to an E208Q substitution was found in one JAK2 V617F- negative patient with essential thrombocythemia (ET), as well as one JAK2 V617F-positive ET patient. Similarly, a P242S substitution was also found in both a JAK2 V617F-negative ET patient, as well as a JAK2 V617F-positive patient with post-polycythemic myelofibrosis. These latter findings suggest that even in the presence of the JAK2 V617F mutation, abrogation of LNK function may be a cooperating pathogenetic mutation. TPO-dependent BaF3-MPL cells transduced with the LNK DEL mutant exhibited augmented and sustained TPO-dependent growth and activation of JAK2-STAT3/5. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. Primary patient samples from MPN patients bearing the LNK DEL and E208Q mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant. The STAT3/5 activation response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Our identification of mutations in LNK, the first reported in human disease, demonstrates that loss of JAK-STAT negative feedback control is a novel mechanism of MPN pathogenesis. As each of these LNK mutations localizes to the PH domain and appears to be heterozygous, mislocalized mutant LNK may exert a dominant negative effect by binding and sequestering wild-type LNK. These findings may also partly explain why some MPN patients lacking JAK2 or MPL mutations respond to treatment with JAK2 inhibitors, and highlight the importance of a more complete understanding of the role of inhibitory pathways in MPN pathogenesis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B6.

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.

Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory

The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.

An Open-Label Study of CEP-701 in Patients with JAK2 V617F-Positive PV and ET: Update of 39 Enrolled Patients.

Abstract 753Since the seminal discovery of the JAK2V617F mutation, small molecule inhibitors of JAK2 to treat patients with myeloproliferative neoplasms have been recently evaluated in a growing number of clinical trials. To date, there has been limited evaluation of the utility of such JAK2 inhibitors in patients with PV or ET. CEP-701 is an orally available, potent, low nanomolar inhibitor of both the wild-type and mutated JAK2 tyrosine kinase, with its inhibitory effect being demonstrated both in enzymatic and cellular assays. The present study is the first JAK2 inhibitor study to test the safety and efficacy of such an agent in high risk JAK2 V617F positive ET and PV patients. The primary endpoint was reduction in JAK2 V617F neutrophil allele burden. Secondary endpoints included reduction in phlebotomy rates; improvement in hemoglobin, white cell and platelet counts; reduction in hydroxyurea (HU) dose and spleen size; and the pharmacokinetics and pharmacodynamics of CEP-701. Enrollment was completed with 39 JAK2V617F-positive subjects, 27 PV and 12 ET, 22 females and 17 males, ages 38 to 80. The PV patients were a high risk group as defined by a median age of 65 years, neutrophil JAK2V617F allele burden of 76%, prevalence of prior thrombotic events of 19%, prior HU use in 44% and an average white cell count of 19,600/ul (range 3,500-78,600). The ET patients were also considered to be a high risk group as defined by a median age of 61.5 years, the prior use of HU in 100% of cases, and prevalence of prior thrombotic events of 25%; the median neutrophil JAK2V617F allele burden was 40%. More than half the patients had had their disease for 5 years or longer. To date, 15 of the patients have completed 18 weeks of treatment and 13 of these patients continue on the extension phase of the trial. Within 18 weeks, responses included a reduction spleen size of > 5cm or to non-palpable in 15/18 (83%) subjects and amelioration of pruritus in 5/5 patients studied. While reduction in phlebotomy requirement occurred in a number of phlebotomy dependent patients (3/5 evaluable at time of report), this effect was not evident in these patients until 6 months of therapy, and was not associated with concomitant reductions in white cell or platelet count. In fact, in many patients, platelet and white cell counts increased while on drug. A reduction in the JAK2 V617F allele burden of 15% or more was observed in 3 of the 15 patients at the 18 week assessment. The most common adverse events were gastrointestinal, which were dose related and improved over time. Serious adverse events have occurred, including 5 patients experiencing thrombotic events (3 venous (1 DVT, 1DVT/PE, 1 PVT), 2 arterial (1 TIA, 1 PAT)), and one non-serious DVT. We conclude that CEP-701 is biologically active agent in patients with JAK2 V617F positive PV and ET. Serious adverse events related to thrombosis, however, have occurred which to date have not been reported with the use of other JAK2 inhibitor trials involving patients with PMF. Though substantial symptomatic improvements have occurred, our data suggest, at a minimum, that JAK2 inhibitor therapy may not prevent thrombosis in high-risk patients with PV and ET. This risk of thrombosis may be a disease specific interaction in PV and ET patients since thrombosis has not been a frequent complication of CEP 701 therapy for other malignancies. Final 18 week data and cumulative extension data will be updated at the annual meeting. Disclosures:Roboz:Cephalon: Consultancy. Carroll:Cephalon consultancy: Consultancy; Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceutical: Research Funding. Bensen-Kennedy:cephalon: Employment.

Diagnosis and Treatment of Essential Thrombocythemia: Assessment of Current Clinical Practice.

Abstract 1903Poster Board I-926 Introduction:The discovery of the JAK2 V617F gene mutation has significantly altered the clinical diagnostic approach to the myeloproliferative neoplasms, reflected in the revised 2008 WHO diagnostic criteria. Both the 2001 and 2008 diagnostic criteria for essential thrombocythemia (ET) require a bone marrow biopsy showing megakaryocytic proliferation to make a diagnosis of ET. Published expert opinion based on clinical studies suggests that ET patients > 60 years old or with history of thrombosis should be characterized as high risk and treated with hydroxyurea. It is unclear whether physicians in clinical practice utilize the WHO diagnostic criteria or follow expert treatment recommendations for ET. Methods:We conducted a retrospective chart review of all patients with a clinical diagnosis of ET made by a hematologist who were seen in clinic from 2006 to 2008 at two university teaching hospitals in Vancouver, Canada. Data collected included demographic information, thrombosis history, diagnostic tests performed and treatment administered. Testing for JAK2 V617F became locally available in 2006, so for assessment of diagnostic tests performed, patients were divided into cohorts of diagnosis pre-2006 and 2006–2008. Patients were characterized as high risk if > 60 y or history of thrombosis at the time of diagnosis. All other patients were considered low risk. Results:Diagnostic information was available for 116 patients diagnosed prior to the availability of testing for JAK2 V617F. 65% (75/116) of patients in this cohort had a bone marrow biopsy performed (table 1). 44 patients received a new diagnosis of ET from 2006–2008. Only 48% (21/44) patients in this cohort had a bone marrow biopsy performed, significantly less than in the historical cohort (p = 0.019). 41/44 had JAK2 V617F testing performed: 41% (17/41) were JAK2 V617F negative, 56% (23/41) positive and 1 equivocal. Bone marrow biopsy was performed in 59% (10/17) of JAK2 V617F negative patients and 39% (9/23) of JAK2 V617F positive patients (p = 0.055) (table 1). Bone marrow biopsy was also performed in 1 patient with equivocal JAK2 V617F testing and 1 patient not tested for JAK2 V617F. 170 patients diagnosed with ET were seen in follow up 2006–2008. 64% (109/170) were high risk due to age > 60 y or history of thrombosis. The remaining 36% (61/170) were considered low risk. Hydroxyurea was used preferentially over anagrelide for treatment of ET (table 2). Only 76% of high risk patients were receiving cytoreductive treatment. 23% of low risk patients received cytoreductive treatment. ASA was prescribed to 89% of high risk and 79% of low risk patients.table 1Frequency of performing bone marrow at time of diagnosis of ETPre-20062006–20082006–2008JAK2 V617F not availJAK2 V617F NegJAK2 V617F PosTotal number of pts1161723Bone marrow performed75 (65%)10 (59%)9 (39%)table 2Treatment of ET patientsNHydroxyureaAnagrelideNo CytoreductionASAHigh risk10970 (64%)13 (12%)26 (24%)97 (89%)Low risk6112 (20%)2 (3%)47 (77%)48 (79%) Conclusion:Despite the requirement for a bone marrow biopsy to meet the WHO criteria for ET, hematologists performed a bone marrow biopsy in less than half of patients they diagnosed with ET since 2006. Hematologists performed bone marrow biopsy less frequently after JAK2 V617F testing became available, particularly in JAK2 V617F positive patients. A substantial portion of high risk patients (24%) were receiving no cytoreductive therapy, contrary to expert recommendation. Further study is required to understand the barriers to implementing treatment recommendations in clinical practice. This study highlights the challenges in translating published diagnostic criteria and treatment guidelines into changes in patient care. Disclosures:No relevant conflicts of interest to declare.

The production of JAK2 wild‐type platelets is not downregulated in patients with JAK2 V617F mutant‐positive essential thrombocythaemia

To clarify the relationship between the levels of JAK2 wild-type (WT) and V617F mutant-positive platelets in patients with essential thrombocythaemia (ET), we quantified mutant levels in purified cells from 10 V617F-positive patients prior to receiving cytoreductive therapy. Mutant levels were significantly higher in platelet than neutrophil RNA (P = 0.002), but the mutation was still only present in a sub-population of platelets (median 54%). When the absolute number of WT platelets was calculated, it was always within or above the normal platelet range, indicating that there is an aberration in the negative feedback to JAK2 WT platelets in ET.

HFEC282Y Mutation as a Genetic Modifier Influencing Disease Susceptibility for Chronic Myeloproliferative Disease

Iron metabolism has been implicated in carcinogenesis and several studies assessed the potential role of genetic variants of proteins involved in iron metabolism (HFE C282Y, TFR S142G) in different malignancies. Few reports addressed this issue with relation to chronic myeloproliferative disorders (CMPD). The aims of our study were (a) to examine the potential associations of CMPD development with genetic modifiers of iron metabolism in a large cohort of CMPD patients; (b) to examine associations of genetic variants of proteins involved in iron metabolism; and acquired JAK2 V617F mutation with clinical characteristics of CMPD. HFE C282Y was genotyped in 328 CMPD patients and 996 blood donors as controls, HFE H63D, and TFR S142G were tested in CMPD patients and 171 first time blood donors. JAK2 V617F mutation was tested in CMPD patients and in 122 repeated blood donors. Decreased C282Y allele frequency (allele frequency+/-95% confidence interval) was found in the CMPD group (1.8%+/-1.0%) compared with controls (3.4%+/-0.8%; P=0.048). TFR S142G allele frequency was reduced among V617F-negative CMPD patients (34.8%+/-7.6%) compared with controls (47.8%+/-5.4%; P=0.02). The frequency of JAK2 V617F was 75.9% (249 of 328) in the CMPD group. At presentation, elevated hemoglobin levels were found in V617F-positive patients compared with V617F-negative counterparts (P<0.000). Vascular complications (26.6% versus 15.2%; P=0.039) as well as female gender (57.4% versus 41.8%; P=0.019) were more common in V617F-positive patients. We found that HFE C282Y might be associated with a protective role against CMPD. Because chronic iron deficiency or latent anemia may trigger disease susceptibility for CMPD, HFE C282Y positivity may be a genetic factor influencing this effect.

Plasma levels of JAK2 mRNA in patients with chronic myeloproliferative diseases with and without V617F mutation: implications for prognosis and disease biology.

The association of V617F JAK2 expression levels with disease behavior has not been studied in patients with nonchronic myelogenous leukemia (CML) myeloproliferative disease (MPD). We found plasma levels of total JAK2 mRNA to be higher in patients with non-CML MPD (n=175) than in CML patients (n=45) and normal controls (n=58) (each P<0.001). Overall survival was studied in 68 patients and showed positive correlation with levels of total and mutant JAK2 mRNA in patients with the V617F mutation, but not those without the mutation. These findings suggest that total JAK2 expression levels play a role in the biology of the disease in V617F-positive patients, and a therapy aiming at downmodulating the expression of the total JAK2 mRNA should be considered. In conclusion, we studied JAK2 total and V6217F mutant mRNA levels in plasma. We show high levels of JAK2 expression in MPD patients and these levels correlate with survival.

An Open-Label Study of CEP-701 in Patients with JAK2 V617F-Positive Polycythemia Vera and Essential Thrombocytosis

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal hematopoietic stem cell disorders characterized by the over production of phenotypically normal circulating blood cells. Most PV and approximately half of ET patients harbor the activating mutation JAK2 V617F. CEP-701 is an orally available, potent low nanomolar inhibitor of both the wild-type and mutated JAK2 tyrosine kinase, with its inhibitory effect demonstrated both in enzymatic and cellular assays and in vivo, where CEP-701 significantly inhibited the growth of JAK2 V617F-positive HEL.92 xenografts in mice. These findings suggest that CEP-701 is an attractive candidate for clinical evaluation in JAK2 V617F-positive myeloproliferative disorders. The purpose of this study is to test the safety and efficacy of CEP-701 administration in JAK2 V617F positive ET and PV patients. The primary endpoint is reduction in JAK2 V617F neutrophil allele burden; secondary endpoints are reduction in phlebotomy rates, improvement in hemoglobin, white cell and platelet counts, reduction in hydroxyurea (HU) dose, and reduction in spleen size. The secondary endpoints include the pharmacokinetics and pharmacodynamics of CEP-701 and the safety of CEP-701 treatment in patients with JAK2 V617F-positive PV and ET. This is a multicenter study with an anticipated enrollment of 40 PV and ET patients. Inclusion criteria include JAK2 V617F-positive PV and ET patients; patients with PV either have a neutrophil count greater than 7,000/ul or are receiving HU, while ET patients are receiving concomitant HU. Other inclusion criteria include an ECOG performance status of 0, 1 or 2, and 18 years of age or older. Exclusion criteria include the active use of anegrelide or interferon, or a recent history of venous or arterial thrombosis. This is an 18 week trial with an optional 1 year extension period; doses will escalate from 80 mg twice daily to a maximum of 120 mg twice daily. To date, 20 subjects, 11 PV and 9 ET, comprised of 11 females and 9 males, ages 34 to 74, have enrolled. Approximately 27% of the PV patients were taking HU. The most common adverse events have been gastrointestinal (GI) and constitutional in nature. No related serious adverse events have been observed. Five patients have discontinued study participation, all for adverse events: 1 due to disease progression, 1 leg cramps, and 3 GI. To date, 7 patients have completed 18 weeks of therapy and 6 of these patients will continue to receive CEP-701 on the extension phase of the trial. Five of 8 subjects with splenomegaly have responded with reductions in spleen size evident within 6 weeks of therapy initiation. Updated results on current and future patients will be presented at the meeting.

Can Peg-Interferon (IFN) α-2a Eradicate JAK2V617F-Positive Bone Marrow Progenitors in Polycythemia Vera (PV)?

Background: In a phase 2 trial of peg-IFN-alpha-2a, we observed disappearance of JAK2V617F in peripheral blood (PB) granulocytes in 7/29 (24%) treated PV patients using an allele specific PCR assay with a 1% sensitivity (Blood online, July 23, 2008). IFN-alpha has been shown to induce cytogenetic remission or reversion to polyclonal hematopoiesis by studies of occasional patients. The 7 cases of complete molecular remissions (CMR) we identified by V617F monitoring constitute the first cohort of patients with measurable reduction of the PV clone. The study of this cohort may allow answering whether such CMR are associated with disappearance of the PV clone.Methods: Bone marrow (BM) was collected at time of CMR after 12 to 30 months of peg-IFN-alpha-2a in 5 V617F-positive (pos) patients. We studied erythroid colonies generated in the presence or in the absence of erythropoietin (Epo) fromtotal BM mononuclear cells in standard plasma clot (SPC) assay, andpurified BM CD34+ cells in methylcellulose (MC) assay.Individual erythroid colonies grown from BM CD34+ cells were harvested and screened for V617F. Results are expressed as mean of multiple assays (2 to 6, according to the number of cells available).Results: At time of CMR, SPC assays revealed the presence of few residual endogenous erythroid colonies (EEC) in the 5 patiens, representing only 0.2% to 3.6% of the total number of colonies grown with Epo (table 1, column 1). MC assays (from BM CD34+ cells) similarly showed few EEC in 4 patients (n°1, 3, 4, 5), representing 2.4 to 6% of colonies grown with Epo, while in one patient (n°2) no EEC was detected (column 2). In 2 of the 4 patients with persisting EEC in MC assays (n°1, 4, column 3), all EEC (n=5 and 26) were V617F-neg, while in the other 2 (n°3, 5) V617F was found in 4/9 and 1/28 EEC, respectively. In the patient with no EEC (n°2), V617F was however detected in 1 of 64 colonies grown with Epo (column 4). V617F was also found in 1.2% to 5.7% of the colonies grown with Epo in 3 pts (n° 3, 4, 5, column 4), while in the last patient (n°1) with V617F-neg EEC, colonies grown with Epo were also completely V617F-neg.Conclusion: In 1 of 5 V617F-positive PV patients who achieved complete disappearance of the mutation in peripheral blood after peg-IFN-alpha-2a (patient n°1 in table 1), no V617F-positive marrow erythroid progenitors were found, either after EPO stimulation or in EEC, while frequency of V617F-positive marrow progenitors was reduced to very low levels in the other patients. This supports the hypothesis that peg-IFN-alpha-2a can eradicate the JAK2-mutated clone in at least a few selected patients, and greatly reduce it in others. In addition, the finding that a few EEC (mostly V617F-negative) were still found in 4/5 patients analyzed, suggests that a subset of erythroid progenitors from those patients retained the ability to form EEC by a mechanism independent of JAK2V617F.PatientSPC assay* n° of EEC/total n° of colonies with EpoMC assay** n° of EEC/total n° of colonies with EpoTotal n° of harvested EEC/n° of V617F-pos EECTotal n° of harvested colonies with EPO/n° of V617F-pos colonies12/10821/415/0109/026/1640/23064/134/4931/179/480/341/3422/4426/0117/255/2354/7528/1156/9* n° of colonies per 250000 BM mononuclear cells (mean of duplicates); ** n° of colonies per 1500 BM CD34+ cells (mean of 2 to 6 assays)

Elevated Leukocyte Alkaline Phosphatase Scores Induced by Jak2 V617F Mutation

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

JAK2V671F, Gender and the Gene Expression Profile of Polycythemia Vera

Polycythemia vera (PV) is a clonal stem cell disorder characterized by the unregulated production of red cells, white cells and platelets. This phenotype was explained by the discovery of an activating point mutation (V617F) in the gene for JAK2, a tyrosine kinase utilized for signal transduction by the erythropoietin, granulocyte colony-stimulating factor and thrombopoietin receptors. The constitutively active kinase enhances the terminal differentiation of erythroid, myeloid and megakaryocytic progenitor cells. However, gene expression profiling of terminally differentiated JAK2 V617F-positive PV neutrophils reflected only their activated state but not differential gene expression specific to PV. Furthermore, PV is a stem cell disorder and JAK2 is not essential for normal stem cell function. Therefore, to define the molecular abnormalities in PV stem cells, and to determine the influence of JAK2 V617F on these, we used DNA microarray technology to examine gene expression in the circulating CD34+ cells of 19 JAK2 V617F-positive PV patients (11 women and 8 men) in comparison to mobilized CD34+ cells from six normal individuals (3 women and 3 men). Differential changes in gene expression were validated by quantitative PCR (QPCR). Both PV CD34+ cells and mobilized CD34+ cells were greater than 97 % CD38+ and 90 % of both CD34+ cell populations were in a G0/G1 state. Principle component analysis revealed that gene expression in the normal controls segregated according to gender and gender also confounded disease effects in the PV patients as well. When differential gene expression in the PV patients was analyzed separately according to gender, women differentially up or down regulated 249 genes and men, 484 genes. This allowed us to identify a core set of 90 genes that were differentially regulated (59 up regulated and 31 down regulated) in both men and women, and thus, specific for PV and likely to be involved in its pathogenesis. When CD34+ cell gene expression was stratified according to both JAK2 V617F status and gender, the gender effect persisted but there was virtually no influence of the JAK2 V617F allele burden on core PV gene expression. Amongst the 59 up regulated genes were the chemokine genes, CCL3, CCL5 and PF4; the hemoglobin genes, epsilon, gamma, alpha and beta; the extracellular matrix genes, collagen types 1, 3, 4 and 6, TIMP1, heparanase, osteonectin, periostin, fibulin 3, ficolin 1 and thrombospondin; the procoagulant genes, PAI-1and fibroleukin; the transcription factor genes NF1B and HES1; the cell cycle gene, CDC20; the DNA synthesis gene, ribonucleotide reductase; and the folate and leptin receptor genes. The 31 down regulated genes included the transcription factor genes, HOXA9, HOP, KLF6 and hepatic leukemia factor; the FLT3 receptor gene and the PDGF-D and the CD133 genes. Overall, there was down regulation of tumor suppressor genes and up regulation of genes involved in Notch, Wnt and TGF-β signaling in the PV patients as compared to the controls. Using the 90 core gene set, unsupervised hierarchical clustering separated the 19 PV patients in two groups that did not differ with respect to age (mean, 63 vs. 65 years), disease duration (median, 10.5 vs. 7 years), gender or JAK2 V617F neutrophil burden (92 % vs. 85 %), but did differ with respect to the frequency of thromboembolic complications (6/6 vs. 1/13, p= 0.01), splenectomy (4/6 vs. 1/13, p=0.001), and a requirement for chemotherapy (5/6 vs. 3/13, p=0.04). Interestingly, based on their gene expression, the 6 PV patients with the more aggressive clinical course also segregated closely with the normal controls. These observations indicate that PV is not a monolithic disorder, that clinical differences between men and women PV patients have in part a genetic basis, that JAK2 V671F does not have a significant impact on gene expression at the CD34+ cell level, and that it may be possible to determine which PV patients would benefit from early therapeutic intervention by examining gene expression in circulating CD34+ cells.

Characterization of JAK2 V617F Allele Burden in Advanced Myelofibrosis (MF) Patients: No Change in V617F:WT JAK2 Ratio in Patients with High Allele Burdens despite Profound Clinical Improvement Following Treatment with the JAK Inhibitor, INCB018424

Background: Activating mutations in the pseudokinase domain of JAK2 occur at a high frequency in Philadelphia chromosome-negative myeloproliferative disorders (MPDs). Increasing JAK2 V617F allele burden has been shown to correlate with disease severity (bone marrow dysfunction, organomegaly and constitutional symptoms), which is consistent with exaggerated JAK2 signaling playing a central role in MPDs. INCB018424 is a potent and selective JAK1/2 inhibitor currently being evaluated in the clinic for the treatment of MPDs including primary myelofibrosis (PMF), post-polycythemia vera and essential thrombocythemia myelofibrosis (Post-PV/ET MF).Methods: The percentage of mutant JAK2 V617F relative to wild-type JAK2 was determined in peripheral blood using a quantitative single-nucleotide extension assay. The assay is based on the single nucleotide extension of a DNA primer that anneals adjacent to the V617F mutation. The percentage of JAK2 V617F in bone marrow was determined using pyrosequencing as previously described. The relationship between JAK2 V617F allele burden and clinical phenotype was assessed with samples obtained at study entry. JAK2 V617F allele burden was assessed in nineteen PMF and Post-PV/ET MF patients treated with 25 mg BID INCB018424 for a minimum of three months using peripheral blood and bone marrow.Results: There is an excellent correlation in the JAK2 V617F allele burden between samples obtained from bone marrow and peripheral blood, suggesting that the fraction of cells bearing the mutant clone remains stable during hematopoiesis in this patient group. The mean %V617F values were 59%, 80% and 86% for Post-ET MF (n=2), Post-PV MF (n=8) and PMF (n=9) patients. V617F allele burden correlated with spleen size, absolute neutrophil count (ANC) and Body Mass Index (BMI) in this patient population (R2 values if 0.61, 0.17 and 0.44, respectively), with a higher allele burden predicting larger spleen size, a higher ANC and a lower BMI. Eighteen of the nineteen evaluable JAK2 V617F-positive patients had baseline JAK2 V617F values above 50%, with the mean %V617F value for all evaluable patients being 81%, implying that the majority of hematopoietic stem cells contain the mutant allele in this patient population. During INCB018424 therapy (at least 3 months of treatment) in these patients, profound improvements in clinical endpoints including reduction or resolution of splenomegaly and constitutional symptoms are observed, yet only a modest decrease in JAK2 V617F allele burden is seen in both bone marrow and peripheral blood (13% and 9 % decrease from baseline, respectively, at 3 months).Conclusions: While the patient group is small and heterogeneous in nature, clinical correlates of V617F allele burden observed in myelofibrosis patients in this study are similar to those reported in PV. In addition, despite profound clinical improvements in these patients, V617F allele burdens do not change dramatically suggesting that the clinical activity of INCB018424 might involve inhibition of downstream signaling in cells harboring activating JAK2 mutations rather than changing the allele burden.

Pegylated Interferon-ALFA-2A (PEG-IFN-α-2A; PEGASYS) Therapy Renders High Clinical and Molecular Response Rates in Patients with Essential Thrombocythemia (ET) and Polycythemia VERA (PV)

Patients with high-risk essential thrombocythemia (ET) and polycythemia vera (PV) are typically managed with cytoreductive agents such as recombinant interferon-alpha (IFN-α), hydroxyurea (HU), and anagrelide (AG). Despite the significant activity of IFN-α in ET and PV, this agent is frequently hindered by poor tolerance and inconvenient dosing schedules. PEG-IFN-α is formulated by covalently attaching polymers of ethylene glycol to the native IFN-α molecule, resulting in decreased renal excretion and increased serum half-life that allows for weekly administration. On this basis, we are conducting a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) for patients with ET or PV. A total of 76 patients have been enrolled and treated thus far (36 ET, 40 PV). Median age is 53 years (range, 18–77), time from diagnosis to PEG-IFN-α-2a 49 months (range, 0–355), WBC count 8.7×109/L (range, 3.7–27.8), hemoglobin 13.5 g/dL (range, 8.9–18.8), and platelet count 554×109/L (range, 140–1641). Prior therapies (median 1; range 0–6) included HU (n=44), AG (n=29), IFN-α (n=11: 5 oral and 6 sc), imatinib (n=7), and dasatinib (n=1). PEG-IFN-α-2a was the initial therapy in 13 patients that refused therapy with HU. The JAK2 V617F mutation was detected in 20 (56%) of 36 ET and in 37 (92.5%) of 40 PV patients. Nine (12%) patients had abnormal cytogenetics. Initial starting dose of PEG-IFN-α-2a was 450 mcg/week, but that was modified to the current starting dose of 90 mcg/week. Dose modifications are allowed according to response or toxicity. Patients are currently receiving 450 mcg (n=1), 270 mcg (n=3), 180 mcg (n=14), 135 mcg (n=8), 90 mcg (n=27), and 45 mcg (n=7). After a median follow-up of 23 months (range, 2–38), 63 (85%) of 74 assessable patients have responded. The median time to response was 4 weeks (range, 0.5–26). Complete response (CR) was achieved by 60 (81%) patients (for ET: platelets <440×109/L, off HU and AG, in the absence of thromboembolic events; for PV: Hb <15 g/dL, off HU and AG, no phlebotomy, with disappearance of splenomegaly) whereas 3 (4%) patients (1 PV, 2 ET) had a partial response ([PR]; no phlebotomy, off HU and AG, but still palpable spleen). Of 5 assessable patients with abnormal karyotype at the start of the study, 2 reverted to diploid cytogenetics. The mutant JAK2 V617F to total JAK2 ratio was determined by quantitative pyrosequencing assay in all 76 patients prior to PEG-IFN-α-2a and was repeated at least once during therapy in 41 JAK2 V617F-positive patients. Overall, 23 (56%) had >10% reduction in JAK2 V617F expression, including 14 (34%) who had a >50% reduction. In 5 (11%) of the latter the mutant allele became undetectable. PEG-IFN-α-2a was well tolerated in most patients. Thirty-nine episodes of grade 3–4 toxicity were reported: neutropenia (n=15), elevated transaminases (n=5), infection (n=4), fatigue (n=3), pain (n=3), cardiac (n=2), and anemia, thrombocytopenia, depression, shortness of breath, pruritus, thrombosis, and dizziness in 1 case each. Sixteen (21%) patients were taken off study after a median of 8 months (range, 2–26) on PEG-IFN-α-2a but only 7 (9%) of them due to due to therapy-related toxicities: grade 3 neutropenia, anorexia, depression, ischemic retinopathy, dyspnea, confusion, and pruritic rash. In conclusion, PEG-IFN-α-2a therapy results in remarkable clinical activity with an acceptable toxicity profile in advanced, previously treated, patients with ET or PV. Clinical responses are frequently accompanied by significant reduction of JAK2 V617F allele burden, which becomes undetectable in a proportion of them, suggesting selective targeting of the malignant clone.