Clonal Myeloproliferative Neoplasm Research Articles

A Bio-Integrative Approach Identifies an Inflammatory Signature in Chronic Myeloid Leukemia (CML) Stem Cells That Is Highly Perturbed in CML Blast Crisis and Involves REL transcription Factor

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression.Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells.Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology.Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. DisclosuresTurhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

Acquired JAK-2 V617F Mutational Analysis in Pakistani Patients with Essential Thrombocythemia.

Essential thrombocythemia (ET) is a clonal hemopoietic stem cell myeloproliferative neoplasm characterized by persistent thrombocytosis along with megakaryocytic hyperplasia. In the last decade following the identification of an acquired JAK2 V617F mutation, there has been acceleration in our understanding of this disease. The rational of this study was to determine the mutational profile of JAK2 V617F in Pakistan patients with ET. In this retrospective cross sectional study, 21 patients with ET were enrolled from January 2011 to December 2014. Patients were diagnosed based on WHO criteria for essential thrombocythemia. Complete blood count was done on an automated hematology analyzer, while JAK2 V617F expression was evaluated by polymerase chain reaction. The mean age was 56.7±19.0 years (range 18-87) and the male to female ratio was 1:1.1. The frequency of JAK2 V617F positivity in our ET patients was found to be 61.9%. The mean hemoglobin was 11.7±2.4 g/dl with a total leukocyte count of 13.3±8.1x109/l and a platelet count of 1188±522x109/l. Positive correlations for JAK2 V617F mutation were established with high TLC count and raised LDH (P<0.05). No correlation of JAK2 V617F could be established with age and gender (P>0.05). JAK2 V617F mutation frequency in our ET patients was similar to those reported previously. Screening for the mutation in all suspected essential thrombocythemia cases could be beneficial in differentiating patients with reactive and clonal thrombocytosis.

Transcriptome analysis of bone marrow mesenchymal stromal cells from patients with primary myelofibrosis.

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM) fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. Within these niches, mesenchymal stromal cells (BM-MSC) play a hematopoietic supportive role in the production of growth factors and extracellular matrix which regulate the proliferation, differentiation, adhesion and migration of hematopoietic stem/progenitor cells. A transcriptome analysis of BM-MSC in PMF patients will help to characterize their molecular alterations and to understand their involvement in the hematopoietic stem/progenitor cell deregulation that features PMF.

Inflammation as a Keystone of Bone Marrow Stroma Alterations in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm where severity as well as treatment complexity is mainly attributed to a long lasting disease and presence of bone marrow stroma alterations as evidenced by myelofibrosis, neoangiogenesis, and osteosclerosis. While recent understanding of mutations role in hematopoietic cells provides an explanation for pathological myeloproliferation, functional involvement of stromal cells in the disease pathogenesis remains poorly understood. The current dogma is that stromal changes are secondary to the cytokine “storm” produced by the hematopoietic clone cells. However, despite therapies targeting the myeloproliferation-sustaining clones, PMF is still regarded as an incurable disease except for patients, who are successful recipients of allogeneic stem cell transplantation. Although the clinical benefits of these inhibitors have been correlated with a marked reduction in serum proinflammatory cytokines produced by the hematopoietic clones, further demonstrating the importance of inflammation in the pathological process, these treatments do not address the role of the altered bone marrow stroma in the pathological process. In this review, we propose hypotheses suggesting that the stroma is inflammatory-imprinted by clonal hematopoietic cells up to a point where it becomes “independent” of hematopoietic cell stimulation, resulting in an inflammatory vicious circle requiring combined stroma targeted therapies.

Survival of Allogeneic Stem Cell Transplantation Vs Conventional Therapies per DIPSS Stratification in Patients with Primary Myelofibrosis Younger Than 65 Years: A Retrospective Analysis on 673 Patients

IntroductionPrimary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm with a high risk of clonal evolution and mortality. Allogeneic stem cell transplantation (ASCT) is currently the only curative treatment approach for myelofibrosis. Due to the inherent complications, careful patient selection is mandatory.Patients and MethodsWith the goal of refining patient selection for ASCT we compared results of ASCT with non-transplant approaches in 673 patients with PMF stratified according the Dynamic International Prognostic Scoring System (DIPSS), which assigns a time-dependent risk on a scale of low, intermediate (int)-1, int-2, and high. Considering a commonly used age-threshold for indication of ASCT, we restricted the analysis to patients aged 65 years or younger. This included 190 PMF patients (median 50 years at diagnosis) who received ASCT (HLA-matched related, n= 88 [46%] HLA-matched unrelated and mismatched related, n= 102 [54%] donors) after reduced intensity (n=93), or high intensity conditioning (n=97), and 248 patients, median age 55 years, from the DIPSS database (PMF patients not receiving any experimental drug at data cut-off and censored at the time of ASCT). DIPSS scores at ASCT, or, for non-transplanted patients, at diagnosis, were low in 22 (11%) and 125 (49%), int-1 in 38 (20%) and 75 (29%), int-2 in 85 (45%) and 52 (20%), and high in 45 (24%) and 3 (1%) patients, respectively. The median time from diagnosis to ASCT was 1.2 years.In line with the dynamic nature of the DIPSS, risk scores in the non-transplanted cohort change with time and, accordingly, were accounted for as time-dependent covariates. The date of diagnosis was considered as origin of the time scale, and patients entered the analysis when receiving ASCT (ASCT cohort) or at the time of acquisition of a specific DIPSS category of a non-transplant cohort. Therefore, left truncated, right censored data were used for both cohorts. Left truncation (begin of observation) for the ASCT cohort was the time to ASCT after diagnosis, while for the non-transplant cohort it was the time of achieving the respective DIPSS risk category.Cox proportional hazard models were built separately for the four DIPSS stages, considering the cohort as a discrete covariate; the corresponding relative risks (RR) between the two cohorts were computed under the proportional-hazards approximation, and Wald tests were used to report significance. Age at diagnosis was not significant in any model and, therefore, was not included in the regressions. The present study could not address the potentially confounding factor of selection bias regarding ASCT.ResultsThe relative risk (RR) of dying among patients receiving ASCT relative to those receiving conventional therapies was 5.6 (95% CI: 1.7-19, p=0.0051) for low risk DIPSS, 1.6 (95% CI: 0.79-3.2, p=0.19) for int-1 risk DIPSS, 0.55 (95%CI: 0.36-0.83, p=0.005) for int-2 , and 0.37 (95%CI: 0.21-0.66, p=0.0007) for high risk DIPSS patients. The proportions of patients with low risk DPSS surviving at 1, 5 and 10 years were 100%, 69%, and 60%, respectively, for the ASCT cohort, and 98%, 95%, and 92% for the non-transplant cohort. The corresponding figures for patients with int-1 risk were 78%, 52%, and 41%, respectively, after ASCT, and 97%, 77%, and 63% for non-transplanted patients. Among patients with int-2 risk survival after ASCT was 82%, 50%, and 32%, respectively, and 77%, 41% and 11% among non-transplanted patients. The corresponding figures for high risk patients were 65%, 37% and 27% for the ASCT cohort, and 67%, 11% and 1%, respectively, for the non-transplant cohort. It should be noted that hazard ratios are not constant over time, and summarizing the risk in a single “average” RR ignores the specifics of the survival trends. Survival differences became pronounced beyond 5 years following diagnosis for int-2 and high risk patients.ConclusionPatients with PMF who, at any age≤ 65 years, have int-2 or high risk disease by DIPSS have superior survival if receiving ASCT during their disease history. Conversely, conventional therapy appears to result in superior outcome in low risk patients, while intermediate-1 risk patients represent a group in whom individual counseling may be particularly important. DisclosuresVannucchi:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.

Spectrum of BCR-ABL Kinase Domain Mutations; A Cohort Study from Saudi Arabia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm (MPN) associated with a characteristic translocation between chromosome 9 and 22 to form Philadelphia chromosome (Ph). The introduction of tyrosine kinase inhibitors (TKIs) imatinib for CML marked a new era for targeted therapy. In general CML patients in chronic phase (CP) have a very good response to Imatinib with a ~85% projected overall rate of survival. However, a significant proportion harbor molecular residual disease and develop acquired resistance after initial response through a number of cellular and molecular mechanisms in which mutations in the Abl kinase domain are the best known cause of resistance. Mutations are detected in 35% of patients with resistance to TKI in Chronic Phase and up to 80% in patients with accelerated phase and blast phase.Given the clinical importance of the mutation status, mutation analysis was established as a clinical service to aid in the clinical decision and identify the subset who would benefit from alternative therapies.In this retrospective study, we assessed the ABL kinase domain mutation in 93 patients referred from our clinics at King Faisal Specialist Hospital and Research centre (General Organization) between 2011-2013 with Ph positive CML displaying either refractory to TKI or resistance displaying increase BCR-ABL1 levels by serial monitoring using Quantitative PCR. Mutation analysis was performed on RNA extracted from either blood or Bone marrows after amplification of the BCR/ABL1 transcript by nested PCR followed by Direct sequencing of the BCR-ABL1 Kinase domain including residues 243-487.In total, 93 patients (46 Females and 47 males), Age between 10-71 years (median age: 38 years) of which 89 were adults and 4 pediatrics were analyzed. Among 93 patients, 20 (21.5%) were positive for 10 different mutations across the ABL1 Kinase domain mutations (7 patients had T315I, 3 patients had each of the following mutation: Y253H, F359I, 2 patients with E255K. Additionally, one patient with each of the following mutations: E355G, V299L, L248V, L298, Y326H and F317L). The duration from diagnosis to mutation detection ranged between 3-144 months with a median duration of 4 years.Despite the retrospective nature and the relatively small sample size of a single center analysis, the mutation frequency is in line with similar studies reported from our region.Further follow up is needed to identify the outcome of these patients acquiring ABL kinase mutations on targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

JAK2 E846D Germline Mutation Associated with Erythrocytosis Causes Increased and Prolonged Epo-Induced Activation of STAT5

Until now, disease-causing JAK2 mutations have been associated with clonal myeloproliferative neoplasms (acquired mutations) or with polyclonal hereditary thrombocytosis (congenital mutations). Here we present the first example of a JAK2 mutation associated with congenital erythrocytosis.We studied a young patient with congenital erythrocytosis who inherited two heterozygous mutations, an E846D mutation in JAK2 from the mother and a Q157H in PHD2 from the father. Each mutation apparently sufficed to induce EPO-hypersensitivity in BFU-E progenitors, since BFU-Es from both parents were hypersensitive to EPO, as were those of the patient. Strikingly, only the patient, but not his parents exhibited an erythrocytosis phenotype.Expression analysis of granulocyte mRNA revealed markers of inappropriately increased signaling mediated by hypoxia sensing pathway (HIF) (Kapralova, Blood, 2014,16;123:391-4) in the propositus and his father. This result suggested that the polycythemia phenotype of the propositus could result from a combined impact of otherwise clinically silent defects co-inherited from both parents and affecting EPOR/JAK2 and HIF signaling pathways.Using a Ba/F3-EPOR cellular model we have characterized the impact of JAK2 E846D on EPO-induced JAK2-mediated signaling, cell cycle progression and cell responsiveness to limited concentrations of EPO. We demonstrated prolonged, EPO-dependent JAK2 and STAT5 activation caused by JAK2 E846D, which was mitigated by a JAK2 inhibitor, AZ-960. However, in contrast to the oncogenic V617F mutation, E846D did not trigger factor-independent constitutive signaling. Also unlike the oncogenic V617F or the JAK2 germline mutations associated with hereditary thrombocytosis, the E846D did not activate STAT1, in agreement with differential signaling of STATs in different clinical phenotypes associated with activating JAK2 mutations. The thrombocytosis phenotype (ET for acquired and hereditary thrombocytosis for germline) is promoted by STAT1 signaling, while STAT5 signaling results in erythroid hyper-proliferation.Using BrdU incorporation assay and growth factor starved Ba/F3 cells we also demonstrated that JAK2 E846D - when compared with JAK2 wild type - stimulates more rapid re-entry of the transfectants into the S phase in response to low concentration of EPO. In agreement with immunoblot analyses JAK2 E846D did not support growth factor-independent proliferation in cytokine-free media; however it provided improved survival in limiting concentrations of EPO. The proliferation advantage of JAK2 E846D transfectants was diminished by AZ-960. These results showed that E846D substitution lacks features of a constitutive gain-of-function mutation and were consistent with its germline transmission and with non-clonal hematopoiesis observed in the JAK2 E846D-positive mother.Structural modeling based on the X-ray crystal structures of inactive and active kinase domain explain the mechanism of hypersensitivity and prolonged signaling. The homologous residue of E846 in the JAK2 pseudokinase domain (JH2) is N542, a residue that is targeted by the exon 12 activating mutations in polycythemia vera, and a close residue to this homologous position in JH2 is R564 recently found to be mutated in hereditary thrombocytosis. Thus the E846D mutation belongs to a conformational hotspot in kinases, but this mutation is only inducing hypersensitivity and prolonged action and not constitutive activation.In conclusion, JAK2 E846D mutation associated with congenital erythrocytosis confers hypersensitivity to EPO, prolonged and selective but not constitutive STAT5 activation, and requires additional cooperating event, likely affecting the HIF signaling, for its full clinical expression.(KK, MH, JK contributed equally to this work.)Funding: Czech Science Foundation P301/12/1503, Czech Ministry of Health NT13587-4, Education for Competitiveness Operational Program CZ.1.07/2.3.00/20.0164 and Palacky University (IGA_LF_2014_011). JK was supported by CZ.1.07/2.3.00/30.0041, EL by a FRIA PhD fellowship. SNC was supported from the F.R.S.-F.N.R.S., Belgium, the Salus Sanguinis Foundation, the Action de Recherche Concertée projects MEXP31C1 and ARC10/15-027 of the University catholique de Louvain, Brussels, the Fondation contre le Cancer, Brussels, the PAI Programs BCHM61B5 and Belgian Medical Genetics Initiative. DisclosuresConstantinescu:Shire: Consultancy, Speakers Bureau; Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity’s Board of Directors or advisory committees; Amgen: Speakers Bureau.

Comprehensive Analysis of JAK2 Mutations: A Single Center Experience

Mutations in Janus kinase 2 (JAK2) genes are the genetic hallmark of BCR-ABL1-negative myeloproliferative neoplasms (MPN). In 2005 several groups described a point mutation in codon 617 of the protein tyrosine kinase (JAK2 gene) in BCR-ABL1-negative MPN. The mutation was found in a vast majority of patients with polycythemia vera (PV), in approximately half of patients with essential thrombocythaemia (ET) and in myelofibrosis (MF). In 2007, an additional exon 12 mutation were found in a small percentage of PV patients. The 2008 World Health Organization classification of haematopoietic neoplasms includes JAK2 mutations as one of the strongest diagnostic criteria for PV and together with the MPLmutations for ET and PMF.In this study, we describe the JAK2 mutation profile in a series of 1811 samples collected over the period of 48-months (2010-2013). The samples were submitted from patients h thrombosis or clinically suspected for having MPN to the Molecular laboratory at King Faisal specialist Hospital & Research center, Riyadh, Saudi Arabia. A comprehensive analysis of exons 12-15 was carried using Sanger sequencing method. Of the 1811 samples, 1706 samples were available for analysis. JAK2 mutations was identified in 271 (16%) of the samples. We evaluated the positive cases for the following: age, gender, type of JAK2mutation, if the patients are evaluated for other genetic mutations and diagnosis.Of these 271 positive cases; 148 (54.6%) were females and 123 (45.4%) males. 103 (38%) cases were from inside the hospital while 168 (62%) cases were from different referring institutions in which the diagnosis was not available. Their age varied between 4 months and 97 years with a median of 54 years. A total of 262 (96.7%) patients were positive for JAK2 V617F; In the remaining 9 (3.3%) samples non V617F mutation were detected, in which 5 (1.8%) cases with JAK2 G571S, 1 case (0.3%) with JAK2 p.E543_D544del, 1 (0.3%) case with JAK2 p.Y570Y, 1 (0.3%) with JAK2 exon 12 deletion (p.R541_E543>K) and 1 (0.3%) with JAK2 del (540I - 542N), ins M. of the 271, 13 (5%) cases were associated with other cytogenetic or DNA Sequence abnormalities together with the JAK2mutation. Of the 13, 4 cases (1.4%) presented with Abnormal karyotype, 5 (1.8%) with a heterozygous Methylenetetrahydrofolate reductase (MTHFR) polymorphism, 3 (1.1%) cases with BCR/ABL1 and 1 case has a missense mutation p.V726A, in exon 10 of the MEFV gene.The positive in-house cases tested for JAK2mutation were 103 (38%). Clinical diagnosis included: 87 (84.4%) MPN cases of which 35 (40.2%) PV, 22 (25.3%) ET, 11 (12.6%) MF, 17 (19.5%) unclassifiable MPN, 2 (2.3%) CML. Moreover, 9 (8.7%) Budd-Chiari syndrome, 6 (5.8%) thrombosis (including DVT, PE and portal thrombosis) and 1 (0.9%) squamous cell carcinoma.In our study 1811 sample were tested in 4 years which means almost 1 sample is submitted daily for JAK2 testing in our institute. The Majority of the in-house patients tested were MPN patients with small percentage having thrombosis which could progress later to full MPN clone. Over 50 different non-V617F mutations have been identified in the literature and were presented by a small portion of our patients cohort. It was shown to have biologic effects similar to those caused by the V617F mutation; however, more research in this area is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Type II Inhibition of JAK2 with NVP-CHZ868 Reverses Type I JAK Inhibitor Persistence and Demonstrates Increased Efficacy in MPN Models

The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development.All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition.We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors.We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity.Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. [Display omitted] [Display omitted] DisclosuresKoppikar:Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

Management of thrombocythemia.

Essential thrombocythemia is a clonal myeloproliferative neoplasm characterized by an elevated platelet count, the potential for both microvascular and macrovascular sequelae, and a risk for transformation to myelofibrosis or acute myeloid leukemia. A systematic and detailed initial analysis is essential for accurate diagnosis of essential thrombocythemia, as many etiologies are reactive and benign. Once a diagnosis has been made, risk stratification and symptom assessment are vital to guide the subsequent therapy. Treatment may be required in high-risk disease, such as in cases of advanced age or prior thrombotic events. Systemic therapy is aimed at reducing the thrombotic risk and includes daily low dose aspirin and in some patients, cytoreductive therapy. Currently, the first line cytoreductive therapy includes hydroxyurea or pegylated interferon, with a phase III clinical trial underway comparing these two important agents. Anagrelide and clinical trials are reserved for refractory or intolerant patients. Looking to the future, new therapies including Janus kinase 2 (JAK2) and telomerase inhibitors are promising and may become valuable to the treatment armamentarium for those afflicted with essential thrombocythemia.

2014 WHO Clinical Molecular and Pathological (WHO-CMP) Diagnostic Criteria for the Classification and Staging of Five Distinct JAK2, MPL and CALR Mutated Myeloproliferative Neoplasms

Somatic mutations in the JAK2, MPL and calreticulin (CALR) genes are the driver causes of clonal myeloproliferative neoplasms (MPN). Applying the WHO Clinical, Molecular and Pathologic (WHO-CMP) classification of MPN, the JAK2 V617F positive ET patients comprise three phenotypes of ET: normocellular ET, hypercellular ET due to increased erythropoiesis (prodromal PV) and ET with hypercellular megakaryocytic-granulocytic myeloproliferation (ET.MGM or masked PV). The percentage of JAK2V617F mutation load is low and stable in heterozygous normocellular ET and increasingly high in hetero/homozygous PV and masked PV. The JAK2 V617F allele burden is related to MPN disease burden in terms of splenomegaly, constitutional symptoms and myelofibrosis. Five distinct clonal MPNs can be distinguished: JAK2 V617F mutated ET and PV; JAK2 exon 12 PV and the JAK2 wild type ET and MF caused by the somatic mutations MPL 515 or CALR. JAK2 mutated trilinear MPN reflects a broad spectrum of ET, prodromal or masked PV and classical PV, but the JAK2 wild type MPL or CALR positive ET and MF lack features of PV at diagnosis and during follow-up. Bone marrow features in JAK2 V617F mutated ET and PV are similar and featured by medium sized to large (pleomorphic) megakaryocytes with only a few giant forms. Bone marrow histology in MPL515 mutated ET and MF is featured by clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow with no features of PV. Bone marrow histology in CALR mutated ET and MF is featured by dense clustered large immature dysmorphic megakaryocytes and bulky (cloude-like) hyperchromatic nuclei similar as described in primary megakaryocytic granylocytic myeloproliferation (PMGM), which are never seen in JAK2

The Pan- JAK Inhibitor Ruxolitinib Impairs T-Cell Activation, Cytokine Production and Proliferation In Vivo and In Vitro

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm, in which the JAK2-V617F mutation is frequently observed. The appearance in up to 50% of the cases makes the JAK2 mutation attractive as therapeutical target. In 2012 Ruxolitinib (Ruxo) a pan-JAK inhibitor was approved for the treatment of MF and showed efficacy in disease treatment, irrespectively of the JAK2V617 mutation status. Currently allogeneic stem cell transplantation (allo SCT) remains the only curative treatment option for MF. To further improve transplant outcome in MF reduction of spleen size and constitutional symptoms prior transplantation is a reasonable target. Harnessing graft versus myelofibrosis post transplantation by immune-modulating drugs may help to reduce the risk of relapse. Ruxolitinib may be used as pre- and post-transplantation drug to improve transplant outcome. However the impact of Ruxolitinib on the immune system, especially on T-cells, is poorly understood. Here we investigated the effects of Ruxolitinib on T-cells in vivo and in vitro.T-cells from healthy donors were isolated by magnetic cell sorting to pan CD3+, CD4+ and CD8+ fraction. All three different cell subsets were cultured with different dosages of Ruxolitinib (100, 250, 500, 750nM and 1µM) for additional 48h. Thereafter cells were analysed for cell growth, cell death, RNA expression, immune phenotype.Additionally, immune profiles of 9 patients were analysed for the changes of the T-cell compartment during the treatment with Ruxolitinib over a period of 3 weeksT –cells from healthy donors showed a dosage dependent impairment in the proliferation capacity compared to non-treated control cells (4.1x106 CD3 cells /ml vs. 1.9x106 CD3 cells /ml, p<0.05), additionally KI67 expression was reduced from 48% in control cells to 12% in 100nM treated CD3 cells and 9% in 500nM treated CD3 cells, p<0.05. Strikingly apoptotic cell death increased from 11% in control cells to 43% and 48% in 100nM and 500nM Ruxo treated cells, p<0.03.Analysing the immune phenotype of Ruxo treated CD3, CD4 and CD8 cells we found a significant reduction in the expression of activation marker like CD25 and HLA-DR (38% vs. 6% and 4.5% respectively, p<0.05 and 63% vs. 47% and 40% respectively, p<0.05). Furthermore, we found that the effector cells, marked by CCR7/CD45RA expression, decreased in the CD8 compartment from 22% to 10.5% and 7.8% respectively, p<0.05. When analysing regulatory T-cells we also observed a decrease in a dose dependent manner (4% vs. 1.2% and 0.8%, p=0.05). While control Treg showed a KI67 expression of >60%, Ruxo (100nM) treated T-reg did not expressed KI67. Likewise to CD8 effector cells and Tregs we found a decrease in pro-inflammatory TH1 and TH17 cells in vitro (27% vs. 14% and 12% for TH1 cells and 6% vs. 4% and 4% for TH17 cells).Next, we analysed mRNA expression and found that pro-inflammatory cytokines like IL23, IL18, IL7 were down regulated after Ruxo treatment. To in contrast to pro- inflammatory cytokines, p53 and cell cycle inhibitor of the cip/waf locus showed to be up regulated in CD3 and CD4 cells suggesting that the observed increase in apoptosis in T-cells is mediated by p53.We next investigated the impact of Ruxolitinib on T-cells in patients. Therefore we analysed the blood of patients treated with Ruxolitinib in weekly intervals. Likewise to in vitro CD3 cells showed a decrease which turned to be significant after two and three weeks of treatment (1560/µl vs. 688/µl and 410/µl, p<0.05), this was mainly through the reduction of CD8+ T-cells (630/µl before treatment vs. 250/µl at week 2 and 200/µl at week 3, p <0.05). We also observed a decrease of CD3+/ HLA-DR+ (as activation marker) from 355/µl before to 130/µl and 70/µl however this did not reached statistical significance. The same was found for Tregs in vivo (5.6% vs. 2.3% and 1.9%, respectively).These data argue that treatment of T-cells by Ruxolitinib impairs their proliferation capacity by inducing apoptosis through an up regulation of p53. This increase of cell death applies all analysed T-cell compartments, and thereby may explains why Ruxo treated T-cells were less able to show a pro-inflammatory as well as regulatory phenotype. Disclosures:No relevant conflicts of interest to declare.

Neutophil Gelatinase –Associated Lipocalin (LCN2) Affects Primary Melofibrosis Hematopoietic Progenitor Cells As Well As Microenvironmental Cells

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34+ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P<0.001) Media conditioned by PMF MNC contained higher levels of LCN2 than media conditioned by normal MNC (p=0.03). The LCN2 receptor level was flow cytometrically analyzed and a significantly greater percentage of normal MNC and CD34+ cells than PMF MNC and CD34+ cells expressed the LCN2 receptor. The effect of increasing concentrations of recombinant LCN2 on CFU-GM and BFU-E derived colony by normal BM CD34+ cells as well as the PMF CD34+ cells was evaluated. The addition of LCN2 inhibited CFU-GM and BFU-E derived colony formation by normal CD34+ cells at a dose of 100 ng/ml (p=0.048 and p=0.043, respectively), while a similar dose of LCN2 did not influence the number of colonies cloned from PMF CD34+ cells. Previously our laboratory has reported that the expression of the chemokine receptor CXCR4 was decreased in PMF CD34+ cells which we hypothesized contributed to abnormal trafficking of CD34+ cells. LCN2 has been reported to affect CXCR4 expression by marrow CD34+ cells (Costa D, et al. 2010, Cytokine). CXCR4 expression by PMF CD34+ cells was lower than that of normal BM CD34+ cells. Incubation with LCN2, however, further reduced the expression of CXCR4 of PMF CD34+ cells by 10 to 50 % (p=0.012). By contrast, LCN2 increased CXCR4+ expression by normal CD34+ cells. We next evaluated the effects of LCN2 on the BM microenvironment. Normal BM mononuclear cells were plated in dishes exposed to the vary doses of LCN2 (10, 50, 100, 200 ng/ml) and the formation of adherent cell layers was monitored. Low doses of LCN2 (10 and 50 ng/ml) promoted the formation and proliferation of adherent cell layers composed of fibroblast-like cells after 1-3 weeks of culture. The fibroblast-like cells expressed vimentin and von Willebrand factor, indicating that they resembled mesenchymal stem cells, fibroblast and endothelial cells. We then examined the formation of adherent cell layers by normal BM MNC co-cultured with PMF, PV or normal MNCs separated by a 0.4 um trans-well for three weeks. The proliferation of confluent fibroblast-like cells was observed solely in BM MNC co-cultured with PMF MNC. These data indicate that LCN2 is generated in increased amounts by PMF mononuclear cells and likely plays a role in PMF biology by promoting malignant hematopoiesis but suppressing normal hemaopoiesis, suppressing CXCR4 expression by PMF CD34+ cells and promoting marrow fibroblast proliferation. Disclosures:No relevant conflicts of interest to declare.

The New Landscape of Therapy for Myelofibrosis

The landscape of therapy for myelofibrosis (MF) is evolving at a pace not previously seen for this clonal myeloproliferative neoplasm. The discovery of the JAK2 V617F mutation in 2005 has led to the rapid development of therapy specifically developed for afflicted MF patients. Indeed, the successful phase III studies of ruxolitinib demonstrating improved symptomatic burden, splenomegaly and survival led to the first approved myelofibrosis drug in the United States and Europe. Multiple additional JAK2 inhibitors are currently in or nearing phase III testing, including SAR302503 (fedratinib), SB1518 (pacritinib) and CYT387 (momelotinib), seeking to offer incremental benefits to ruxolitinib in regards to cytopenias or other disease features. In parallel, phase III testing of pomalidomide is ongoing, with the goal of solidifying the role of immunomodulatory therapy in MF-associated anemia. Multiple single agents strategies are ongoing with histone deacetylase inhibitors, hedgehog inhibitors and hypomethylation agents. Incremental advances are further sought, either in additive or synergistic fashion, from combination strategies of ruxolitinib with multiple different approaches ranging from allogeneic stem cell transplant to current therapies mitigating anemia and further impacting the bone marrow microenvironment or histology. Transitioning from a pre-2011 era devoid of approved MF therapies to one of multiple agents that target not only disease course but symptomatic burden has indeed changed the platform from which MF providers are able to launch individualized treatment plans. In this article, we discuss the diagnostic and therapeutic milestones achieved through MF research and review the emerging pharmacologic agents on the treatment horizon.

Role of Methylenetetrahydrofolate Reductase (Mthfr), Glutathione S-transferases (Gsts M1 and T1) and Haptoglobin (Hp) Gene Polymorphisms in Susceptibility to Chronic Myeloid Leukemia (Cml)

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm derived from an abnormal pluripotent hematopoietic stem cell that underwent a chromosomal translocation t(9;22)(q34;q11) acquiring the BCR-ABL1 fusion gene, also known as the Philadelphia chromosome. Although the clinical and biological aspects are well documented, little is known about individual susceptibility to CML. There are no known hereditary, familial, geographic, ethnic, or economic associations with CML, and the mechanisms behind disease progression are not fully understood. However, while a specific chromosomal translocation is the common oncogenetic mechanism of CML, it may have environmental causes such as irradiation and chemical (e.g., benzene) exposure, which exert toxicity via pro-oxidant mechanisms. Oxidative stress has been observed in several hematopoietic malignancies, including acute and chronic myeloid leukemias. It is known that BCR/ABL expression stimulates reactive oxygen species (ROS) production in hematopoietic progenitor cells, and studies have suggested that ROS may contribute to increased DNA damage in CML cells. Leukemia cell lines expressing BCR-ABL1 kinase accumulate ROS and oxidative DNA damage, resulting in genomic instability, which leads to the disease relapse and/or malignant progression to a fatal blast phase. These suggest a probable genetic susceptibility to this disease related to the body’s ability to defend itself from external aggression such as chemicals, ionizing radiation and others, through functional polymorphic variant enzymes that metabolize toxicants and/or protect against oxidative stress. Thus, this review aims to address a comprehensive summary of CML, including the physiological function of the normal proteins encoded by the ABL1 and BCR genes and the oncogenetic mechanism of the BCR-ABL1 fusion proteins, as well as to discuss the role of polymorphisms in methylenetetrahydrofolate reductase (MTHFR), glutathione S-transferases (GSTs M1 and T1) and haptoglobin (Hp) genes in the susceptibility to Chronic Myeloid Leukemia (CML).

Thrombotic disease in the myeloproliferative neoplasms

Abstract Thrombosis is a leading cause of morbidity and mortality in patients with Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), particularly polycythemia vera and essential thrombocythemia. Mechanisms involved in the pathogenesis of the acquired thrombophilic state associated with these diseases include abnormalities of MPN clone–derived blood cells, which display prothrombotic features, and abnormalities of normal vascular cells, which become procoagulant in response to inflammatory stimuli. Ultimately, the release into the blood of elevated levels of procoagulant microparticles by platelets and vascular cells and the increase in the global thrombin generation due to an acquired activated protein C resistance result in a highly prothrombotic scenario in patients with polycythemia vera and essential thrombocythemia. The acquired point mutation in the pseudokinase domain of JAK2 (JAK2V617F) in these disorders is variably associated with thrombosis and, more consistently, with elevations in WBC counts and alterations in biomarkers of blood-clotting abnormalities. The predictive value of these biomarkers for thrombosis remains to be established to identify subsets of patients at elevated risk who may benefit from prophylaxis with antithrombotic drugs.

Abstract 1352: Shp2 is required for CML initiation and maintenance

Abstract Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm (MPN) caused by the constitutively activated BCR-ABL tyrosine kinase. CML progresses from an initial chronic phase, through an accelerated phase, to a final and fatal blast crisis. Current first-line therapy for CML involves tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinibs. However, TKIs don't completely cure CML, as revealed by low complete molecular response rate and frequent disease relapse upon drug withdrawal. Studies of patient samples and mouse models indicate that this most likely results from lack of dependence of CML stem cells (CML-SCs) on BCR-ABL for survival. It is important to further identify potential targets of CML-SC. Shp2 is a non-receptor protein tyrosine phosphatase involed in RTK, cytokine, and integrine signaling pathways, and is a target of Abl kinase during cell proliferation. It is required for survival and maintenance of normal hematopoietic stem cells (HSCs). In the context of CML, BCR-ABL constitutively phosphorylates Gab2, which then binds to Shp2, resulting in its inappropriate activation of downstream pathways, including the Ras-Erk cascade. Gab2 is required for CML initiation in vivo. We hypothesized that Shp2 is required for CML development from the stem cell level and might be an effective target for treatment of CML. Using our induced Shp2 deletion tools, we found that Shp2 is required for CML initiation and maintenance in a bone marrow transplant model of CML. Moreover, primitive CML cells undergo elevated cell death upon Shp2 knockout. This implicates Shp2 as a potential target for CML therapy in future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1352. doi:1538-7445.AM2012-1352

Case of polycythemia vera with unusual organizing pneumonia mimicking the clinical features of military tuberculosis and possibly caused by the involvement of neoplastic megakaryocytes

Polycythemia vera (PV) is a clonal myeloproliferative neoplasm (MPN) of hematopoietic stem cells. Although the management of MPN patients generally focuses on the prevention of thromboembolic events caused by hypercoagulability, it is true that the patients with hematological malignancy often suffer from pulmonary diseases with atypical radiological patterns. We present here a 56-year-old woman with PV harboring a JAK2(V617F) mutation that had a diffuse reticulonodular pattern on chest radiography and was initially suspected of having military tuberculosis. Pathological assessment of a video-assisted thoracoscopic surgery lung biopsy revealed that the lesions were in fact organizing pneumonia (OP). Interestingly, pulmonary extramedullary hematopoiesis with a diffuse plugging of the alveolar blood capillaries by numerous atypical megakaryocytes was also observed around the granulation components. The histological findings of our case of unusual OP suggest that local activated neoplastic megakaryocytes and platelets played an important role in the development of spreading fibrotic lesions. JAK2 mutation or the preleukemic phase of MPN may accelerate the activation of megakaryocytes and result in the proliferative process of fibrosis.

Leukemic Hierarchy and Systemic Factors Dictating Disease Evolution In Chronic Myelogenous Leukemia

AbstractAbstract 886Chronic myelogenous leukemia (CML) is a clonal myeloproliferative neoplasm (MPN) characterized by the t(9;22)(q34;q11) reciprocal translocation, which leads to the expression of the BCR/ABL fusion protein. CML is clinically characterized by the abnormal expansion of the myeloid lineage leading to progressive peripheral neutrophilia. This indolent disease has the propensity to evolve into an acute leukemia-like blastic phase involving either myeloid or B lymphoid cells. As such, CML constitutes a paradigm for understanding cellular and molecular events controlling chronic diseases and their evolution toward frank leukemia. CML arise from a small pool of leukemic stem cells (LSC) that can be operationally defined by their ability to sustain tumor growth over time and to transplant leukemia to recipient mice. Multiple lines of evidence indicate that LSCs originate from abnormally behaving hematopoietic stem cells (HSCs), although it is still largely unknown how BCR/ABL affects the biology of HSCs and the fate of downstream progenitor cells. To study the impact of BCR/ABL expression on these compartments, we used a transgenic mouse model that allows for inducible expression of BCR/ABL in HSCs and downstream progeny (Blood 105: 324, 2005). Induction of BCR/ABL expression in this model recapitulated many features of human CML such as myeloid bone marrow (BM) hyperplasia, myelofibrosis, splenomegaly and myeloid cell infiltration in non-hematopoietic organs. Disease development also correlated with a dramatic reorganization of the stem and progenitor compartments in the BM and their relocation/expansion in the spleen. In the BM, we observed a marked reduction in the number of Lin– Sca1+ c-Kit+ Flk2– CD48– CD150+ long-term (LT) and Lin– Sca1+ c-Kit+ Flk2– CD48– CD150– short-term (ST) HSCs associated with an expansion of several types of non-self-renewing multipotent progenitors (MPP) and myeloid committed progenitors. We confirmed by limited dilution transplantation experiments that BCR/ABL+ LT-HSCs were true LSCs as they were the only cells capable of transferring sustained CML disease in recipient mice with as few as 50 cells injected. We also found that BCR/ABL expression impacts on the biology of the ST-HSC and Lin– Sca1+ c-Kit+ Flk2+ MPP compartments. Transplantation of both populations induced dramatic but transient hyperplasia, which could eventually mimic a leukemic phenotype with high doses of cell injected (4,000 cells per mouse). However, while transplantation of BCR/ABL+ ST-HSCs led to the expected myeloid hyperplasia, transplantation of BCR/ABL+ MPPs led to a massive accumulation of B-cell progenitors in the BM that resembles lymphoid blast crisis. Strikingly, co-transplantation of 4,000 BCR/ABL+ LT- or ST-HSCs with 4,000 BCR/ABL+ MPPs almost always resulted in myeloid hyperplasia suggesting an active inhibition of MPP-derived lymphoid progeny by the leukemic myeloid compartment. We reasoned that the molecular effectors for this lymphoid inhibitory effect could be extracellular signaling molecules that will be detectable in the serum of CML-developing BCR/ABL mice. Using antibody arrays and enzyme-linked immunosorbent assays (ELISA), we found that the serum concentration of the proinflammatory cytokine interleukine-6 (IL-6) correlates with CML progression both in primary and transplanted mice. Moreover, we showed in vitro that IL-6 controls lineage fate decision of leukemic progenitors by promoting myeloid differentiation from MPPs at the expense of the B lymphoid lineage differentiation. As such, IL-6 targets both normal and malignant MPPs thereby providing a positive feedback loops that promote CML development. In summary, our results identify and functionally characterize a pathological hierarchy in CML that includes LSC and immature leukemic progenitors. They uncover a novel fate-regulatory mechanism at the systemic level that controls the differentiation outcome of the leukemic progenitors and can have key implication for disease progression. Taken together, they demonstrate that CML evolution is the result of a balance between BCR/ABL cell intrinsic effects and environmental cues and provide a rational for the paradoxical myeloid-lymphoid conversion that can be observed during lymphoid blast crisis. Disclosures:No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS) Transformation From Essential Thrombocythemia (ET) In Patients Treated with Hydroxyurea or Anagrelide: A Single Center Series of 17 Cases

AbstractAbstract 4102 Background:ET is an acquired clonal myeloproliferative neoplasm rarely transforming into AML or MDS. Such transformations have been reported to occur in patients treated with alkylating agents primarily. Recently, concern has arisen about the long term safety of the 2 mainstream cytoreductive therapies, i.e. hydroxyurea (HU) and anagrelide (AG), in terms of leukemogenesis and myelofibrosis. This large series of 17 cases of transformed ET aims to compare clinicopathologic features and outcomes of MDS or AML that developed in patients treated with HU or AG. Design:Cases of secondary MDS or AML evolved from ET from January 2000 to February 2010 were retrieved from institutional databases. Results:Eighteen patients with MDS or AML transformed from ET in our institute were evaluated. One patient who was previously treated with an alkylating agent was removed from the study. General characteristics and clinical outcomes of 17 cases are summarized in Table 1. Ten out of 17 patients were diagnosed with AML at the time of transformation while 7 were diagnosed as high grade MDS. Results showed that the median time to MDS/AML development in 17 cases was 87 months; the median survival from the time of ET diagnosis was 124 months and the median survival after transformation was 12 months. There were 16 patients that were mainly treated with long term HU or AG alone. One patient treated with a combination of HU and AG, was excluded from the comparative studies. All patients were founded to have moderate (3/16) to severe (13/16) reticulin fibrosis (2+ to 3+), regardless of treatment. Dyspoiesis including MDS or AML with myelodysplasia related changes (AML-MRC) were more commonly noted in patients treated with AG than in those with HU (6/8 vs. 2/8, p=0.046). The time to transformation was similar in the 2 groups: 76 months (AG) vs. 82 months (HU). The survival since ET diagnosis also was not significantly different, with median survival of 146 months in AG group vs. 96 months in HU group (p= 0.51). However, although not statistically significant (p=0.24), the AG group had a median survival after transformation of 21 months vs. 10 months in the HU group (Figure 1). Thirteen of 14 available karyotypes were abnormal. Complex cytogenetic abnormalities were found in 8/14 cases (5 in AG group vs. 3 in HU group). The common abnormalities, listed in the order of their frequencies, were: -20 or 20q- (5), 5q-(4), 17p- (2), -7 (2), 15q-(2),-18(2), +8(1). Conclusion:There were no differences in degree of fibrosis or time to onset of secondary MDS and/or AML between HU and AG therapy in ET. There was a trend that the median survival after transformation was longer in AG group, which was thought to be related to a predominance of MDS at the time of transformation in AG group. We attributed the shorter median duration of survival from the time of ET diagnosis in our study, in comparison to the literature, to our focus on selection of transformed cases. Complex cytogenetic abnormalities with loss of chromosomal materials in 20, 5, 7, and 17, could play a critical role in disease transformation and progression independent of the specific drug use (HU or AG) in ET. [Display omitted] Disclosures:No relevant conflicts of interest to declare.