JAK2V617F-positive Myelofibrosis Research Articles

Paroxysmal Nocturnal Hemoglobinuria in the Context of a Myeloproliferative Neoplasm: A Case Report and Review of the Literature

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolytic anemia and thrombosis and is notoriously associated with aplastic anemia and myelodysplastic syndromes. Rarer associations include myeloproliferative neoplasms (MPNs), which are also burdened by increased thrombotic tendency. The therapeutic management of this rare combination has not been defined so far. Here, we describe a 62-year-old man who developed a highly hemolytic PNH more than 10 years after the diagnosis of MPN. The patient started eculizumab, obtaining good control of intravascular hemolysis but without amelioration of transfusion-dependent anemia. Moreover, we performed a review of the literature regarding the clinical and pathogenetic significance of the association of PNH and MPN. The prevalence of PNH clones in MPN patients is about 10%, mostly in association with JAK2V617F-positive myelofibrosis. Thrombotic events were a common clinical presentation (35% of subjects), sometimes refractory to combined treatment with cytoreductive agents, anticoagulants, and complement inhibitors. The latter showed only partial effectiveness in controlling hemolytic anemia and, due to the paucity of data, should be taken in consideration after a careful risk/benefit evaluation in this peculiar setting.

Next-Generation Sequencing with a 54-Gene Panel Identifies Unique Mutational Profile and Prognostic Markers in Chinese Patients with Myelofibrosis

Introduction and objectives: Myelofibrosis (MF) has the worst outcome amongst various myeloproliferative neoplasms. Its prognosis is determined by clinicopathologic features and mutations in key driver genes. An increasing number of gene mutations involving various biological pathways in myeloid malignancies has been discovered. The prognostic significance of these mutations have not been clearly defined. In this study, we aim to describe the genomic characteristic in a large cohort of MF patients and identify clinical and molecular predictors of outcome.Methods: We evaluated the genetic profile of 101 patients with MF (primary, N=70; secondary, N=30) using next-generation sequencing with a 54-gene panel comprising: ABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA, CSF3R, CUX1, DNMT3A, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2. Multivariate cox regression analysis was used to determine prognostic factors for overall survival (OS) and leukemia-free survival (LFS).Results: We identified mutations in 39 genes implicated in myeloid malignancies (Figure 1A). 96 patients (95%) with MF had a mutation in 1 or more genes: 14 patients (13.9%) had 1 mutation, 38 patients (37.6%) had 2 mutations, 18 patients (17.8%) had 3 mutations, 15 patients (14.9%) had 4 mutations, 7 patients (6.9%) had 5 mutations and 4 patients (4%) had 6 or more mutations. TET2 / JAK2V617F (16 patients, 15.9%), ASXL1 / JAK2V617F (12 patients, 11.9%) and ASXL1/CALR (10 patients, 9.9%) were the most frequently co-mutated genes (Figure 1B). Other JAK2 variants occurred concomitantly with JAK2V617F in 10 patients (9.9%) and CALR mutations in 4 patients (4%) mutations. Other frequently concomitant mutations included CUX1 / JAK2V617F (6 patients, 5.9%), EZH2 / JAK2V617F (6 patients, 5.9%), RUNX1 / JAK2V617F (5 patients, 5%), SF3B1 / JAK2V617F (5 patients, 5%), SETBP1 / JAK2V617F (4 patients, 4%) and ZRSR2 / JAK2V617F (4 patients, 4%).The median follow-up of the cohort was 49 (1-256) months. The 5-year and 10-year OS were 66.3% and 35.4%. The 5-year and 10-year LFS of were 84% and 63.3%. There were no statistically significant differences in OS and LFS between primary and secondary MF. Significant negative prognostic indicators were identified on multivariate analysis, including male gender (P=0.044), age > 65 years (P=0.044), Hb < 10g/dL (P=0.001), mutated CUX1 (P=0.003) and mutated TP53 (P=0.043) for OS, and Hb < 10g/dL (P=0.007), mutated TP53 (P=0.043) and mutated IDH2 (P=0.001) for LFS. In primary MF, inferior prognostic indicators included male gender (P=0.031), Hb < 10g/dL (P=0.002), platelet count < 100 x 109/L (P=0.021), mutated TET2 (P=0.011) and mutated CUX1 (P=0.011) for OS; and Hb < 10g/dL (P=0.027), mutated RUNX1 (P=0.019) and mutated DNMT3A (P=0.004) for LFS. In JAK2V617F positive MF, inferior prognostic indicators included mutated ASXL1 (P=0.006) and mutated SRSF2 (P<0.001) for OS; and mutated U2AF1 (P=0.037) for LFS.Conclusion: Our study demonstrated unique molecular profiles and prognostic predictors of outcome in Chinese patients with MF. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic Effect of Imatinib and Ruxolitinib in a Patient with JAK2V617F positive Myelofibrosis and Concomitant BCR-ABL1 positive Chronic Myeloid Leukemia

Introduction:JAK2V617Fmutation is present in majority of polycythemia vera (PV) and in 50-60% of patients with primary myelofibrosis (MF). JAK1/2 inhibitor ruxolitinib (RUX) is approved for treatment of both PV and intermediate/high risk MF. Two independent randomized studies showed the benefit of RUX in MF with significant reduction in spleen volume and improvement in constitutional symptoms compared to best supportive care. Clonal evolution may occur in PV and MF upon acquisition of new mutations and transformation to acute myeloid leukemia. Rarely, patients with JAK2V617Fpositive PV or MF have also concomitant BCR-ABL1 fusion gene with phenotype of chronic myelogenous leukemia (CML). Imatinib, a BCR-ABL1 inhibitor, induces long-term cytogenetic and molecular remissions in CML. Single agent imatinib has shown no clinical benefit in MF patients despite in vitro efficacy (Gaikwad et al Exp. Hematol. 2007). We describe here a patient with JAK2V617F positive post-PV MF who later developed concomitant BCR-ABL+ CML. Splenomegaly was initially resolved by RUX but returned coincidently with the diagnosis of CML and was again normalized by imatinib alone. Massive splenomegaly reappeared along with elevated hematocrit on imatinib and was resolved by the addition of low dose RUX.Case report: A 71-year old woman initially presented in September 2011 with isolated erythrocytosis and splenomegaly. A diagnosis of PV was established based on the presence of the JAK2V617Fmutation and hypercellular marrow. Bone marrow karyotype was normal (Table 1). The patient was randomized to pegylated interferon arm in the MPD Consortium study. A complete hematologic response was achieved after nearly 2 years on maximal dose 180 mcg weekly. In early 2015, she developed constitutional symptoms, and progressive splenomegaly. Progression to post-PV MF was confirmed on bone marrow evaluation. The patient was taken off study and started on RUX 20 mg BID. She developed severe anemia 4 months later requiring dose reduction to 10 mg BID. The anemia markedly improved, while JAK2V617F allele burden remained high at 88% with normal cytogenetics. In December 2017, the patient developed anemia, fatigue and rapid regrowth of spleen. Marrow karyotype showed Philadelphia (Ph) chromosome in 25% of the cells analyzed, PCR was positive for BCR-ABL1 p210, and JAK2V617F allele burden was reduced to 56.4%. In January 2018, she started imatinib 400 mg daily and RUX was discontinued due to toxicity concerns. In April 2018, her hematocrit increased and massive splenomegaly returned. Restart of initial RUX 20 mg BID was followed by severe pancytopenia which normalized when RUX was reduced to 5 mg BID with continuing imatinib regimen. After 8 weeks, splenomegaly completely resolved (Figure 1). A complete cytogenetic response and major molecular response were achieved after 3 months and 6 months of combination therapy respectively.Methods: Mononuclear cells from peripheral blood collected in June 2018 were plated in methylcellulose without erythropoietin or other cytokines. After 14 days, colonies were plucked, with ½ of each colony used for JAK2V617F allele-specific PCR and ½ prepared as cytospins for BCR-ABL fluorescent in situ hybridization (FISH) with the Vysis LSI BCR/ABL Dual Color, Dual Fusion Translocation Probe from Abbott Laboratories.Results: Analysis of 3-6 EPO-independent colonies of each CFU-E, G, M, GM, GEMM, BFU-E showed no BCR-ABL1 positive cells, while a single CFU-GEMM colony was positive and also homozygous for JAK2V617F. Frozen marrow cells from the time of the original diagnosis are being analyzed.Conclusion: The recurrence of splenomegaly in this patient following single agent RUX indicates emergence of novel BCR-ABL1+ clone. While single agent imatinib temporarily normalized the splenomegaly and leukocytosis for several months, MF phenotype reoccurred. Splenomegaly resolved completely in 8 weeks with the addition of very low dose RUX to standard dose imatinib, providing the first clinical observation of in vivo synergism of RUX and imatinib. We are investigating serial samples during her course, which will be presented at the meeting. The analysis of a single clone demonstrate that BCR-ABL1 mutation did not occur in previously normal dormant stem cell but in JAK2V617F positive progenitors and represent in this pts a novel subclone of previous PV/MF stem cell. DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

AML-Fated Clones Arise in Stem and Progenitor Cells in Myelofibrosis Patients Several Years Prior to AML Diagnosis

In myeloproliferative neoplasms (MPN), somatic mutations in recurrently mutated myeloid leukemia genes, apart from MPN driver genes, are common and can increase transformation risk to acute myeloid leukemia (AML). However, the temporal acquisition of these mutations, the properties of the cells bearing these mutations, and how each mutation cooperates to promote leukemic transformation remains largely unknown. We therefore examined blood and bone marrow samples collected serially from patients with myelofibrosis (MF) who developed AML to further elucidate the genetic basis of leukemic transformation. Specific aims include: 1) determining the timeline of acquisition of mutations that contribute to AML in chronic phase and 2) defining the cell type that harbours the clone fated to initiate AML.Ten patients who transformed to AML following a diagnosis of MF (6 PMF, 3 post-PV MF, 1 post-ET MF) were studied. The time interval from the first MPN collection to AML diagnosis ranged between 1.5 to 6.6 years; and in 4 patients additional MPN samples collected between these time points were available for study. Somatic mutations were identified from whole genome sequencing (WGS) data of AML blasts (CD45dim cells), the MPN clone (CD45highCD33+ and CD34+ cells), and germline references (T-cells or Buccal DNA); and from targeted sequencing results of 54 recurrently mutated myeloid leukemia genes performed on AML samples as part of another study. Stem and progenitor populations (HSC, MPP, LMPP, CMP, MEP, GMP), as well as, mature cell populations (myeloid, erythroid, T-lymphoid, B-lymphoid and NK) were sorted from MPN time points and whole genome amplified using the REPLI-g Single Cell Kit (Qiagen). Somatic mutations were tracked by droplet digital PCR (ddPCR) and/or targeted sequencing.We identified an average of 5 (range 1-8) recurrently mutated myeloid leukemia genes that were somatically mutated in each patient at AML diagnosis, with an average of 5.5 (range 1-11) different mutations affecting these genes. Many of these mutations were present at ≥5% variant allele frequency (VAF) in the MPN clone at the earliest chronic phase time point. In 8 patients, the MPN driver mutation was detected in the MPN clone and AML blasts (MPNdriver-concordant), while in 2 other patients there was discordance as JAK2V617F was present in the MPN clone, but not the AML blasts. Myeloid leukemia genes that were mutated in 2 or more patients included SRSF2 (n=5), ASXL1 (n=4), TET2 (n=4), IDH1/2 (n=4), RUNX1 (n=4), NRAS (n=4), KRAS (n=2), U2AF1 (n=2), PHF6 (n=2), and STAG2 (n=2). We tracked mutations by ddPCR in populations sorted from MPN time points and demonstrated that 10 out of 13 mutations not identified by WGS were in fact present at low VAF in one or more cell populations. Moreover, in 5 patients with MPNdriver-concordant AML, the AML-fated clone was detectable 1.5 to 3 years before AML was diagnosed; and in 4 of these patients the AML-fated clone resided among cells with a HSC or MPP immunophenotype. In order to better establish the reservoir harbouring AML-fated clones, clonal tracking within the stem and progenitor populations by targeted sequencing of a greater number of AML-specific variants is underway. Among the two patients with JAK2V617F-negative AML, we observed in one patient the emergence of the AML-fated clone within one year of AML diagnosis.In summary, we show that clones fated to intiate AML in patients with MF often reside among cells with a HSC or MPP immunophenotype and can appear several years prior to the diagnosis of AML. This strongly suggests that mechanisms beyond the acquisition of somatic driver mutations (including but not limited to epigenetic alterations, acquisition of non-coding mutations, inflammation) are necessary to effectively promote full leukemic transformation. Interestingly, in one patient with JAK2V617F-negative AML (following JAK2V617F-positive MF), the latency period between the appearance of the AML-fated clone and AML diagnosis was much shorter, suggesting that the mechanisms underlying transformation are likely different for these 2 clonal evolutionary pathways. DisclosuresGupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding.

A phase 1 study of the Janus kinase 2 (JAK2)V617F inhibitor, gandotinib (LY2784544), in patients with primary myelofibrosis, polycythemia vera, and essential thrombocythemia

Mutations in Janus kinase 2 (JAK2) are implicated in the pathogenesis of Philadelphia-chromosome negative myeloproliferative neoplasms, including primary myelofibrosis, polycythemia vera, and essential thrombocythemia. Gandotinib (LY2784544), a potent inhibitor of JAK2 activity, shows increased potency for the JAK2V617F mutation. The study had a standard 3+3 dose-escalation design to define the maximum-tolerated dose. Primary objectives were to determine safety, tolerability, and recommended oral daily dose of gandotinib for patients with JAK2V617F-positive myelofibrosis, essential thrombocythemia, or polycythemia vera. Secondary objectives included estimating pharmacokinetic parameters and documenting evidence of efficacy by measuring clinical improvement. Thirty-eight patients were enrolled and treated (31 myelofibrosis, 6 polycythemia vera, 1 essential thrombocythemia). The maximum-tolerated dose of gandotinib was 120mg daily, based on dose-limiting toxicities of blood creatinine increase or hyperuricemia at higher doses. Maximum plasma concentration was reached 4h after single and multiple doses, and mean half-life on day 1 was approximately 6h. Most common treatment-emergent adverse events were diarrhea (55.3%) and nausea (42.1%), a majority of which were of grade 1 severity. Best response of clinical improvement was achieved by 29% of myelofibrosis patients. A ≥50% palpable spleen length reduction was observed at any time during therapy in 20/32 evaluable patients. Additionally, ≥50% reduction in the Total Symptom Myeloproliferative Neoplasm Symptom Assessment Form Score was seen in 11/21 (52%) and 6/14 patients (43%) receiving ≥120mg at 12 and 24 weeks respectively. Gandotinib demonstrated an acceptable safety and tolerability profile, and findings at the maximum-tolerated dose of 120mg supported further clinical testing. Clinicaltrials.gov identifier: NCT01134120.

Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms

Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells.Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN.Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki.Results:CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001).The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05).Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004).Conclusion:CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients. DisclosuresNo relevant conflicts of interest to declare.

A Thrombopoietin Antagonist Is Capable of Affecting Stem and Progenitor Cells of Patients with Myelofibrosis

Aberrant thrombopoietin (TPO)/MPL signaling has been hypothesized to contribute to the pathogenesis of myelofibrosis (MF) (Kaushansky K. J Clin Invest. 2005; 115: 3339; Moliterno AR, et al. N Engl J Med.1998; 338:572). Agents that would be capable of inhibiting this signaling pathway are possible novel therapeutic agents that might be effective for MF treatment. A peptide antagonist of TPO, LCP4, has been created which is highly antagonistic to CB CD34+ cell proliferation and differentiation induced by TPO. In this report we examined the effect of LCP4 on the proliferation of MF CD34+ cells and their differentiation to megakaryocytes (Mk).Elevated levels of TPO (345±114ng/ml, n=13) were detected in MF plasmas as compared to that detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF hematopoietic stem cells (HSC) and progenitor cells (HPC). MF splenic CD34+ cells (2.5×104/mL) were incubated in serum free expansion media (SFEM) alone, with 50 ng/ml SCF+ varying doses of TPO (0, 10, 30,100 ng/mL) or 50 ng/ml SCF +varying doses of TPO+ varying concentrations of LCP4 (0, 10, 50, 100, 500, 1000nM) for 1 or 2 wks. Cells were then enumerated and stained with CD34, lineage cocktail, CD15 and CD41a mAbs. When splenic MF CD34+ cells were cultured in the presence of SCF and varying concentrations of TPO, 100ng/ml TPO resulted in the generation of the greatest numbers of CD34+Lin-, CD41a+CD34-CD15- and CD15+CD34-CD41a- cells, suggesting that TPO promotes the proliferation of MF HSCs/HPCs and the production of MF MKs and myeloid cells. By contrast, after 1 wk of treatment of MF CD34+ cells (n=8; 5 MF spleens and 3 MF PB) with 100nM and 500nM LCP4, the numbers of MF total cells, CD34+Lin- (MF HSCs/HPCs), CD34+CD41a+ (immature Mks), CD41a+CD34-CD15- (mature Mks), CD15+CD34-CD41a- cells (myeloid cells) as well as hematopoietic colonies (HC), including CFU-Mk, CFU-GM, BFU-E/CFU-E, CFU-GEMM, were all significantly reduced (P all <0.05), as compared with cells cultured with SCF and TPO alone (Table 1). Two days of treatment of MF splenic CD34+ with 100nM LCP4 led to a greater degree of apoptosis (15.4% ± 3.5%) as compared with cells treated with cytokines alone (6.75% ± 1.7%, P =0.05; n=4). These findings suggest that LCP4 is able to inhibit the proliferation of MF HSCs/HPCs and the generation of immature and mature Mks as well as myeloid cells in a dose dependent fashion. Furthermore, the treatment of JAK2V617F-positive MF CD34+ cells from 4 JAK2V617F-positive MF patients (JAK2V617F allele burden: 78-90%) with 100nM LCP4 for 7 days reduced the percentage of JAK2V617F-positive HCs by 9-20% and JAK2V617F homozygous HCs by 1-20%. Since LCP4 treatment of MF CD34+ cells resulted in a reduction in the number of HPCs, LCP4 treatment reduced the absolute numbers of JAK2V617F-positive HCs generated by 53.0±4.0% (P<0.0002) and the absolute number of JAK2V617F homozygous HCs by 50.3±4.3% (P<0.0003). These data suggest that LCP4 treatment is able to impair the in vitro generation of MF HPCs and thereby leads to a depletion but not elimination of the number of malignant HPCs.We next examined the effect of LCP4 on MF HSCs by transplanting NSG mice with cells generated after splenic MF CD34+ cells were cultured in the presence of cytokines alone or cytokines plus LCP4 for 1 week. Two months after the transplantation, hCD45+ cells were detected in the BM of recipient mice receiving splenic MF CD34+ cells treated with cytokines alone and were reduced by 28% in mice receiving grafts treated with LCP4. MF CD34+ cells treated with or not treated with LCP4 had similar multilineage differentiation patterns (myeloid, lymphoid and erythroid). These data suggest that TPO antagonist therapy is capable of depleting MF HSCs and HPCs and that therapeutic strategy utilizing such strategies might serve as novel approaches to treating MF.Abstract 819. Table 1Inhibitory effects of LCP4 on MF HSC/HPC proliferation and generation of Mks and myeloid cellsTreatmentCells or HCs Generated (% of Cytokines Alone)Total CellsCD34+ Lin- CellsCD34+CD41a+ CellsCD41a+CD34-CD15- CellsCD15+CD34-CD41a- CellsCFU-MkCFU-GMBFU-E /CFU-ECFU-GEMMSCF+TPO100±0100±0100±0100±0100±0100±0100±0100±0100±0SCF+TPO+LCP4 (100nM)61.3±4.661.6±10.357.3±11.454.9±8.768.9±4.554.4±8.054.0±8.752.6±15.12.1±2.1SCF+TPO+LCP4 (500nM)53.3±8.146.8±7.059.1±17.142.8±8.062.5±7.343.6±10.044.6±5.750.5±12.90±0 DisclosuresWang:The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS): Research Funding.

Effects of Imetelstat on CD34+ Cells of Patients with Myelofibrosis

At present, there is no known therapeutic agent capable of altering the natural history of myelofibrosis (MF); this can only be achieved by allogeneic stem cell transplantation. Imetelstat is a potent and specific inhibitor of telomerase. An investigator initiated clinical trial in MF showed that imetelstat can achieve complete clinical remissions by IWG criteria. This includes the reversal of bone marrow fibrosis and induction of morphologic and molecular remissions in a subset of patients with MF. This suggests that imetelstat has disease-modifying activity in MF (Tefferi A, et al. 55th ASH Annual Meeting; December 7-10, 2013; New Orleans, LA). However, the mechanism by which imetelstat achieves these beneficial effects remains poorly understood. Therefore we investigated the effects of imetelstat on MF CD34+cells.MF splenic or CB CD34+ cells (2.5×104/mL) were incubated in serum free medium (SFEM) alone or SFEM supplemented with 50 ng/ml SCF, 100 ng/ml FLT-3 ligand (FL), 100 ng/ml TPO, and 50 ng/ml IL-3 in the presence of imetelstat or a mismatched oligonucleotide (Mismatch, 1.8uM, 3.75uM, 7.5μM, Geron) or vehicle alone for 2-14 days. Cells were then enumerated and stained with CD34, a lineage cocktail, CD38, CD15, and Glycophorin A (GlyA) mAbs and analyzed by flow cytometry. After the treatment of cultured normal CB CD34+ cells for one week with imetelstat (1.8uM, 3.75uM, 7.5μM), mismatch or cytokines alone, numbers of total cells, Lin-CD34+ (HSCs/HPCs), Lin-CD34+CD38- (primary HSCs), Lin-CD34+CD38+ cells (HPCs), assayable hematopoietic colonies (HC, CFU-GM, BFU-E) as well as CD34-GlyA+ (erythrocytes) and CD34-CD15+ cells (myeloid cells) were not significantly changed (P all>0.05). Moreover, the amount of CB CD34+cells that expressed aldehyde dehydrogenase (ALDH), a functional stem cell marker, was not significantly changed with imetelstat or mismatch treatment (P all >0.05). These findings suggest that imetelstat at the doses studied has minimal effects on normal CB hematopoiesis.By contrast, imetelstat was capable of selectively inhibiting in vitro MF hematopoiesis. After treatment of MF CD34+ cells (n=7; 6 MF spleens and 1 MF PB) for one week with 7.5 uM imetelstat, the total number of MF cells (36.4%, P=0.058), Lin- CD34+ (50.1%, P=0.02), Lin- CD34+CD38- (41.9%, P=0.07), CD34+ALDHhigh (49.1%, P=0.01), Lin-CD34+CD38+ cells (48.1%, P=0.04), CFU-GM (34.6%, P=0.02) and BFU-E (15%, P=0.2) as well as CD34-CD15+ (47.1%, P=0.09) and CD34-GlyA+cells (47.7%, P=0.02) was reduced compared to treatment with mismatch. After one week of culture, individual colonies (CFU-GM and BFU-E/CFU-E) from 3 JAK2V617F-positive MF patients (JAK2V617F allele burden: 78-90%) were plucked and analyzed for the JAK2V617Fusing a nested allele-specific PCR. Two out of three patients did not have any wild type (WT) JAK2 HCs before or following treatment with imetelstat. In one patient with a reservoir of JAK2V617F WT HCs, the percentage of JAK2V617F-positive HCs was reduced to 9% by imetelstat treatment as compared to 41% observed in cultures exposed to cytokines alone and 57% in cultures exposed to mismatch. In this single patient the JAK2V617Fhomozygous HCs were completely eliminated by imetelstat treatment, while 29% and 50% of the HCs remained homozygous after exposure to cytokines alone or mismatch, respectively. To explore the mechanisms by which imetelstat might reduce MF CD34+ cell counts, the ability of the drug to induce apoptosis was studied. After two days of treatment with 7.5uM of imetelstat (n=4), the percentage of MF CD34+ Annexin V+ PI− cells was significantly greater (32.2% ± 6.0%) than cells treated with mismatch (11.21± 1.4%; P=0.002) or cytokines alone (4.7% ± 0.2%, P =0.003). The absolute number of CD34+ Annexin V+ PI− cells present in imetelstat containing cultures was 3.4±1.0-and 5±1.4-fold greater than in cultures treated with mismatch or cytokines alone, respectively. Furthermore, imetelstat treatment did not induce apoptosis of CB CD34+ cells. These findings suggest that imetelstat is capable of selectively promoting the apoptosis and inhibiting the proliferation of phenotypically and functionally defined MF but not normal CD34+ cells. Furthermore, imetelstat in some patients can preferentially deplete malignant MF HPCs. Imetelstat, therefore, represents a potentially promising drug for the treatment of MF which appears to affect primitive MF HSCs. DisclosuresWang:Geron Corporation: Research Funding. Lam:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.

Phase I Study of the JAK2 V617F Inhibitor, LY2784544, in Patients with Myelofibrosis (MF), Polycythemia Vera (PV), and Essential Thrombocythemia (ET)

Abstract▪2814▪This icon denotes a clinically relevant abstract Background:The majority of primary and secondary MF, PV, and ET neoplasms are characterized by gain-of-function activation of JAK2 V617F/STAT5 signaling. Given that wild-type JAK2 plays a pivotal role in multiple stages of hematopoiesis it is desirable to treat JAK2 V617F-positive cases by selectively inhibiting JAK2 V617F while minimizing inhibition of wild-type JAK2 in order not to impede normal marrow function. In murine MPN models and in vitro cell based assays, a novel JAK2 inhibitor, LY2784544, demonstrates selective inhibition of JAK2 V617F/STAT5 signaling and mutant cell proliferation (Ma et al. Abstract 4087 ASH 2010). These results provided the basis for the LY2784544 Phase I trial in MF, PV, and ET patients (I3X-MC-JHTA, NCT01134120). Methods:The primary objectives are to determine the safety and tolerability of LY2784544 and define a recommended oral daily dose for further study in patients with JAK2 V617F-positive MF, ET, or PV. Secondary objectives include determination of pharmacokinetics and evaluation of response using the IWG-MRT response criteria, European Leukemia Net response criteria for PV and ET (EULN), and symptom burden using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) (Scherber et. al. Blood 2011). The study has a standard 3+3 dose-escalation design to define the maximum tolerated dose (MTD). The study was recently amended to allow cohort expansions at lower than MTD doses demonstrating potential clinical merit. Results:To date, 19 patients have been treated: 1 PV, 1 post-ET MF, and 17 MF patients. Four patients discontinued therapy, 1 for each of the following: Adverse Event, progressive disease, investigator decision, and subject's decision. Reported drug-related serious adverse events were seen in 4 patients, and include: creatinine increase (n=4 grade 2), hyperuricemia (2 grade 4), and hyperkalemia (1 grade 1). All resolved within 14 days with temporary treatment interruption. Analysis of collected laboratory studies using the definitions of Cairo and Bishop (BJH 2004, 2010) demonstrated that 1 instance of laboratory tumor lysis syndrome (LTLS) and 3 instances of Grade 2 clinical tumor lysis syndrome (CTLS) occurred. Hyperuricemia and creatinine increases in cases of TLS were reported as dose-limiting toxicities at 240 and 200 mg with 1 and 3 instances at each respective dose. The most frequently reported drug-related AEs across all grades were diarrhea (3 grade 2, 5 grade 1), nausea (3 grade 2, 4 grade 1), transient increased creatinine (4 grade 2), anemia (3 grade 2, 1 grade 1), and hyperuricemia (2 grade 4).A palpable spleen length reduction of at least 35% was seen in 13 of 17 evaluable patients (76% including 16 MF and 1PV). A partial response by EULN for PV involving 100% reduction in spleen size was observed for >10 months. In MF, 7 patients had a ≥50% reduction, including 4 with sufficient stability or duration of follow-up to qualify as a clinical improvement by IWG-MRT. After 5 cycles of therapy, a reduction in bone marrow fibrosis grade was observed in 3 of the 5 MF patients for whom follow-up biopsy results are available. Using the MPN-SAF questionnaire, patients reported improvements in symptom burden within the first 2 cycles of therapy. Fifty-nine percent (59%) reported a ≥50% improvement in the Key MPN Related Symptoms, and 47% reported a ≥50% improvement in the categories of Fatigue-Enjoyment of Life, Fatigue-Walking Ability, itching, and early satiety. A similar level of improvement was reported by 65% of patients for inactivity, while 35% had improvements in bone pain and night sweats. At tested doses, a reduction in mutant allele burden has not been observed. Conclusions:An MTD for daily dosing of LY2784544 was identified as 120 mg. Based on PK analysis, exposure from doses associated with TLS appear to overlap the lower boundary of the predicted biologically efficacious dose (BED) range established in murine JAK2 V617F models. To allow testing of doses within the BED, the dosing regimen has been amended to include a lower dose lead-in period, intended to reduce tumor burden, prior to further testing of dose escalation. This trial amendment will also further evaluate the bone marrow fibrosis response. Disclosures:Verstovsek:Eli Lilly: Research Funding. Off Label Use: Study of LY2784544 - Purpose is to present results of a study. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding. Kloeker Rhoades:Eli Lilly: Employment, Equity Ownership. Giles:Eli Lilly: Employment, Equity Ownership. Pitou:Eli Lilly: Employment, Equity Ownership. Jones:Eli Lilly: Employment, Equity Ownership. Walgren:Eli Lilly: Employment, Equity Ownership.

A Phase-2 Trial of Low-Dose Pomalidomide In Myelofibrosis with Anemia

AbstractAbstract 4109 Background:In a previous study, we reported safety and efficacy of low-dose (0.5 mg/d) pomalidomide and prednisone and pomalidomide alone (2 mg/d) for the treatment of anemia associated with myelofibrosis (J Clin Oncol 2009; 27: 4563). The current study examined the value of low dose pomalidomide alone (ClinicalTrials.gov No.NCT00669578). Methods:Persons with primary (PMF) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis were studied. The main eligibility criterion was RBC-transfusion-dependence or hemoglobin <10 g/dL. Subjects failing lenalidomide or thalidomide were eligible whereas those with deep vein thrombosis or pulmonary-embolism <1 y pre-study entry were not. The primary endpoint was anemia response assessed by the International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria (Blood 2006; 108: 1497). Results:Pomalidomide (0.5 mg/d) was given to 58 subjects (median age 68 y; 44 males). 46 (79%) were RBC-transfusion-dependent and 42, JAK2V617F-positive. Baseline disease-stage at time of study-entry according to the Dynamic International Prognostic Scoring System (DIPSS; Blood 2010; 115: 1703) was high-risk in 16 (28%) and intermediate-2 risk in 42 (72%). Cytogenetic findings were favorable in 46, unfavorable in 9 and not evaluable in 3. 46 (79%) had PMF and the remainder post-PV/ET MF. 49 (84%) patients completed ≥3 mo of therapy. Treatment was well-tolerated with no instances of thrombosis. There was grade-1 neuropathy possibly related to drug in 1 subject. ≥Grade-3 thrombocytopenia/neutropenia occurred in 2 subjects. There was an anemia response in 10 (17%) subjects including 9 who became RBC-transfusion-independent. Median response-duration was 8+ mo (range, 6–13 mo). 14 of 24 subjecst (58%) with platelets ≤100 × 10e9/L had a >50% increment. There were no spleen responses. Anemia response occurred only in subjects with JAK2V617F (24% vs. 0; p=0.03) but was not correlated with mutant allele burden (p=0.39). Anemia response in JAK2V617F-positive subjects was predicted by the presence of pomalidomide-induced basophilia in the 1st month of therapy (38% vs. 6%; p=0.02) or absence of marked splenomegaly (38% vs. 11%; p=0.05); response was not affected by cytogenetic risk-category (p=0.96) or WBC (p=0.27). Conclusions:These data suggest that low-dose pomalidomide is effective in the treatment for anemia associated with JAK2V617F-positive myelofibrosis, especially in the absence of marked splenomegaly. Response appears to be predicted by early drug-induced basophilia. Pomalidomide also appears to improve thrombocytopenia in most subjects with baseline platelets <100 × 10e9/L but is not active in controlling disease-associated splenomegaly. Disclosures:Mesa:SBio: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Roche: Research Funding; eisai: Research Funding; telik: Research Funding.

A phase-2 trial of low-dose pomalidomide in myelofibrosis

In a previous study, we reported on the safety and efficacy of low-dose (0.5 mg) pomalidomide and prednisone and pomalidomide alone (2 mg/day), for the treatment of anemia associated with myelofibrosis (MF). The current study examined the value of low-dose pomalidomide alone. The main eligibility criterion was transfusion-dependency or hemoglobin <10 gm per 100 ml. Anemia response was assessed by International Working Group criteria. Pomalidomide (0.5 mg/day) was given to 58 patients (median age 68 years); 46 (79%) were transfusion-dependent and 42 were JAK2V617F positive. Anemia response was documented only in the presence of JAK2V617F (24 vs 0%; P=0.03) but was not further affected by mutant allele burden (P=0.39); 9 of the 10 anemia responders became transfusion independent. Anemia response in JAK2V617F-positive patients was predicted by the presence of pomalidomide-induced basophilia in the first month of therapy (38 vs 6%; P=0.02) or absence of marked splenomegaly (38 vs 11%; P=0.05). A total of 14 (58%) of 24 patients with a platelet count of ≤ 100 × 10(9) cells/l experienced a >50% increment in platelet count. There were no spleen responses. Grade 3 or 4 thrombocytopenia/neutropenia occurred in 2%/0% of patients. Low-dose pomalidomide is effective in the treatment of anemia associated with JAK2V617F-positive MF; response is predicted by early drug-induced basophilia.

Phase 2 study of CEP-701, an orally available JAK2 inhibitor, in patients with primary or post-polycythemia vera/essential thrombocythemia myelofibrosis

AbstractFew treatment options exist for patients with myelofibrosis (MF), and their survival is significantly shortened. Activating mutation of the JAK2 tyrosine kinase (JAK2V617F) is found in approximately 50% of MF patients. CEP-701 is a tyrosine kinase inhibitor that inhibits JAK2 in in vitro and in vivo experiments. We conducted a phase 2 clinical study of CEP-701 in 22 JAK2V617F-positive MF patients (80 mg orally twice daily), and 6 (27%) responded by International Working Group criteria (clinical improvement in all cases): reduction in spleen size only (n = 3), transfusion independency (n = 2), and reduction in spleen size with improvement in cytopenias (n = 1). Median time to response was 3 months, and duration of response was more than or equal to 14 months. No improvement was seen in bone marrow fibrosis or JAK2V617F allele burden. Phosphorylated STAT3 levels decreased from baseline in responders while on therapy. Eight patients (36%) experienced grade 3 or 4 toxicity, and 6 (27%) required dose reduction. Main side effects were myelosuppression (grade 3 or 4 anemia, 14%; and thrombocytopenia, 23%) and gastrointestinal disturbances (diarrhea, any grade, 72%; grade 3 or 4, 9%; nausea, grade 1 or 2 only, 50%; vomiting, grade 1 or 2 only, 27%). In conclusion, CEP-701 resulted in modest efficacy and mild but frequent gastrointestinal toxicity in MF patients. The study was registered at http://clinicaltrials.gov as NCT00494585.