Citations

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

Abstract Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML. i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status. ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively. Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.

Abstract Background: One of the utilities of molecular markers in hematological malignancies is their potential to predict response to drug therapy. Accordingly, we inquired about the effect of both cytogenetic profile and JAK2V617F mutational status on drug therapy response in myelofibrosis with myeloid metaplasia (MMM). Methods: Mutation analysis for JAK2V617 was performed in DNA derived from peripheral blood mononuclear cells, granulocytes, or both. Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification (Levin et al. Cancer Cell2005;7:387). Abnormal cytogenetic findings were considered to be either favorable (sole 13q- or 20q- abnormalities) or unfavorable (other abnormalities) (Tefferi et al. BJH2001;113:763). Results: i. Patients and treatment: A total of 69 patients with MMM (median age 62 years, range 38–75; 47 males) received the following drugs as first-line therapy for either anemia or symptomatic splenomegaly; erythropoietin (Epo) alone or in combination with hydroxyurea (n=25), hydroxyurea alone (HU; n=17), interferon alpha (IFN; n=11), combination of thalidomide and prednisone (ThalPred; n=7), androgen preparations (Andro; n=5), and etanercept (n=4). ii. Results of cytogenetic studies and JAK2 mutation screening: Cytogenetic findings were abnormal in 35 patients (51%); favorable in 9 and unfavorable in 26. JAK2V617 mutation analysis revealed wild-type allele in 31 patients (45%) and either heterozygous (n=31) or homozygous (n=7) mutation in the remaining 38 patients (55%). iii. Correlation of response to cytogenetic/molecular markers: Overall, 17 patients (25%) achieved either a major (n=8) or minor (n=9) response in anemia from treatment with one of the aforementioned drugs. Regardless of either JAK2V617 mutational status or cytogenetic profile, treatment-induced responses in anemia were poor for IFN (0%) and HU (12%). In contrast, the best anemia responses were documented for ThalPred (57%), Andro (40%), and Epo-based therapy (32%). Among the 25 patients that received Epo-based therapy, all 3 patients with favorable cytogenetic abnormalities achieved major responses in anemia whereas such responses were seen in none of the 9 patients with unfavorable cytogenetic abnormalities and only 1 of 13 patients with normal cytogenetics (p=0.004). Furthermore, 3 of the 4 major responders were heterozygous for JAK2V617 and one carried the wild-type allele. In contrast, none of the 3 JAK2V617 homozygotes showed any type of response but all 3 also displayed unfavorable cytogenetics. None of the 7 ThalPred-treated patients carried favorable cytogenetics and yet 3 achieved major responses including one with unfavorable cytogenetics. JAK2V617 mutational status was heterozygous in 1 and wild-type in the other two. There were no major responses among the 5 Andro-treated patients despite the presence of favorable cytogenetic abnormalities in 2 patients. In order to investigate the relationship between cytogenetic profile and JAK2V617 mutational status further, we referred to an expanded database of 116 patients and found the incidence of unfavorable cytogenetics to be higher in JAK2V617 homozygotes compared to non-homozygotes (57% vs. 31%; p=0.16) Conclusion: The current study suggests clustering of favorable cytogenetic abnormalities in MMM with anemia response to Epo therapy and unfavorable cytogenetic abnormalities with homozygosity for JAK2V617 mutation.

Abstract Background: The occurrence of an activating JAK2 mutation (JAK2 V617F) has been reported in a variable proportion of patients with bcr/abl-negative myeloproliferative disorders including myelofibrosis with myeloid metaplasia (MMM). In the current study, we report mutational frequencies in each of the 3 MMM variants as well as examine the prognostic relevance of the mutation in a large group of patients from a single institution. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from peripheral blood mononuclear cells (PBMC) from all study patients (n=157). Concomitant analysis was also performed in DNA derived from purified granulocytes (n=57), CD34-positive cells (n=25), and T cells (n=19). Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study cohort included 157 consecutive patients with MMM including 117 with agnogenic (AMM), 22 with post polycythemic (PPMM), and 18 with post thrombocythemic (PTMM) myeloid metaplasia. The overall detection rate for JAK2 V617F in PBMC was 51% (homozygous in 5.7%) and was significantly (p=0.0002) higher in PPMM (91%; homozygous in 18%) compared to either AMM (45.3%; homozygous in 2.6%) or PTMM (38.9%; homozygous in 11.1%). In AMM (n=117), the presence of JAK2 V617F was significantly associated with an older age at diagnosis (p=0.03), and a history of both thrombosis (p=0.01) and pruritus (p=0.005). No other statistical associations were evident and the presence of the mutation did not predict leukemic transformation. Furthermore, multivariate analysis for overall survival of patients with AMM identified only age and the Dupriez prognostic score as independent prognostic factors; JAK2 V617F had no prognostic significance. Finally, the mutation was not found in T cells and its detection rate in either granulocytes or CD34-positive cells was similar to that in PBMC. Conclusion: JAK2 V617F is a myeloid lineage-specific event, its incidence in MMM is significantly higher with an antecedent history of polycythemia vera, and its presence in AMM does not affect prognosis but is associated with PV-characteristic clinical features.

Abstract Background: Conventional cytogenetic studies in myeloproliferative neoplasms (MPNs) have provided pathogenetic, diagnostic and prognostic information and in certain instances guide therapeutic decisions. It is therefore reasonable to hypothesize that a higher resolution examination of chromosomes might reveal additional changes that are scientifically as well as clinically relevant. Methods: Granulocyte DNA from 70 patients (median age 56 years, range 18–84; 43 males) with classic BCR-ABL-negative MPNs were studied: 27 with primary myelofibrosis (PMF), 22 with polycythemia vera (PV), 12 with essential thrombocythemia (ET), 6 with post-ET MF and 3 with post-PV MF. Twelve healthy volunteers were used as controls. Cytogenetic information, collected at variable times before the current study, was available in 57 patients. Samples were analyzed by comparative genomic hybridization (CGH) with a gender matched normal control using oligoarrays with either 44,000 or 105,000 oligonucleotides. Data were analyzed with CGH Analytics (Agilent). Copy number changes were considered significant (CNCs) if they were defined by 4 or more adjacent oligonucleotides spanning at least 150,000 base pairs, contained at least one gene, and were not identified in the Database of Genomic Variants. Results: Array CGH results were equivocal in 6 cases, which were therefore excluded from further analysis. Among the 12 normal controls, granulocyte DNA displayed no CNCs. The percentage of patients with CNCs was 52% for PMF, 32% for PV and 17% for ET. In addition, 2 of 6 patients with post-ET MF and 1 of 2 patients with post-PV MF displayed CNCs. There was no correlation between the occurrence of CNCs and JAK2V617F mutational status in PMF and ET. Array CGH revealed CNCs in 16 of 49 patients (33%) with normal and 7 of 8 patients (88%) with abnormal cytogenetic findings; one patient with t(2;12)(p13;q14) in 19 of 20 metaphases detected at 9 months prior to sample collection for the array CGH study displayed no CNCs. In patients with abnormal cytogenetic findings, array CGH also detected imbalances at chromosomal translocations, including t(1;6), t(9;11), and t(1;18), as well as additional abnormalities that were not detected by cytogenetic analysis. A total of 23 single occurrence CNCs were documented and included all the ET [del(2)(q31.3q32.1), del(10)(q11.21q11.22)] and post-ET MF [del(2p12), del(Xq12)] associated CNCs. In contrast, several specific abnormalities were shared between PV and PMF including del(6p22) and dup(15q11.2). As expected, del(13q) and del(20q) abnormalities were recurrent in PMF. Conclusions: Conventional cytogenetic analysis underestimates the extent of genomic gains and losses in BCR-ABL-negative MPNs. The current study suggests that oligonucleotide array CGH is more sensitive to detect small genomic alterations and we are currently studying higher resolution arrays in a larger group of patients to detect additional genomic alterations as well as assess overall clinical utility. The current data also suggest that chromosomal translocations are commonly associated with imbalances.

Abstract Background: Myelofibrosis with myeloid metaplasia (MMM) currently has limited therapeutic options. BAY 43-9006 (sorafenib; Bayer Pharmaceuticals) is a oral multikinase inhibitor with both inhibitory properties against the Raf/ MEK survival pathway and VEGF that are a potential therapeutic targets for the myeloproliferation and apoptotic resistance seen in this disorder. We evaluated the activity of this agent through inhibition of aberrant in vitro myeloid colony growth, as we have previously successfully utilized in this disorder (Leukemia2003;17(5):849). Methods: Peripheral blood mononuclear cells (PBMC) were isolated and plated on the Methocult™ (StemCell Technologies) colony assay media. Dilutions of BAY 43-9006 (0–10 μM) (Phase I trial sustained concentrations of 10 μM (JCO2005;23:965) was added for either a 24 hour incubation, or for 14 days. Myeloid colonies were then quantified according to standards established by the manufacturer. Subsequent assays were performed combining BAY 43-9006 with the pro-apoptotic agents arsenic trioxide (ATO) or adaphostin (ADA) (both at 1.25μM). Finally, immunoblotting was performed on protein extracted from treated primary MMM cells to quantify the downregulation of Mcl-1 (shown to be crucial for the pro-apoptotic effects of BAY 43-9006; Yu et. al. Oncogene 2005). Results: PBMC were isolated from 12 MMM patients and 10 controls (normal n=5, other myeloproliferative (MPD) n=5). Twenty-four hour exposure of cells to the agent was insufficient to suppress aberrant colony growth. Continuous exposure (for 14 days) of the agent led to a median inhibitory concentration-50% (IC50) of 12 μM for MMM (compared to 7.1μM for normals, and 5μM for MPD controls). Although no specificity could be deduced from these latter results, 4 MMM patients had an IC50 of <10μM (range 1.25 to 8.6μM). In contrast, the other 8 patients were quite resistant IC50 of > 10μM (higher than clinically achievable ranges). Subsequently we evaluated whether there might be benefit to use of BAY43-9006 in combination with a pro-apoptotic agent (i.e. ATO or ADA). No significant activity was seen combining ADA with BAY43-9006. When BAY43-9006 was combined with ATO, the median IC 50 for the BAY compound decreased to 2μM (range 1.25 to 16), however there was not a significant increase in colony inhibition over ATO alone. In order to evaluate whether the lack of activity of BAY43-9006 observed in colony formation assays was secondary to a lack of downregulation of the established target Mcl-1, we immunoblotted for Mcl-1 (after in vitro incubation) in a subset of MMM patients. Western blots demonstrated successful downregulation of Mcl-1 by Bay 43-9006 in MMM samples, even in samples with clear in vitro resistance to the drug (i.e. IC50 > 10 μM). Conclusions: The multi-kinase inhibitor BAY43-9006 appears to have minimal activity against primary cells isolated from MMM patients. Although, suppression of aberrant colony growth was noted in a subset of patients the sensitivities would not support specificity when compared to controls. Although BAY 43-9006 was observed to down regulate the anti-apoptotic target Mcl-1, in patient samples, no detectable inhibition of colony formation was observed in 8/12 MMM samples. These preliminary results do not appear to support a clinical trial of BAY 43-9006 in MMM, however combination of this multi-kinase inhibitor with other pro-apoptotic agents should be evaluated.

Abstract In a prospective study of 100 subjects, an ELISA assay was used to measure platelet-rich plasma (PRP) serotonin levels in patients with polycythemia vera (PV; n=26), essential thrombocythemia (ET; n=14), myelofibrosis with myeloid metaplasia (MMM; n=26), secondary or spurious polycythemia (SP; n=20) and controls (n=14). Neutrophil PRV-1 expression was concurrently assayed by real-time PCR in 69 patients (23 PV, 17 SP, 12 ET, 7 MMM, 10 controls). Table 1 outlines the median and range of measured values across the different study groups. Table 1 Controls SP PV ET MMM Age in years N/A 49 (18–77) 64 (32–83) 52 (18–81) 60 (22–80) Plt count x 109/L N/A 249 (157–371) 509 (191–940) 613 (284–1100) 259 (64–955) PRP serotonin; ng/109 plts 567 (360–1071) 609 (369–1780) 205 (0–496) 385 (137–1026) 90 (0–278) PRV-1/GAPDH ratio 1.28 (1.20–1.43) 1.23 (1.13–1.38) 1.03 (0.84–1.28) 1.24 (0.99–1.44) 1.25 (1.11–1.40) A highly significant difference (p < 0.0001), in PRP serotonin levels, was seen among the different disease categories and control group (table 1). The lowest levels were registered for MMM and PV with a statistically significant difference between the two groups (p = 0.015) but a large degree of overlap. The results in both were significantly as well as markedly lower than those of controls as well as SP with only slight overlap in the PV group and no overlap in the MMM group (p < 0.0001; table 1). The PRP serotonin levels in patients with ET were significantly reduced compared to both controls (p = 0.019) and SP (p = 0.002) and significantly increased compared to both PV (p = 0.0008) and MMM (p < 0.0001). However, there was a substantial degree of overlap in values between ET and other disease categories and controls. Neither aspirin nor cytoreductive therapy correlated with PRP serotonin levels (p = 0.116 and p = 0.148, respectively). Similarly, within a specific disease category, PRP serotonin level did not significantly correlate with platelet count, hemoglobin level, leukocyte count, spleen size, disease duration, or gender. However, an inverse correlation with age was noted only in MMM. In general, there was not a significant correlation between PRP serotonin level and neutrophil PRV-1 expression in each of the three myeloproliferative disease variants. Tests of sensitivity, specificity, and accuracy in distinguishing PV from SP were 92, 95, and 93% for the PRP serotonin assay and 78, 95, 86% for neutrophil PRV-1 assay, respectively. The current study suggests that PRP serotonin concentration might be considered as one of several biological markers that complement each other for the diagnosis of PV.

Abstract BACKGROUND: The potential risks of tumor growth promotion and thromboembolism associated with erythropoietin (Epo) therapy warrant cautious use of erythropoiesis stimulating agents (ESA). Recent data suggest a surprising and significant association between ESA use and increased incidence of leukemic transformation in primary myelofibrosis (PMF) (data from an analysis separately submitted for American Society of Hematology meeting, 2007). It is prudent, therefore, to avoid unnecessary ESA exposure in PMF, especially in patients with low likelihood of treatment response. METHODS: Diagnosis of PMF was based on World Health Organization criteria. Study inclusion required availability of treatment history with ESA and adequate follow-up. We excluded patients who were receiving hemoglobin-improving agents concomitantly with an ESA, such as prednisone, androgens, danazol, or thalidomide. Transfusion dependency was defined by a history of minimum 2 units of red blood cell transfusions in a 1-month period for a hemoglobin < 8.5 g/dL, not associated with bleeding. An adequate treatment response was defined as a minimum 2.0 g/dL increase in hemoglobin level or becoming transfusion-independent over a minimum 1-month period. RESULTS: A master database of PMF patients seen at the Mayo Clinic from 1976 through 2006 was queried to identify 43 patients (median age 67 years; 35% females) treated with ESAs and in whom adequate follow-up was available to determine response. Prior to ESA treatment, median hemoglobin was 9.2 g/dL (range 7.0–11.9), and 16 (37%) were red blood cell transfusion-dependent. In 25 patients, pre-treatment serum Epo level was available (median 34 mU/mL; range 2–323). Among the 43 study patients, 16 (37%) were previously treated with other PMF-specific therapies, and 18 (42%) were receiving cytoreductive treatment concurrent with an ESA. Overall, 23% of patients (10 of 43) responded to treatment with ESA. The response rate was 0% (0 of 16) in the transfusion-dependent group compared to 37% (10 of 27) in the transfusion-independent group (p=0.007). Among the transfusion-independent patients, the proportion of ESA responders was significantly higher in the presence of a baseline hemoglobin level < 10 g/dL prior to treatment (p=0.009). ESA response was not correlated with baseline serum Epo level in the context of either all 43 study patients (p=0.68) or the 27 transfusion-independent patients (p=0.39). ESA response was not affected by PMF-specific treatment history (p=0.46), use of concurrent cytoreductive therapy (p=0.28), cytogenetic findings (p=0.62), JAK2V617F presence (p=0.69) or its allele burden at time of initial PMF diagnosis (p=0.16). CONCLUSIONS: Clear benefit of ESAs in PMF is limited to red blood cell transfusion-independent patients and those with baseline hemoglobin levels less than 10 g/dL.

Abstract Background: Systemic mastocytosis (SM) has a varied presentation ranging from indolent forms with limited morbidity over many years to the rapidly fatal mast cell leukemia (MCL). This heterogeneity complicates clinical decision making regarding choice and timing of therapy. The aim of this study was to evaluate the prognostic value of the 2001 World Health Organization (WHO) classification of SM, and to refine risk stratification within SM sub-groups to facilitate clinical decision making. Information on KITD816V and JAK2V617F mutation analysis and clinical correlates is also provided. Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those patients where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. KITD816V mutation analysis was performed by DNA sequencing. JAK2V617F was screened by allele-specific quantitative PCR analysis. Results: i. Clinical characteristics at presentation: A total of 342 SM patients were identified (154 female; median age at diagnosis=57 years; range 19 to 87 years). Per the 2001 WHO classification, 159 had indolent SM (ISM; median age=49 years), 41 aggressive SM (ASM; median age=65 years), 138 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD; median age=65 years), and 4 MCL (median age=55 years) (p-value for age <0.0001). 140 (41%) patients had urticaria pigmentosa (UP) (63% with ISM; p<0.0001), 160 (47%) mast cell (MC) mediator release symptoms (69% with ISM; p<0.0001), 107 (31%) skeletal symptoms, and 142 (42%) constitutional symptoms (61% with ASM/SM-AHNMD versus 19% with ISM; p<0.0001). 123 patients (36%) had palpable splenomegaly (57% with SM-AHNMD; p<0.0001). 56 patients (16%) exhibited prominent eosinophilia (absolute eosinophil count >1500/mcl) – 31% and 22% with SM-AHNMD and ASM, respectively, versus 3% with ISM (p<0.0001); 12 of 23 patients (52%) with eosinophilia who were tested carried the FIP1L1-PDGFRA mutation. Serum tryptase (normal <11.5 ng/mL) was measured in 160 patients (47%) and almost all (96%) had an elevated level (median=64 ng/mL; range=4 to 2000 ng/mL; 33 patients had >200 ng/mL). ii. Disease course and prognostic factors After a median follow-up of 20.7 months (range=0–417), 153 deaths were recorded. 17 patients (5%) transformed to acute myeloid leukemia (AML; n=14) or MCL (n=3), most frequently with SM-AHNMD (11% versus 0% for ISM; p<0.0001). The median overall survival was 63 months (ISM=198 months, ASM=41 months; SM-AHNMD=24 months, and MCL=2 months; p<0.0001). Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and WHO sub-type (p<0.0001), age at diagnosis (p<0.0001), history of weight loss (p=0.01), anemia (p=0.007), thrombocytopenia (p=0.0008), hypoalbuminemia (p=0.0008), and excess BM blasts (>5%; p=0.004). A similar analysis in ISM identified anemia (p=0.04), hypoalbuminemia (P=0.002), and BM MC ≥ 30% (p=0.03) as independent predictors of inferior survival; the corresponding parameters in ASM were age at diagnosis (p=0.002) and history of weight loss (p=0.003). iii)KITD816V andJAK2V617F analysis Archived bone marrow was available in 165 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 28%: ISM 20%, ASM 29%, SM-AHNMD 33% (p=0.4). In patients with ASM (p=0.003) and SM-AHNMD (p=0.001), detection of KITD816V was associated with inferior survival. Greater than 1% (range 1–57) JAK2V617F allele burden was detected in 6 patients (4%); all belonged to SM-AHNMD including 4 with non-MC lineage myeloproliferative neoplasm. Conclusions: The current study confirms the prognostic value of the WHO subclassification of SM and identifies advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess BM blasts as additional adverse prognostic factors. In ISM, presence of anemia, hypoalbuminemia, or BM MC ≥ 30% is associated with inferior survival. In ASM and SM-AHNMD, BM KITD816V allele burden might influence survival. JAK2V617F is rare in SM and when found, it is almost always in the context of SM-AHNMD.

Abstract Background: Systemic mastocytosis (SM) and hypereosinophilic syndrome (HES) are JAK2V617F-negative orphan diseases without effective long-term therapeutic options. Both of these diseases share clinical and laboratory features with FIP1L1-PDGFRA-positive chronic eosinophilic leukemia (CEL). SM is associated with KITD816V or other KIT mutations. HES is currently not molecularly characterized. The exquisite sensitivity of FIP1L1-PDGFRA to inhibition by imatinib mesylate (IM) underlies the efficacy of this agent in treating FIP1L1-PDGFRA-positive CEL. In contrast, KITD816V is resistant to inhibition by IM, and is only moderately sensitive to dasatinib (cellular IC50=micromolar concentrations), which may explain the relatively disappointing clinical responses in SM with these agents. TG101348 is an orally bioavailable JAK2-selective inhibitor that is currently being tested in a Phase 1 clinical trial for the treatment of myelofibrosis. The aim of the current study was to evaluate TG101348 (relative to the tyrosine kinase inhibitors IM, dasatinib, and sorafenib) for its ability to: inhibit growth of leukemic cell lines that carry KITD816V and FIP1L1-PDGFRA; and inhibit the in vitro growth of eosinophil colonies derived from progenitor cells from HES patients. Methods: The following cell lines were used for in vitro experiments: HMC-1, a mast cell leukemia line (KITD816V-positive); EOL-1, a CEL derived line (FIP1L1-PDFRA-positive), Ba/F3 T674I, a Ba/F3 line that express the IM-resistant FIP1L1-PDFRAT674I mutation, and HEL, a human erythroleukemia line (JAK2V617F-positive). Cell proliferation assays were performed in triplicate using the XTT assay (Leukemia. 2007 21:1658). Drug concentrations in these experiments ranged from 2.9 × 10−12 M to 10−4 M. JAK Inhibitor I (Calbiochem) is a non-selective JAK inhibitor tool compound. Eosinophil colonies were obtained by plating PBMCs from healthy controls or HES patients in methylcellulose, in the presence of IL-3, IL-5, and GM-CSF. Effects of IM and TG101348 on eosinophil colony growth were studied in parallel experiments. Results: Consistent with prior observations, the HMC-1 cell line was resistant to inhibition by IM (IC50>10 mM), but was moderately sensitive to dasatinib (Table). In contrast, TG101348 inhibited HMC-1 growth at nanomolar concentrations, similar to its effect on HEL cells that harbor the JAK2V617F mutation. TG101348 inhibited HMC-1 growth 53-fold and 4-fold more potently than IM and dasatinib, respectively (Figure). Sorafenib, a multikinase inhibitor that reportedly has activity against KIT kinase (enzyme IC50=68 nM), had only a limited effect on HMC-1 growth (IC50=4000 nM). In contrast to HMC-1, EOL-1 growth was inhibited by IM, dasatinib, sorafenib, and TG101348 at picomolar concentrations (Table). The T674I mutation (in the context of FIP1L1-PDGFRA) corresponds to the T315I mutation in BCR-ABL – it occurs infrequently in CEL and is usually found in the context of transformation to acute leukemia. Growth of Ba/F3 cells harboring FIP1L1-PDFGRA-T674I has been shown to be inhibited by sorafenib (confirmed in this report; Table) and nilotinib, at nanomolar concentrations. TG101348 is less potent at inhibiting growth of these cells, with cellular IC50 of ~2 mM – pharmacokinetic data from the ongoing Phase I study of TG101348 shows that this concentration is achievable in plasma with once daily dosing. Drug Cellular IC50 HMC-1 (nM) EOL-1 (pM) Ba/F3 T674I (nM) HEL (nM) Imatinib 18800 4 10700 - Dasatinib 1300 <1 30900 - Sorafenib 4000 2 16 - Jak Inhibitor I 926 30 4300 ? TG101348 355 <1 1950 300 Figure Figure Conclusions: The JAK2-selective inhibitor TG101348 is a potent inhibitor of KITD816V and FIP1L1-PDGFRA, warranting clinical trials using this drug in SM and FIP1L1- PDGFRA-positive CEL. Data regarding TG101348 effects on signaling intermediates in leukemic cell lines and on eosinophil colony growth will be presented at the meeting – data from the latter experiments may indicate a therapeutic role for TG10134 in the treatment of patients with HES.