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Supplementary Figure 4 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 5 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 2 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 3 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Supplementary Figure 1 from Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor–induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment. [Cancer Res 2007;67(12):5658–66]

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

Foetal spleen is described as a transient focus of haematopoiesis between the 3(rd) and 5(th) month of gestation: this function is however entirely replaced by the bone marrow before the end of pregnancy. This study identifies haematopoiesis in foetal spleen by exploring changes of echogenicity during its development throughout gestation. Two intervals of pregnancy were studied: Mid-Pregnancy (Mid-P, 19-23 weeks) and End-Pregnancy (End-P, 37-41 weeks). The foetal spleen was investigated in 80 pregnant women (41 vs 39). Due to quality criteria the comparison was made between 60 images (30 Mid-P vs 30 End-P). The acquisition of splenic parenchyma was followed by clustering segmentation. We identified two new parameters resulted from the clustering segmentation: Dark Ratio (DR) and Light Ratio (LR). These are related to splenic echogenicity expressing the percentage of dark and light signal in the clustered image, influenced by blood cellularity. The mean of DR value was different among the 2 groups (0.0631 vs 0.0483, P = 0.014), while LR did not show any significant differences. We conclude that DR may represent a reliable radiomic parameter in the determination of extramedullary haematopoiesis in the spleen.

We present a 47-year-old, South-African origin, woman with a background of stable monoclonal gammopathy of unknown significance (MGUS) who attended A&E with a history of coryzal symptoms associated with persistent fever, lymphadenopathy and a new onset of rash, not responding to antibiotics and paracetamol. A trial of high-dose steroids resolved symptoms. Bone marrow biopsy confirmed a progression of MGUS into multiple myeloma and her axillary lymph node biopsy analysis supported a diagnosis of Kikuchi-Fujimoto disease (KFD). This is an unusual presentation where KFD has been noted alongside MGUS progression to multiple myeloma. Haematology follow-up is underway.

Myelodysplastic syndrome and myeloproliferative neoplasms are clonal myeloid disorders arising from haematopoietic stem cells that have the tendency to progress into acute myeloid leukaemia. Multiple prognostic scoring systems have been proposed and utilised in clinical practice to predict disease evolution, however none of them can predict treatment response. In solid tumours, the relationship between the pro-phagocytic calreticulin and the anti-phagocytic CD47 is repeatedly investigated. Overexpression of calreticulin has been documented to produce a pro-phagocytic signal in solid tumour and it is often counteracted by a concomitant expression of the anti-phagocytic CD47 as they act in response to one another, reflecting an apoptosis vs survival mechanism in response to chemotherapy. The role of both calreticulin and CD47 are currently poorly understood in myeloid malignancies including myelodysplastic syndrome and myeloproliferative neoplasms. The aim of this review is to elaborate on the current understanding round the roles and implications of calreticulin and CD47 signalling with in solid and haematological cancers, discuss potential roles for calreticulin and CD47 expression in transformation of myeloid cells in patients with MDS or MPN into AML and how these advances are starting to be used to design new therapeutic strategies to determine disease progression and treatment response in both solid cancer and myeloid malignancies.

Introduction Current treatment options for myelofibrosis (MF) are diverse; apart from rare patients who are eligible for allogeneic transplantation, active treatments are focused on controlling MF-related symptoms and are not disease modifying. Patients who are initially asymptomatic and those at low risk for progression are usually observed without active treatment (watch and wait) until the appearance of symptoms or progression. There are limited real-world data on the management of patients with MF in the United Kingdom (UK). The REALISM UK study documented the early management pathways for patients with MF in routine clinical practice. Method REALISM UK was a multi-centre, retrospective, non-interventional study in 15 UK secondary care centres. Eligible patients were those aged ≥18 years at diagnosis of MF, with diagnosis >6 months and ≤5 years prior to data collection and with ≥1 follow-up visit. Data on patient demographics, clinical characteristics, MF management strategies and outcomes were collected from patients' medical records; the observation period was from the date of MF diagnosis until data collection. The primary endpoint was the time from diagnosis to active treatment. Watch and wait strategies included supportive therapies (blood transfusions, erythropoiesis-stimulating agents, and steroids). Results A total of 200 patients were recruited (63% [n=126/200] with primary MF (PMF); 37% [n=74/200] with secondary MF). The median age at MF diagnosis was 69.7 years (interquartile range [IQR] 63.5-75.7) and 71% (n=141/200) had a JAK2 mutation. At diagnosis, 52% (n=104/200) of patients had International Prognostic Scoring System (IPSS) categories of intermediate-2 (Int-2) or high. Common documented features of disease at diagnosis included an enlarged spleen (47%, n=94/200), anaemia (44%, n=88/200), and constitutional symptoms (e.g. night sweats, unexplained fever, or weight loss [30%, n=60/200]). The median (IQR) time to first active treatment was 46 days (range 0-350) in the total population; patients with higher risk disease were prescribed active treatment sooner: IPSS low, 136.5 days (range 0-625; n=26); IPSS Int-1, 90 days (range 0-494; n=70); IPSS Int-2, 0 days (range 0-252; n=65); IPSS high: 0 days (0-216; n=39). The choice of first management strategy by IPSS at diagnosis is shown in Table 1. During the study period, watch and wait was the first management strategy for 54% (n=107/200) of patients, with the median (IQR) time from diagnosis to active treatment in this group being 322 (130-610) days. Constitutional symptoms were recorded in 20% (n=21/107) of patients for whom watch and wait was the first management strategy. Active treatment was started at diagnosis for 47% (n=93/200) of patients, with the most commonly used medications being hydroxycarbamide (22.5% [n=45/200]) and ruxolitinib (17.5% [n=35/200]). The median (IQR) treatment duration during the study observation window was 346 days (range 121-768) for hydroxycarbamide and 375 days (range 172-691) for ruxolitinib. In total, 5.5% (n=11/200) of patients were recorded as having undergone allogenic stem cell transplantation during the observation period. Conclusion Clinicians in the UK apply a broad range of management strategies for the treatment of patients with MF. Many patients were observed without active treatment following diagnosis, despite having symptomatic disease. The most common active treatments as first management strategies were hydroxycarbamide and ruxolitinib. Disclosures Mead: Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; Bristol Myers-Squibb: Consultancy. Somervaille:Novartis: Consultancy, Honoraria. Butt:Novartis: Consultancy, Honoraria, Speakers Bureau. Whiteway:Novartis: Consultancy, Honoraria, Speakers Bureau. Kirkpatrick:Novartis: Consultancy, Honoraria. Roughley:Novartis: Consultancy, Honoraria. Ewing:Novartis: Honoraria, Other: Meeting attendance sponsorship ; Bristol Myers-Squibb: Other: Meeting attendance sponsorship . Garg:Novartis: Consultancy, Honoraria. Harrison:Promedior: Honoraria; Shire: Speakers Bureau; Sierra Oncology: Honoraria; Celgene: Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; Incyte: Speakers Bureau; Gilead: Speakers Bureau; CTI: Speakers Bureau; AOP: Honoraria; Janssen: Speakers Bureau. Bains:Novartis: Employment. Chiu:Novartis: Employment. Hickey:OPEN VIE: Employment.

Janus-activated kinase 2 (JAK2) mutations are common in myeloproliferative disorders; however, although they are detected in virtually all polycythemia vera patients, they are found in ∼50% of essential thrombocythemia (ET) patients, suggesting that converging pathways/abnormalities underlie the onset of ET. Recently, the chromosomal translocation 3;21, leading to the fusion gene AML1/MDS1/EVI1 (AME), was observed in an ET patient. After we forced the expression of AME in the bone marrow (BM) of C57BL/6J mice, all the reconstituted mice died of a disease with symptoms similar to ET with a latency of 8 to 16 months. Peripheral blood smears consistently showed an elevated number of dysplastic platelets with anisocytosis, degranulation, and giant size. Although the AME-positive mice did not harbor Jak2 mutations, the BM of most of them had significantly higher levels of activated Stat3 than the controls. With combined biochemical and biological assays we found that AME binds to the Stat3 promoter leading to its up-regulation. Signal transducers and activators of transcription 3 (STAT3) analysis of a small group of ET patients shows that in about half of the patients, there is STAT3 hyperactivation independently of JAK2 mutations, suggesting that the hyperactivation of STAT3 by JAK2 mutations or promoter activation may be a critical step in development of ET. [Cancer Res 2009;69(1):262–71]

A letter to the editor is presented related to the case where GATA1 is overexpressed in patients with essential thrombocythemia and polycythemia vera, but not in patients with primary myelofibrosis or chronic myelogenous leukemia.

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor–induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment. [Cancer Res 2007;67(12):5658–66]

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Background: Cytotoxic regimens used in induction treatments for acute myeloid leukemia (AML) almost always include standard or high-dose cytarabine (Ara-C). During or soon after induction therapy, leukemic patients frequently develop gastroenteric complications, characterized by abdominal pain and diarrhea. The association ofthese symptoms with f and melena is typical ofnecrotizing enterocolitis (NE), a life-threathening condition that can be documented by ultrasound abdominal scan. Patients and methods: We analyzed retrospectively the clinical course of169 adult patients with AML treated by standard dose Ara-C-containing induction regimens, either by continuous venous infusion (group 1) or subcutaneous injection (group 2). Ultrasonography was employed as early diagnostic tool in a majority ofpatients with gastroenteric complications. Bowel wall thickening was accurately measured and used to confirm the diagnosis of necrotizing enterocolitis. Results: In the first group of 115 patients (median age, 51 years), gastroenteric complications were observed in 55 patients (48%), and 10 patients (9%) received diagnosis ofNE, which was fatal in four. Patients with NE had a median age older than that of patients without gastroenteric symptoms, and a more prolonged neutropenia. In the second group of54 patients (median age, 60 years), gastroenteric events were observed in 14 patients (26%), and no case of NE was recorded. Conclusions: This retrospective analysis shows that NE is a serious complication occurring mainly in patients treated by Ara-C administered as continuous i.v. infusion. The Hematology Journal (2003) 4, 346–350. doi:10.1038/sj.thj.6200304

Introduction: Over the past year, 3 agents have been approved for the treatment of melanoma by the Food and Drug Administration. These include pegylated interferon α-2b for stage III melanoma, vemurafenib for unresectable or metastatic melanoma with BRAF V600E mutation, and ipilimumab for unresectable or metastatic melanoma. Case Presentation: We present here the case of a 65-year-old Caucasian male diagnosed with advanced melanoma in April 2011 and treated with ipilimumab (Yervoy®), a monoclonal antibody targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), as second-line treatment after progression with dacarbazine, for (wild-type BRAF) metastatic melanoma. The patient was referred to us for several painful lumps on his right arm. A biopsy of one of them revealed melanoma. CT and PET scans did not show any other lesions or a primary site. The patient was started on first-line chemotherapy with dacarbazine 850 mg/m2 on day 1, every 3 weeks. After 3 cycles, the patient showed disease progression with an increase in size of the skin metastasis. Second-line treatment was started with ipilimumab 3 mg/kg on day 1, every 3 weeks. At the end of the treatment, after 4 cycles, we documented a complete clinical response with total resolution of the skin metastasis. At the time of writing this paper, our patient had finished his treatment more than 9 months earlier and is still in complete remission. Conclusion: This is a paradigmatic case where, despite extensive metastatic disease, treatment with ipilimumab has confirmed its efficacy. It is still an open question why only a minority of patients have such a remarkable response, and further trials are warranted to address this important question.

Intracranial extramedullary haematopoiesis (EMH) is very rare. We report the case of EMH with intracranial involvement which developed after trauma in a 73-year-old lady affected by JAK2 mutated myelofibrosis (MF). She was diagnosed with MF in 2005 and treated with low-dose-steroids (prednisolone and oxymethalone) for about 5 years with anexcellent response on full blood count and splenomegaly. In June 2011, she fell from the stairs and sustained minor trauma to the forehead. Six weeks later, the patient had a gradually enlarging and moderately painful lump in the centre of her forehead. A contrast-enhanced CT scan of the head was performed, showing a small fracture in the frontal bone infiltrated and surrounded by a soft tissue mass extending intracranially and invading the skin extracranially (Figures 1 and 2). A metastatic deposit from an occult tumour was suspected. In August 2011, we performed a CT-guided trucut biopsy of the forehead mass. The histology report confirmed extramedullary haematopoiesis according to the patchy expression of myeloid (CD45, CD43, myeloperoxidase) and megakaryocytic markers (CD68, CD42b). We concluded that the trauma caused a small fracture which triggered the bone marrow tissue reaction resulting in production of extramedullary haematopoiesis in the site of the trauma. The patient underwent local radiotherapy resulting in complete resolution of the lesion.

Objective: Recently, fine needle cytology (FNC) of the thyroid has been combined with biomolecular analysis. In particular, there has been detailed study of the V600E-BRAF mutation. The aim of our study is to demonstrate that with a single thyroid sample it is possible to obtain enough cellular material for both cytological diagnosis and a V600E-BRAF molecular test. Study Design: FNC was carried out under ultrasound guidance aided by an echographist and cytopathologist. We acquired one biopsy for each nodule with a 23-gauge needle without suction. The preparations were smeared by the pathologist onto one glass slide, air dried and stained with Diff-Quick. Cell adequacy was evaluated for each patient. The needle was washed by aspirating 2 ml of physiologic solution which was collected into a tube. The material was collected for molecular testing. Results: The following cytological diagnoses were made: not neoplastic, Tir2 (n = 227); indeterminate, Tir3 (n = 15); suspicious, Tir4 (n = 4), and malignancy, Tir5 (n = 12). The V600E-BRAF mutation was found in 0 of 227 Tir2 specimens, 2 of 15 (13.3%) Tir3 specimens, 2 of 4 (50%) Tir4 specimens and 9 of 12 (75%) Tir5 specimens. Conclusions: Our data showed that, in a routine clinical setting, FNC specimens can be handled properly to provide both morphological and molecular information. In fact, our tests show that with a single specimen it is possible to set up a slide for the cytological diagnosis and to obtain enough residual cellular material for DNA extraction (>70 ng) and for the identification of the V600E-BRAF mutation.

The myeloproliferative neoplasia (MPNs) are a spectrum of clonal disorders of the hematopoietic system. The distinct clinical manifestations are dictated by the primary cell type affected, and thus chronic myeloid leukemia (CML) is a proliferation of mature granulocytes, polycythemia vera (PV) is an expansion or red blood cells, essential thrombocythemia (ET) results in an increase of platelets, etc.. The natural history of MPNs is generally chronic in nature, and patients come to medical attention either by coincidence (abnormal blood findings during routine exam) or by signs and symptoms related to the expansion of the hematopoetic system (e.g., an enlarged spleen). Common to most MPNs is a small but finite risk of disease evolution to an acute leukemia, where hematopoetic development is blocked at an early stage of differentiation, leading to the accumulation of poorly functioning myeloid blasts at an expensive of critical depletion of normal white blood cells and platelets, leading to morbidity and mortality from infections and bleeding complications. If they do not progress to an acute leukemia, the natural history of MPNs often results in fibrosis of the bone marrow, migration of hematopoesis to other organs (spleen and liver), and eventual complications of this secondary organ involvement, as well as from decreased normal blood counts from marrow fibrosis. A unifying theme in the pathogenesis of MPNs is the activation of tyrosine kinases. The ‘‘poster child’’ is CML, where the BCR-ABL translocation is found in all cases; the fusion BCR-ABL activates proliferative and antiapoptotic pathways; and most importantly, inhibition by tyrosine kinase inhibitors (TKIs) can markedly reverse the natural history of the disease. The molecular lesions responsible for PV, ET, and myelofibrosis (MF) were unknown until relatively recently. From 2005, a flurry of reports found that a point mutation in JAK2, resulting in a valine for phenylalanine substitution at codon 617 (JAK2V617F), occurred at a high prevalence in these disorders. The mutation was found in roughly half of MF and ET cases and nearly all PV cases. Constitutive activation of JAK2 activates STAT and MAPK proliferative signaling pathways, leading to transformation of hematopoetic progenitors. Curiously, not all hematopoetic stem cells in cases with the JAK2V617F harbor the mutation. Moreover, the data suggested a differential dosage effect in the different diseases. Whereas in most cases the JAK2V617F is heterozygous with a normal JAK2 allele, in many cases of

International audience; Transformation to acute myeloid leukemia (AML) is a known complication of myeloproliferative neoplasia (MPN). Recent studies reported the high incidence (53%) of JAK2 negative blasts from transformed JAK2V617F-MPN. We collected, by cell sorting, blast cells and mature cells (GRA) from total bone marrow (BM) of 34 patients newly diagnosed of secondary AML. At MPN diagnosis (PMF n = 18; PV n = 9; ET n = 7), JAK2 was mutated in 22 of 34 patients. Twenty of 22 JAK2V617F-MPN (91%) maintained the mutation in blasts and GRA after leukemic switch, while in 2 of 22 patients the selected compartments lost the mutation. Surprisingly we also found the first case of JAK2V617F-AML from a wild type (WT)-MPN. In contrast to the previous study, we conclude that JAK2V617F-MPN yields rarely (9%) a JAK2WT-AML and any JAK2-status modification/persistence involves always the entire BM during leukemic transformation.

The involvement of the small bowel in systemic forms of amyloidosis may be diffuse or very rarely focal.? Some cases of focal amyloidomas of the duodenum and jejunum without extraintestinal manifestations have been reported. The focal amyloidomas consisted of extensive amyloid infiltration of the entire intestinal wall thickness. Radiological barium studies, ultrasound and computed tomography (CT) patterns of diffuse small bowel amyloidosis have been described: the signs are non-specific and may include small-bowel dilatation, symmetric bowel wall thickening, mesenteric infiltration, and mesenteric adenopathy. No data are available about the positron emission tomography (PET)/CT and magnetic resonance imaging (MRI) patterns of intestinal amyloidosis. We report two cases of small bowel amyloidosis: the former characterized by focal deposition of amyloid proteins exclusively within blood vessel walls of the terminal ileum, the latter characterized by diffuse intestinal involvement observed on MRI and PET/CT studies.

EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of ∼10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro. [Cancer Res 2009;69(4):1633–42]

RUNX1 (AML1, CBFalpha2, PEBP2alphaB) is a transcription factor essential for the establishment of the hematopoietic stem cell. It is generally thought that RUNX1 exists as a monomer that regulates hematopoietic differentiation by interacting with tissue-specific factors and its DNA consensus through its N terminus. RUNX1 is frequently altered in human leukemia by gene fusions or point mutations. In general, these alterations do not affect the N terminus of the protein, and it is unclear how they consistently lead to hematopoietic transformation and leukemia. Here we report that RUNX1 homodimerizes through a mechanism involving C terminus-C terminus interaction. This RUNX1-RUNX1 interaction regulates the activity of the protein in reporter gene assays and modulates its ability to induce hematopoietic differentiation of hematopoietic cell lines. The promoters of genes regulated by RUNX1 often contain multiple RUNX1 binding sites. This arrangement suggests that RUNX1 could homodimerize to bring and hold together distant chromatin sites and factors and that if the dimerization region is removed by gene fusions or is altered by point mutations, as observed in leukemia, the ability of RUNX1 to regulate differentiation could be impaired.

Our retrospective experience underscores the ability of Doppler echocardiography to detect the cardiotoxicity of chemotherapy (functional and pericardial abnormalities, heart involvement) and points out the need for an accurate echocardiographic follow-up of hematologic patients.

Background: Polycythemia vera (PV) is characterized by erythrocytosis, thrombocytosis, and/or leukocytosis and a broad range of disease-related symptoms. In high-risk patients, the most common first-line treatment is hydroxyurea (HU). The open-label RESPONSE trial demonstrated that ruxolitinib (RUX), a JAK1/JAK2 inhibitor, provided superior efficacy compared with best available therapy in patients with PV who were resistant to or intolerant of HU according to modified European LeukemiaNet (ELN) criteria. This study (RELIEF) was conducted in patients receiving a stable dose of HU and who were generally well controlled but reporting disease-associated symptoms, comparing the change in PV-related symptom burden in patients continuing their HU therapy with those switching to RUX treatment. Methods: RELIEF was a randomized, multicenter, double-blind, double-dummy, phase 3b study of patients with PV aged ≥18 years on a stable dose of HU monotherapy and reporting PV-related symptoms. Patients were required to be receiving HU for ≥12 weeks prior to enrollment and on the same dose level for the last 4 weeks, and have a score ≥8 on the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) cytokine total symptom score (TSS-C). The TSS-C comprised symptoms of itching, tiredness, muscle ache, night sweats, and sweats while awake; each symptom was rated on a scale of 0=absent to 10=worst imaginable, with a maximum TSS-C score of 50. Patients also had to meet one of the following: ≤1 phlebotomy in the previous 6 months or no palpable splenomegaly. Those eligible were randomized 1:1 to receive RUX 10 mg BID and HU-placebo, or HU at the same dose/schedule and RUX-placebo. Dose adjustments were permitted for safety and efficacy. After Week 16, patients could receive open-label RUX until Week 48. The primary endpoint was the proportion of patients with a ≥50% reduction in TSS-C at Week 16; secondary endpoints included proportion of patients with a ≥50% reduction in individual TSS-C symptoms and safety. Results: Overall, 54 and 56 patients were randomized to RUX and HU, respectively; 87.0% and 89.3% remained on treatment through Week 16. At baseline, the median age (range) was 64 (36-87) in the RUX group and 66 (19-85) in the HU group; 44% and 61% were men. The majority of patients in the RUX and HU groups did not have baseline platelet counts or WBC above ELN thresholds: platelets >400 and ≤600 x 109/L (RUX 31.5%, HU 28.6%), >600 x 109/L (3.7%, 8.9%); WBC >10 and ≤15 x 109/L (16.7%, 16.1%), >15 x 109/L (11.1%, 14.3%). In the RUX and HU groups, the mean TSS-C at screening (22.4, 23.1) was higher than that at baseline (16.7, 18.0); the ratio of screening to baseline TSS-C was 1.7 and 1.6. The proportion of patients achieving a ≥50% reduction from baseline in TSS-C at Week 16 (primary endpoint) was 43.4% in the RUX group and 29.6% in the HU group (P=0.139; OR, 1.82; 95% CI, 0.82-4.04). The proportions of patients in the RUX vs HU groups achieving a ≥50% reduction in scores for itching and tiredness at Week 16 were 40.0% vs 26.4% and 54.2% vs 32.0%, respectively. Median percentage changes in individual TSS-C symptoms are shown in Table 1. Additional analyses found no correlation between individual changes in HU dose from baseline to Weeks 13-16 and percentage change in TSS-C in the HU arm (r2=0.030). Even patients maintaining the same HU dose from prior to study entry through Week 16 reported symptom improvement: 12/35 (34.3%) with no dose change, 4/12 (33.3%) with a dose increase, and 0/9 (0%) with a dose decrease had a ≥50% reduction in TSS-C. The most common nonhematologic adverse events in the RUX arm on randomized treatment were fatigue (20.4% RUX vs 10.7% HU), headache (16.7% vs 5.4%), and dizziness (13.0% vs 8.9%). The most common adverse events on HU were diarrhea (9.3% RUX vs 19.6% HU) and constipation (7.4% vs 12.5%); most events were grade 1 or 2. Grade 3 or 4 anemia or thrombocytopenia (lab values) were not reported in the RUX group; two patients in the RUX group had grade 3 or 4 neutropenia. Conclusion: In generally well controlled PV patients receiving a stable dose of HU, there was a positive trend in symptom improvement for patients switched to RUX therapy versus those continuing on HU therapy, although this was not statistically significant. The 34% response rate among patients who continued to receive a stable HU dose suggests a placebo effect that led to an underpowered study. Further analyses are required to better interpret these findings. Disclosures Off Label Use: Ruxolitinib is a JAK1/JAK2 inhibitor approved for the treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Vannucchi:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Yacoub:Alexion Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Novartis Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrne:Novartis Pharmaceuticals: Honoraria. Verstovsek:Incyte Corporation: Research Funding. Hunter:Incyte Corporation: Employment, Equity Ownership. Jones:Incyte Corporation: Employment, Equity Ownership. He:Incyte Corporation: Employment, Equity Ownership. Morozov:Novartis Pharmaceuticals: Employment, Equity Ownership.

GATA1, founding member of the GATA transcription factor family, is essential for cell maturation and differentiation within the erythroid and megakaryocyte (MK) lineages. Disruption of DNA- or protein-binding capacity of GATA1 causes severe hematopoietic dysfunction and plays a role in blood disorders such as thrombocytopenia, anemia or leukemia. GATA1 expression seems to be related to the MK commitment both in mice and in humans; indeed, similarly to the murine myeloid M1 cell line, in which the enforced expression of GATA1 induces the c-Mpl appearance and MK differentiation, transduction of human hematopoietic stem cells with a GATA1 highly expressing vector results in self-renewal block and in the exclusive generation of Meg-E lineages. More recently, a role for GATA1 also in myeloproliferative disorders (MPDs) was indicated by the “GATA1-low” mouse model which develop a disease closely resembling the human idiopathic myelofibrosis. Interestingly, patients affected by myelofibrosis was also shown to express decreased GATA1 levels by immunostaining of BM sections. In this study, we investigated by Real Time PCR the levels of GATA1 in a myeloproliferative disorders such as essential thrombocytemia (ET) tipically characterized by a neoplastic megakaryocitic proliferation. We have studied BM samples of 40 newly diagnosed patients (M:F ratio 1:1 - median age 53 years, range 18–84) affected by ET, as for the PVSG group criteria. These patients were selected from a cohort of 65 ET patients considering a similar erythroid/myeloid ratio at the FACS analysis to reduce a possible bias for the RT-PCR results due to the erythroid compartment interference. The median platelets count of the selected patients was 670,000/mL (range 493,000–1,400,000/mL), myelofibrotic index 0/1, and 18 out of 40 patients (45%) showed mild splenomegaly both at the physical examination and US scan (median spleen vol 550 ml - range 430–1400 mL). No chromosomal abnormalities were detected by cytogenetic analysis. JAK2 sequencing in 21/40 patients indicated that 9/21 patients (43%) were positive for the JAK2 V617F genomic mutation. At the end of observation time (median 18 mo.) no patients had evidence or signs of thrombotic or hemorrhagic complications. BM cells from six healthy donors were used as normal controls in the study. The relative GATA1 quantification was calculated in according to the DCt method with GAPDH as internal control. The results showed a significant increase of GATA1 expression in BM cells from ET patients (median DCt + 6,11 ; range −0,41/+18,11) compared to the controls (median DCt + 0,172 ; range −4,03/+1,7) (p < 0,003). Interestingly, the GATA1 overexpression is not a mere consequence of the proliferation and activation of MKs, indeed samples from three patients affected by idiopathic thrombocytopenic purpura, whose BM smears had the typical secondary megakaryocytic hyperplasia, showed GATA1 levels much lower than the ET patients (median DCt − 0,6 ; range − 3,21/−0,9). No significant difference in GATA1 level was found between patients harbouring a JAK mutation (median DCt +5,86 ; range 0,85/16,12) and those with wild type alleles (median DCt +4,75 ; range −0,41/10,21). In conclusion, our results suggest that GATA1 overexpression could be a trigger for MK neoplastic commitment and proliferation and, consequently, seems to have a central role for ET pathogenesis both in JAK2 mutated and in JAK2 WT patients.

EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes. Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.

Blast localization in the brain occurring at diagnosis or at relapse in AL and in NHL patients is considered an adverse prognostic event. The management of meningeal or CNS disease includes intrathecal (IT) injection of cytotoxic drugs (methotrexate, cytarabine, steroids), cranial radiotherapy and high dose chemotherapy. IT therapy is currently carried out by weekly or biweekly drug administration, until clearance of leukemic cells from CSF. Liposomal cytarabine (Depocyte®), a newcomer in the strategy of lipid incapsulation of cytotoxic drugs, is a molecule registered exclusively for IT administration in lymphomatous meningeal diseases. Because of its long-lasting clearance profile, this drug can be administered every two weeks, thus resulting in better patient compliance. From September 2004 to April 2006, we treated 8 patients by IT Depocyte at the standard dose of 50 mg every two weeks, for a total of 25 doses. Three patients had acute leukemia: 2 ALL, 1 AML; 24, 54, and 55 year-old, respectively. At time of meningeal involvement, two patients were also in hematological relapse, while the other was in complete hematological remission. The patients had been previously treated by cytotoxic chemotherapy according to the current protocols; intrathecal prophylaxis with MTX and methylprednisolone had also been administered to ALL patients. A 46 year-old patient, had CML in blast crisis (CML-BP), with blast cells expressing both myeloid and lymphoid markers (CD 34+, CD19+, CD10+, CD24+, CD79a+, TdT+, CD33+, CD13+, CD11b+, CD7+). The blast crisis, involving simultaneously bone-marrow and CNS, had occurred during treatment with imatinib. Two elderly patients (> 60 year-old) suffering from NHL (mantle-cell and diffuse large-B-cell histotypes, respectively, had showed CNS localization (meningeal and meningeal + right orbital mass) during their disease progression. Two young patients, aged 28 and 39 yrs, suffered from primary cerebral (PCNS) lymphoma. 7/8 patients had blast cells detectable at CSF examination (mean, 857/μL; range, 23 – 2500/μL). AL patients received four doses of Depocyte as unique IT drug, and all showed early and complete clearance of blast cells from CNS. In the patient with CML-BP, CSF was not cleared after two doses of the drug, and the treatment was interrupted. As for NHL patients, the one with mantle cell lymphoma received the four scheduled Depocyte injections, and showed complete remission of the meningeal localization, while the other stopped the treatment after the 2nd dose because of CNS disease progression. Both patients with PCNS lymphoma received only two doses of Depocyte because of rapid neurological deterioration. No patient showed toxicity or adverse events due to the IT administration of liposomal cytarabine. In responding patients (4/8), CSF leukemic cell count fell rapidly after the first IT injection, and none of them had further meningeal involvement at hematological disease progression. Intrathecal therapy with Depocyte appears feasible, well tolerated and effective in patients with meningeal leukemic/lymphomatous disease, while it was ineffective, at least in our hands, in patients suffering from primary CNS lymphoma, for which the gold standard of treatment remains systemic high dose methotrexate and cytosine arabinoside followed by radiation therapy.

The molecular etiology of subgroups of essential thrombocythemia (ET), as well as some other myeloproliferative disorders (MPDs) has been recently better understood by the identification of an activating somatic point mutation (Valine 617 to Phenylalanine) in Jak2 detected in patients with MPDs. Approximately 23%–57% of ET patients harbor this mutation, which is thought to be one of the dominant factors contributing to the disease. However, the pathways disrupted in at least half of ET patients who have wild type Jak2 are not known. The fusion gene AML1/MDS1/EVI1 (AME), a product of the t(3;21)(q26;q22) translocation, is associated with several hematopoietic disorders, primarily chronic myelogenous leukemia and acute myeloid leukemia, and to a lesser extent with ET. We have investigated the role of AME by generating C57BL/6J mice that express this fusion oncogene in their bone marrow after bone marrow infection and transplantation. After a latency of 12 ± 1.8 months, all the reconstituted mice invariably developed an ET-like disease. The disease was fatal and the animals died of anemia probably caused by bleeding. Until 1–2 weeks before death, at which time the mice were severely cytopenic, the peripheral blood hemoglobin and leukocyte counts of the reconstituted AME mice were normal. The only striking difference was the platelet counts, which were consistently above the normal range (1456–3153 and 940–1608 K/ml for AME and control mice, respectively, p=0.00029). Platelets appeared dysplastic with anisocytosis, various degrees of degranulation, and giant size. The bone core biopsies of AME mice were especially remarkable for large and giant megakaryocytes with a tendency for clustering. Touch preparations of the biopsies showed progressive maturation in the granulocytic and erythroid precursor cell lines, a normal myeloid to erythroid ratio and no evidence of dyshematopoiesis. These finding coupled with markedly increased levels of TPO but normal concentrations of c-Mpl and Epo-R are concordant with the morphologic and laboratory features of patients with ET. The sequence analysis of exon 12 of Jak2 demonstrated no guanine-to-thymine transversion at nucleotide 1849 in all AME mice tested. These data are valuable because they provide a novel mouse model for understanding the deregulated pathways in ET patients with wild-type Jak2.

Essential thrombocytemia (ET) is a myeloproliferative disorder characterized by a primary increase in platelets count often associated with splenomegaly and/or hepatomegaly often developing/evolving in thrombosis. The t(3;21)(q26;q22) resulting in the AML1/MDS1/EVI1 (AME) fusion gene is associated with several hematological disorders often characterized by severe dysmegakaryopoiesis, suggesting that AME alters the megakaryocytic program. By BM infection and transplantation we generated two groups of mice that express either wild type AME or a point/internal-deletion mutant of AME (AME-D-C). This mutant lacks the eighth zinc finger motif and is unable to interact with the CtBP corepressor. Both groups of mice developed a fatal disease with features of human ET, however Kaplan-Meyer analysis showed a significant difference (p < 0.0032) in survival between the two groups with the AME-D-C-mice living considerably longer. The morphological analyses of the peripheral blood smears did not show significant differences between the AME- and AME-D-C-mice. However while the AME-mice were anemic, the AME-D-C-mice had normal counts. In both groups the platelet number was increased and atypical features like macro-platelets, agranular platelets and small aggregates were observed. Both groups of mice developed thrombocytosis with median platelet values consistently above the normal whereas the neutrophil counts were normal. It is possible that death resulted from bleeding complications maybe due to an intrinsic platelet dysfunction more evident in the AME population. Peripheral blood smears showed evidence of megakaryocytic fragments, large cytoplasm pieces containing pro-platelet, and nucleus scraps in both groups of mice. Seven out of 12 AME-mice and 8 out of 12 AME-D-C-mice showed slight neuthrophil dysplastic aspects such as hypersegmentation and open nuclei. The red blood cells were normocromic and normocytic in all mice however minimal anisocytosis was observed during the course of disease, with a variation in degree of polycromatophilia at the time of death. The bone core biopsies were normocellular in eight AME mice, three were hypocellular and only one was hypercellular. In contrast the cellularity in all the AME-D-C samples was normal. The most prominent aspects of the AME-mice were an increased number of BM megakaryocytes often grouped in clusters of four to six elements located in proximity of sinuses or in paratrabeculae and rare megakaryocytes that contained intact white cells in their cytoplasm. These two features previously described as endosteal dislocation and emperipolesis, often seen in patients with MPD or ET, were absent in AME-D-C-mice, in which only a minimum increase of megakaryocyte without atypical aspects was observed. No significant differences were found in spleen size, weight and histological features aside from a slight increase in fibrosis in AME-D-C-mice. These results suggest that a mutation in the eighth zinc finger motif and the abrogation of CtBP binding could attenuate the myeloproliferative phenotype caused by AME particularly in terms of histological features and overall survival.

The prognostic significance of the response to initial prednisone treatment in adult ALL has been recently emphasized. Prednisone response is usually defined on the basis of the peripheral leukemic blast count. The threshold value for the defintion as good or poor prednisone response is 1000 blasts/mmc on day 8 of prednisone pre-treatment. The drawback of this definition is the difficulty of classifying patients with less than 1000 blasts at diagnosis. In the LAL2000 GIMEMA study we therefore evaluated whether the blast reduction rate, which is not affected by the initial blast level, could be a factor with comparable prognostic value. The protocol design provided a 7-day (−6 to 0) pre-treatment phase with an escalating dose of prednisone up to 60 mg/sqm. On day 1 before starting the induction the response was assessed both according to the absolute blast count (< versus ≥ 1000/mmc) (criterion 1) and according to the blast reduction rate ≥ 75% (criterion 2) in the peripheral blood. The induction included high dose Daunorubicin; for patients in complete remission (CR) this was followed by consolidation with high dose ARA-C, chemo and radio prophylaxis of the central nervous system, and periodical reinduction over a three years maintenance period. Patients with adverse cytogenetic features [i.e. t(9;22), t(4;11), t(1;19)] who achieved a CR were treated according to the HAM protocol that included high dose ARA-C and Mitoxantrone followed by Imatinib for Ph+ ALL and by allogeneic or autologous hemopoietic stem cells transplantation for the others. Between September 2000 and December 2003 a total of 368 patients were evaluable for response to induction. The median age was 35 years (15–60) and median WBC count 15′109/L (0.3–872); 72 (20%) were T ALL and 121 (33%) had cytogenetic high risk features (104 (86%) Ph+, 4 (3%) t(4;11) and 13 (11%) t(1;19)). Eighty-seven percent of the patients were evaluable for response to steroid pre-treatment: ’responders’ were 75% according to criterion 1 (blast =/

Abstract 4090 Transformation to acute myeloid leukemia (AML) is a known complication of MPN but the role of JAK2V617F mutation is still undefined. In 2006, Campbell described a possible model for the development of a JAK2WT-AML in a patient with JAK2V617F-MPN and more recently, Theocharides et al. reported that in up to 53% of the patients who developed secondary AML from a JAK2-mutated MPN the mutation was no longer detectable; however the results obtained with DNA extracted from cells scraped or laser-capture-microdissected from bone marrow (BM) or peripheral blood (PB) smears, were confirmed in fresh samples only in few cases. In this study, we collected, by cell sorting, blast cells and mature myeloid cells (granulocytes, GRA) from whole BM aspirates of 40 newly diagnosed patients with AML secondary to MPN (18 derived from PMF; 13 from PV and 9 from ET) and analyzed the JAK2 status before and after leukemic transformation in selected cell compartments. To evaluate the modification in the JAK2 status before and during leukemic transformation we performed ASO-PCR and (QRT)-PCR assay on total BM of the MPN phase and sorted cell populations from AML phase. At the time of MPN diagnosis, JAK2V617F was detectable in 28 of 40 patients (70%) (10 of 18 PMF; 12 of 13 PV and 6 of 9 ET). No cytogenetic abnormalities or MPL and JAK2-exone 12 mutations were detected at this stage. Median time to AML progression (TTP) was 5.09 years (yrs) (range 0.38 – 27.81). A significant difference (p= 0.02) in TTP was found grouping patients according to JAK2 status during the MPN phase [JAK2WT-MPN n=12, TTP median 15.10 yrs (0.38-16.32); JAK2 mutated-MPN n=28, TTP median 4.07 yrs (0.67-27.81)]. Eight patients showed additional abnormalities involving chromosomes 1, 5, 7, 8, 9, 12, 14, 17 and 20 while no other AML-associated mutations (FLT3, NPM, CEBPA, RUNX1) were detectable at this stage. In our cohort of patients we found that JAK2V617F mutation was still present at the blast transformation in both compartments: CD34+ cells (blasts) and CD15+ cells (GRA) in 24 of 28 JAK2 mutated MPN (86%). Four of 28 patients (14%) developed JAK2V617F negative AML starting from a mutated PV with a mean TTP of 5.14 yrs. Interestingly, the WT status was confirmed in blast cells but also in GRA. Surprisingly we found 2 cases of JAK2V617F mutated AML transforming from a WT-PMF. Also in this case the JAK2V617F positivity in the AML phase occurred in both GRA and blast compartments. No differences (p= 0.3) in the allele burden were found comparing MNCs from chronic phase with MNCs of leukemic transformations or comparing GRA with blasts in AML phase. In conclusion, these results contrast with the previous study in which the JAK2 mutation was lost in 53% of blasts during leukemia transformation. In our work, the loss of JAK2V617F mutation during AML progression is a rare event (14%). Additional studies in larger patient series and multivariate analysis are needed before a prognostic role of JAK2V617F mutation regarding time to leukemia transformation can be definitely assessed. Disclosures: Vannucchi: Novartis: Consultancy.