Powell, Heather L.

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: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534), and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET). METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide (THAL) daily, etanercept (ETAN) 25 mg subcutaneously twice-a-week, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide and etanercept. RESULTS: 15 patients were enrolled to the trial (median age 66 years, range, 54–77; 14 males). Twelve patients (80%) completed the planned three cycles (i.e. 3 months) of treatment (progression (n=2) and patient choice (n=1) accounted for the withdrawals), nine (60%) patients with evidence of response continued on an additional three months of THAL/ETAN alone (of which 3 progressed prior to study completion). At enrollment, 11 patients (73%) were anemic (7 (47%) were red cell transfusion dependent), 7 (47%) were at least moderately thrombocytopenic (platelet count < 100 x 109//L), and 6 had severe constitutional symptoms (night sweats or disease related fever). Anemia response were seen in 6 (of the 11 eligible; 54%) patients (2 in the non-transfusion dependent (sustained increases of 1.5 and 4.9 g/dL hemoglobin); 4 in transfusion dependent (1 transfusion independent, 3 >50% decrease in transfusion requirements)). All thrombocytopenic patients experienced an increase in platelet counts (median 55% increase (range(10–229) with 2 returning to the normal range (>150 x 109/L)). A greater than 50% decrease in organomegaly was documented in 3 patients with splenomegaly (25% of eligible), and 1 patient with hepatomegaly (25%). Constitutional symptoms resolved, or signifcantly improved in all afflicted. Therapy overall was well tolerated with the majority of toxicities being transient. Neuropathy (grade 1; n=3; grade 2; n=1) and hyperglycemia (grade 2; n=1) were minimal. The only greater or equal to grade 3 toxicities were (grade 3: elevated bilirubin, infection, and blurred vision; grade 4: anemia). Follow-up bone marrow examination, when available, did not show post-treatment histological changes. CONCLUSIONS: This phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.

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 BACKGROUND: Bone marrow fibrosis might represent either a primary myeloid malignancy such as myelofibrosis with myeloid metaplasia (MMM) or a reactive phenomenon associated with other malignancies or inflammatory/infectious processes. The development of myelofibrosis in the context of a plasma cell dyscrasia is uncommon and incompletely understood from the standpoint of both pathogenesis and clinical implications. We sought to determine the origin and clinical ramifications of this phenomenon based on a consecutive series of patients with plasma cell disorders (PCD). METHODS: A retrospective search of institutional databases was performed for clinical cases of PCD with the concomitant observation of myelofibrosis. Bone marrow aspirates and trephines from the time of diagnosis of both PCD and myelofibrosis were reviewed. Clinical history was abstracted for clinical manifestations as well as disease course. DNA from archived cytogenetic pellets, when available, from the time of diagnosis, was screened for the presence of JAK2V617F. RESULTS: Among 7587 patients with a PCD seen over the last 30 years at our institution, 29 (0.3%) displayed concomitant myelofibrosis (median age 59 years, range 27–77; 52% males). The specific PCD was multiple myeloma in 25 patients, smoldering myeloma in 2, plasmacytoma in 1, and heavy chain disease in 1. Four patients (14%) had organomegaly and bone marrow histological features most consistent with MMM. Otherwise, myeloproliferative disorder (MPD)-characteristic features were infrequent and included leukocytosis and/or thrombocytosis in only 2 patients. None of the patients displayed thrombohemorrhagic complications. Archived DNA was available in 7 cases without either clinical or bone marrow histological evidence of a MPD and yet revealed the presence of JAK2V617F in 6 of the 7 cases (86%). In addition, one other patient without MPD phenotype displayed a monosomy 5 abnormality. To date, 2 patients, one with and one without a MPD phenotype underwent leukemic transformation. All other causes of death were related to PCD-associated complications. Median survival (Kaplan-Meier) for the patients with multiple myeloma was 50 months. Survival stratified by the myeloma international staging system (ISS) was similar or better than expected (published controls of 62, 44, and 29 months): median survivals for this series of 73, 51 and 37 months for stage I, II, and III disease, respectively. CONCLUSIONS: Myelofibrosis that accompanies a PCD often represents an underlying MPD and does not negatively impact the natural history of the associated PCD.

Abstract BACKGROUND: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534) and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET). METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide daily, etanercept 25 mg subcutaneously twice-a-week, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide and etanercept. RESULTS: To date 12 patients have been enrolled to the trial (median age 67 years, range, 54–77; 11 males). At enrollment, all patients were severely anemic (6 were red cell transfusion dependent), 6 were at least moderately thrombocytopenic (platelet count < 100 x 109//L), and 4 had severe constitutional symptoms. The responses to date included resolution of constitutional symptoms in all 4 pertinent patients, a 12–326% improvement in thrombocytopenia in 4 of 6 pertinent patients, and anemia response in 4 patients including one patient who became red cell transfusion independent. A more than 50% decrease in splenomegaly was documented in 3 patients. Only one patient experienced greater or equal to grade 3 toxicity (i.e. elevated bilirubin and infection). Follow-up bone marrow examination, when available, did not show post-treatment histological changes. CONCLUSIONS: The preliminary findings from the current ongoing phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, at least preliminarily the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.

AbstractA somatic mutation in the JH2 autoinhibitory domain of the Janus kinase 2 (JAK2) tyrosine kinase was recently described in polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. The prevalence of this mutation in either “atypical” myeloproliferative disorders (MPDs) or the myelodysplastic syndromes (MDSs) is unknown. Bone marrow–derived genomic DNA from 245 patients—119 with chronic myelomonocytic leukemia (CMML), 101 with MDS, 11 with hypereosinophilic syndrome (HES), 8 with systemic mastocytosis (SM), and 6 with chronic neutrophilic leukemia (CNL)—was screened for the JAK2 V617F mutation. A mutant allele was detected in 11 patients: 3 with CMML (3%), 5 with MDS (5%), 2 with SM, and 1 with CNL. Interestingly, one of the patients with SM and the patient with CNL with JAK2 V617F had a history of lymphoma, and this patient with SM also had associated myelofibrosis and CMML. The current observation strengthens the specific association between JAK2 V617F and classic MPD, but also suggests an infrequent occurrence in other myeloid disorders.

Previous studies demonstrated that ataxia telangiectasia mutated– and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.