Atypical Chronic Myelogenous Leukemia Research Articles

Atypical Chronic Myelogenous Leukemia, BCR-ABL1 Negative: Diagnostic Criteria and Treatment Approaches.

Atypical chronic myelogenous leukemia (aCML), BCR/ABL1 negative is a rare myelodysplastic/myeloproliferative neoplasm, usually manifested with hyperleukocytosis without monocytosis or basophilia, organomegaly, and marked dysgranulopoiesis. In this review, we will discuss the classification and diagnostic criteria of aCML, as these have been formulated during the past 30 years, with a focus on the recent advances in the molecular characterization of the disease. Although this entity does not have a definitive molecular profile, its molecular characterization has contributed to a better understanding and more accurate classification and diagnosis of aCML. At the same time, it has facilitated the identification of adverse prognostic factors and the stratification of patients according to their risk for leukemic transformation. What is more, the molecular characterization of the disease has expanded our therapeutic choices, thoroughly presented and analyzed in this review article.

Management of Patient Primigravida 36-37 Weeks with Chronic Myeloid Leukemia, Anemia, and Thrombocytopenia: A Case Report

<p>Chronic myelogenous leukemia (CML) is a type of cancer caused by a disturbance in the hematopoietic stem cells. CML itself rarely occur on women who are in labor and an advanced procedure in this event has become a special challenge for medics, especially an anesthesiologist. This limits the development of standard anesthesia guidelines, so in this case we describe the incidence of CML in pregnancies performed by Cesarean section with general anesthesia.</p><p>The first pregnant patient was 36 weeks pregnant; the patient was first diagnosed with Chronic myelogenous leukemia (CML) at the age of 26-28 weeks, at that time the patient complained of frequent dizziness, abdominal pain and weakness, then the patient complained of bleeding gums, and currently the patient complained of nosebleeds. The Bone Marrow shows Conclusion an accelerated phase chronic myeloid leukemia (CML) (suspected atypical CML) with nutritional deficiency. We perform General Anesthesia technique Rapid Sequence Intubation with Regimen Fentanyl 100 mcg, Propofol 80 mg and Rocuronium 50 mg.</p><p>The patient was admitted to the ICU for 2 days before transferring to intensive care and the patient received intravenous paracetamol 1 gram four times, cefazolin 1 gram twice a day, lansoprazole 30 mg once a day, tranexamic acid 1gr three times a day, and 15 mcg per hour fentanyl contionously. Hemodynamic patients in the ICU are in a stable condition. On the second postoperative day of care, the patient was transferred to the High care ward, then at the third postoperative day the patient's hemodynamics was stable and the patient was transferred to a normal room.</p>

The Colony-Stimulating Factor 3 Receptor M696T Mutated in a Patient with Ph+ Acute Lymphoblastic Leukemia

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders, such as severe congenital neutropenia (SCN), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and atypical chronic myelogenous leukemia (aCML). Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. We report a case of philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) with the M696T mutation in CSF3R gene and assess the pathogenicity of the CSF3R M696T mutation in Ph+ ALL. Experimental Design: Here we report on a 32-year-old female who presented with asthenia. The initial hematological workup revealed white blood cell (WBC) count of 97 x 109/L (normal range 4-10 x 109/L). There was 84% prolymphocyte in the bone marrow. The immunophenotype of the blasts as judged from flow cytometry was in accordance with a B-ALL. The fusion gene for BCR-ABL P210 was positive. Hot mutation closely related to diseases was: CSF3R (nucleotide change c.2087 T>C, amino acid change p.M696T, mutation frequency 50.4%). Cytogenetic analysis showed 46, XX, t (9;22) (q34;q11). The patient was diagnosed as Ph+ ALL with the CSF3R M696T mutation and achieved Long-term survival after unrelated donor hematopoietic stem cell transplantation. Meanwhile we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. The phosphorylation of STAT3 was analyzed by G-CSF dependence assays and immunoblot analysis to evaluate the CSF3R M696T mutation contribution to the tumor transformation ability of Ba/F3 cells. Results: This patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. We confirmed the presence of a CSF3R M696T germline mutation in this patient, and the mutation was inherited from her mother. The experiments in vitro result showed the CSF3R M696T mutation harbors marginal contribution to the tumor transformation ability of Ba/F3 cells. CSF3R M696T mutation was neutral in tumor transformation ability. Conclusions: We believe that TKI is still effective in patients with the CSF3R M696T mutation in Ph+ ALL. Donor with CSF3R M696T mutation might still be selected. Disclosures No relevant conflicts of interest to declare.

Treatment Free Remission for Chronic Phase Chronic Myelogenous Leukemia Patients Who Stopped Tyrosine Kinase Inhibitor Therapy Due to Intolerance

Introduction: Chronic Myelogenous Leukemia (CML) is a myeloproliferative neoplasm that is often diagnosed in adults between the ages of 25-60. The outcome of the chronic phase CML has dramatically changed due to Tyrosine Kinase Inhibitor (TKI) therapy. There are well established guidelines from NCCN and ESMO on stopping TKI for patients achieving prolonged remissions with TKIs. We report clinical outcomes from a single tertiary care center in patients who stopped TKI therapy for reasons other than prolonged remission status. Methods: We retrospectively reviewed all the CML patients who were treated at our institution in the past 10 years (January 1st,2009 - December 31st,2018). We excluded patients who had accelerated or blast phase CML, atypical CML, patients on non-TKI therapy, and patients who received an allogeneic stem cell transplant. Results: A total of 117 patients were diagnosed with chronic phase CML at our institution in the past 10 years. Among the 117 patients, 12 of these discontinued TKI therapy. Six patients stopped TKI after achieving prolonged remission with TKI therapy and the remaining patients discontinued due to intolerance to treatment, fear of side effects, and loss of insurance. The median age of the whole cohort is 66 years (range 42-85). Six patients were male and 6 were females. Six patients were diagnosed with CML prior to year 2009 and rest after 2009. Prior to stopping, six patients received only 1 kind of TKI, 2 patients were treated with 2 types of TKIs, 2 patients received 3 types of TKIs, and 2 patients had 4 lines of TKIs (See Table). Cohort 1: 6 patients who stopped due to prolonged remission, median major molecular remission - MMR4 (BCR-ABL <0.01% IS by RT-PCR testing) prior to stopping TKI is 6 years (range 3-13 years). Of the six, only 1 relapsed (within 1 month of stopping) and was initiated back on the same TKI (imatinib). The relapsed patient has not achieved MMR4 level remission to date. Median treatment free remission for this cohort is 13 months (range 1-24 months). Cohort 2: Of those 6 patients who stopped TKI for other reasons: 4 stopped due to side effects/intolerance, 1 stopped due to fear of side effects after FDA label was updated, and 1 patient discontinued due to a loss of insurance. Median duration of MMR4 prior to stopping is 4 years (range 1-11 years). 5 of these 6 patients relapsed in the median time of 6 months (range 3-16 months). Of these 5, 4 were started back on the TKI therapy (three on the same TKI and one on a different TKI). Median treatment free remission for this cohort is 4 months (range 2-16 months). Conclusion: In this small cohort from a single institution's experience, CML patients who discontinued TKI therapy after achieving MMR4 for reasons other than prolonged remission have experienced poor outcomes including a higher rate of relapse and a shorter treatment free remission. We need studies with larger samples sizes and longer follow up to assess outcomes in patients stopping TKI therapy for various reasons. Disclosures No relevant conflicts of interest to declare.

Inhibitory effect of RNA interference targeting GFI-1 on the proliferation of atypical chronic myelogenous leukemia NT1 cells

To investigate the inhibitory effects of RNA interference targeting GFI-1 on growth and proliferation of atypical chronic myelogenous leukemia (aCML) NT1 cells. NT1 cells were transfected with PBS and liposome complex (vehicle group), scrambled siRNA and liposome complex (negative control, NC group), and GFI-1 siRNA and liposome complex (GFI-1 siRNA group), respectively. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression levels of GFI-1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Quantitative real-time PCR data showed that the expression level of GFI-1 mRNA in GFI-1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (P<0.05)]. Western blot results showed that the GFI-1 protein expression level in the GFI-1 siRNA group was also significantly reduced, compared with those of the NC group and vehicle group (P<0.05 for both). From MTT assay data, the absorbance value of NT1 cells in the GFI-1 siRNA group (0.667±0.059) was significantly lower than those of the NC group (1.096±0.049) and vehicle group (1.193±0.064, P=0.023). Flow cytometry data showed that sub-G1 and G0/G1 phase proportions of the GFI-1 siRNA group were significantly higher than those of the NC and vehicle groups [sub-G1: (8.2±2.5)% vs. (1.9±1.3)% and (2.0±3.6)%, respectively, (P<0.05); G0/G1: (66.7±3.8)% vs. (53.3±4.5)% and (48.6±3.2)%, respectively, (P<0.05)]. Furthermore, the tumor weight in the GFI-1 siRNA group [(0.37±0.02) g] was significantly lower than those in the NC group [(0.83±0.06) g] and vehicle group [(0.92±0.04) g] (P<0.05). RNA interference targeting GFI-1 inhibits the growth and proliferation of NT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.

Chronic myelogenous leukemia-like hematological malignancy with t(8;22) in a 26-year-old pregnant woman: A case report.

t(8;22)(p11;q11) is a rare but recurrent genetic alteration in various hematological disorders. Patients with t(8;22)(p11;q11) may be misdiagnosed with chronic myelogenous leukemia (CML), due to the similar clinical features. Thus, the current study presents a patient with t(8;22)(p11;q11) who was previously misdiagnosed with CML in the chronic phase. The current patient was a 26-year-old woman who was 4-weeks pregnant and in whom an increased white blood cell count (4.0×1010/l) was found upon physical examination. The patient had no history of hematological disease. Although cytogenetics showed a normal karyotype and no breakpoint cluster region/Abelson murine leukemia viral oncogene homolog 1 (BCR/ABL) fusion gene was detected by reverse transcription-polymerase chain reaction, a diagnosis of chronic myelogenous leukemia (CML) was initially made according to the clinical and morphological features. Another 6 weeks later, t(8;22)(p11;q11) rearrangement was present in 9 out of 10 analyzed metaphases. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction indicated a negative result for the BCR/ABL fusion, but gave a positive result for the BCR-fibroblast growth factor receptor 1 fusion. A hematological diagnosis of atypical CML was again formed.

DNMT3A mutations exert a dominant adverse effect in De novo acute myeloid leukemia with concurrent FLT3 and NPM1 mutations

S188 of AML, MDS, and MDS/MPN with t(v;5q33-34) remain poorly characterized. Methods: We queried our files for all myeloid neoplasms associated with t(v;5q33-34). Therapy-related myeloid neoplasms and cases with peripheral eosinophilia fulfilling criteria of myeloid/lymphoid neoplasms with abnormalities of PDGFRB were excluded. The clinicopathologic features of the study group were reviewed. Results: The study group (n1⁄417) included 12 men and 5 women with a median age of 64 years (range, 19 to 86 years). At presentation, 12 patients had AML, 4 had MDS, and 1 had MDS/MPN. In addition to t(v;5q3334) among patients with AML, 3 (25%) had neither myelodysplasia nor other MDS-related cytogenetics, 2 (17%) had myelodysplasia only, 5 (83%) had other MDS-related cytogenetic changes only, and 2 (17%) had myelodysplasia and other MDS-related cytogenetic changes. Among patients with MDS, 3/4 had only t(v;5q33-34) and 1 patient had a diploid karyotype and acquired t(v;5q33-34) at the time of transformation to AML. The patient with MDS/MPN had atypical chronic myelogenous leukemia and only t(v;5q33-34). Three patients with AML had a prior history of MPN (essential thrombocythemia, polycythemia vera, and MPN not otherwise specified), and 1 patient had chronic myelomonocytic leukemia. The latter case was the only patient with a history of prior cytotoxic therapy. A median follow up of 25 months (range, 1-108) was available on 14 patients. At last follow up, 10/14 patients were dead (all with AML) and 4 patients were alive (3 with MDS; 1 with MDS/MPN). Conclusions: AML with t(v;5q33-34) is associated with poor outcome but is not always associated with myelodysplasia or MDS-related cytogenetic features. Although a larger dataset is needed, in this study group patients with MDS or MDS/MPN and with t(v;5q33-34) seem to have a favorable outcome.

Acute Lymphoblastic Leukemia evolving from atypical Chronic Myelogenous Leukemia: Case Report and Review of the Literature

Atypical chronic myeloid leukemia (aCML) is a rare chronic myeloproliferative disorder characterized by leukocytosis, absence of Philadelphia chromosome or BCR-ABL rearrangement, and marked myeloid dysplasia. The diagnosis of aCML is difficult and challenging, more so if the initial presentation is with blast crisis. In the absence of characteristic mutational profile, blast crisis of aCML can lead to significant diagnostic dilemma. We describe a case of refractory acute lymphoblastic leukemia (ALL), needing critical evaluation of hemopathological and cytogenetic abnormalities. It was hypothesized that patient had undiagnosed aCML and presented with lymphoid blast crisis. To our knowledge, this is the first described case of aCML presenting with lymphoid blast crisis.

Identification of the Novel Fusion Gene, SPAG9-JAK2, in Adolescent Acute Lymphoblastic Leukemia (ALL) with t(9;17)(p24;q21), and Its Association with Gene Alterations Involved in Ph1/BCR-ABL1-like ALL

Introduction: JAK2, which is located on chromosome 9p24, encodes a cytoplasmic tyrosine kinase involved in cytokine-mediated signal transduction, and is fused to 10 partner genes in hematological malignancies, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia, atypical chronic myelogenous leukemia, myelodysplastic syndrome/ myeloproliferative neoplasms (MPN), and Hodgkin lymphoma with 9p24 translocations. Furthermore, a somatic acquired activating mutation of JAK2 (V617F) was noted in a majority of patients with MPN, and mutations at R683 of JAK2 was detected not only in ALL with Down syndrome, but also in high-risk pediatric B-cell progenitor (BCP)-ALL.Case presentation: A 14-year-old boy presenting with a persistent fever was admitted to our hospital because of hyperleukocytosis and thrombocytopenia. Cytogenetic analysis demonstrated the 46,XY,t(9;17)(p24;q21) in 17 of 20 bone marrow cells. He was classified as extremely high risk BCP-ALL with myeloid marker and treated on TCCSG L95-14 HEX/SCT protocol. He obtained a complete remission after the induction therapy, relapsed after 7 months. Although he underwent an auto bone marrow transplant because he had no matched donor, 19 months after the initial diagnosis, he died due to progressive disease.Materials&Methods: To characterize the breakpoints, FISH analysis was performed on metaphase chromosome using BAC clones closely flanking JAK2. Genomic DNA and total RNA were isolated from bone marrow cells at diagnosis and relapse. For the mRNA-Seq sample preparation, sequencing libraries were generated according to the standard Illumina protocol for high-throughput sequencing. Using a HiSeq 2000 sequencer, 100 bp-paired end reads were obtained. Mapping reads to genes were performed by using a Bowtie software and fusion transcript discovery was done by a deFuse data analysis software. We evaluated DNA copy number changes by multiplex ligation-dependent probe amplification (MLPA) analysis and Single nucleotide polymorphism (SNP) array analysis.Results: FISH showed a fusion signal on normal chromosome 9, and split signals on der(9) and der(17). We identified a novel JAK2 fusion gene by paired-end mRNA-seq. This fusion transcript was identified. RT-PCR followed by direct sequencing confirmed the in-frame fusion of SPAG9 exon 26 and JAK2 exon 19. Moreover, the homozygous deletion of CDKN2A, TRG, TRA/D, and IGH and hemizygous deletion of CDKN2B, PAX5, BTG1, CDK6, TRB, ADARB2, IGL and IKZF1were identified by MLPA and SNP array.Discussion: This ALL having both rearrangement activating tyrosine kinase and genomic lesions affecting lymphoid transcription factors is similar to the Philadelphia chromosome (Ph1)/BCR-ABL1 like ALL subgroup. In conclusion, we have identified SPAG9 as a novel fusion partner of JAK2 gene in adolescent BCP-ALL as a consequence of a t(9;17)(p24;q21). The further examination of this fusion gene may be worthwhile to consider that JAK2 would be a good target for treatment in refractory ALL patients with rearrangements in JAK2. DisclosuresNo relevant conflicts of interest to declare.

G-CSF receptor ( CSF3R ; CD114 ) mutations to guide diagnosis and treatment of atypical chronic myelogenous leukemia (aCML) and chronic neutrophilic leukemia (CNL)

G-CSF receptor ( CSF3R ; CD114 ) mutations to guide diagnosis and treatment of atypical chronic myelogenous leukemia (aCML) and chronic neutrophilic leukemia (CNL)

Fighting back against chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder with features of both myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS). Thus, CMML is classified as a mixed MDS/MPN disease by the World Health Organization (WHO). CMML is primarily a disease of the elderly with a median age of 65-75 years at diagnosis, and accounts for approximately 10% of all MDS cases. Patients with CMML suffer mainly from cytopenias, splenomegaly, or organ infiltration by monocytes [1]. The prognosis of this disorder is generally poor. Treatment with hypomethylating agents such as azacitidine and decitabine elicit hematologic responses and provide survival advantages in several patient populations. The responses, however, are usually not long lasting or curative [2, 3]. Recently, prognosis scoring systems have been proposed for CMML [4, 5], and relevant discoveries have been made in its molecular pathogenesis. However, these developments have not yet improved the clinical outcome for CMML patients. Although allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment, it is rarely feasible because of the nature of this disorder. Thus, therapy for CMML remains a challenge. Studies on the efficacy of HSCT for treatment of CMML are quite limited. Although a few case reports have been published, no prospective studies have been reported thus far. Recently, a French group reported the outcome of allogeneic HSCT in 73 patients with CMML [6]; this study included the largest patient group among such studies published to date. In this study, the 3-year overall survival, non-relapse mortality, event-free survival, and cumulative incidence of relapse were 32%, 36%, 29%, and 35%, respectively. They concluded that allogeneic HSCT is an effective treatment option for patients with CMML, and the outcome of patients undergoing HSCT has improved in recent years. Data on HSCT in Asian populations with CMML are even more limited. One report from China described the clinical outcome of 12 patients with CMML [7]. In this report, the overall survival, disease-free survival, and relapse rate after the median follow-up of 17.5 months (12-32 months) were 66.7%, 66.7%, and 16.7%, respectively. Therefore, the authors concluded that allogeneic HSCT could improve the survival of patients with CMML. In this issue of Blood Research, Lim et al. report the outcome of allogeneic HSCT in 10 Korean patients with mixed MDS/MPN [8]. Although this report includes 2 patients with atypical chronic myelogenous leukemia and 1 patient with unclassifiable MDS/MPN, of particular interest are the 7 patients with CMML. Of these 7 patients, 3 maintained disease-free survival for 4.6, 31.2 and 47.5 months, respectively. Four patients died from cytomegalovirus pneumonia, graft versus host disease (GVHD), and CMML relapse, at 2.7, 8.7, 10.1, and 36.2 months. The relapses were observed in patients who received low-intensity conditioning regimens and did not develop chronic GVHD. Although the results of this study appear promising, it is important to note that the number of patients analyzed was small, and there was no uniformity in the therapies patients received prior to HSCT, the degree of HLA mismatch, or the type of conditioning regimens. Therefore, we should be cautious when drawing conclusions from this study. However, despite the limitations, this study suggests that allogeneic HSCT may be efficacious for the treatment of patients with CMML. Because of the rarity of this disorder and the advanced age of most patients with CMML, a single medical center cannot easily perform a prospective study to answer all questions regarding allogeneic HSCT. However, it is crucial to determine how to best select transplant candidates, treat patients prior to transplant, find stem cell sources, discuss and identify issues involving HLA disparities, and determine optimal conditioning and GVHD prophylaxis regimens. Therefore, it is vital for multicenter and/or multinational prospective studies involving HSCT in CMML patients to be conducted in the future.

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated. TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro. Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis. This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice.

Abstract 1437Poster Board I-460Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation.Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2. DisclosuresNo relevant conflicts of interest to declare.

Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11) in a patient with acute myeloid leukemia

We report the occurrence of a BCR-JAK2 fusion gene in a case of acute myeloid leukemia (AML) resulting from a t(9;22)(p24;q11) translocation as the sole cytogenetic abnormality. The BCR-JAK2 fusion gene has the same breakpoint in BCR as is found in the BCR/ABL p210. The chimeric gene is the result of a reciprocal translocation between chromosomes 9 and 22, which implies a double break on chromosome 9; this has allowed generating an in-frame fusion transcript. Previously, BCR-JAK2 rearrangement was observed in a single case with atypical chronic myelogenous leukemia (CML), but in that case the breakpoint in the BCR was different.

Dimerization by a Cytokine Receptor Is Necessary for Constitutive Activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis

A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.

Increased Expression of CD47 Is a Constant Marker in Mouse and Human Myeloid Leukemias.

CD47, also known as integrin associated protein, is a ubiquitously expressed cell surface glycoprotein that interacts with a number of integrins, modulating leukocyte adhesion, migration, cell motility and platelet activation. CD47 is also the ligand for the macrophage inhibitory receptor signal regulatory protein α (SIRP α) and thus, impairs macrophage-mediated phagocytosis. Recent reports suggest that increased CD47 expression may play a role in the pathogenesis of lymphoproliferative disorders such as CLL (Mateo et al, Nat Med. 1999; 5:1277) and multiple myeloma, and that a bivalent single-chain antibody fragment against human CD47 induces apoptosis of myeloma cells (Kikuchi et al, Leukemia Research 2005; 29:445). However, little is known about the role of CD47 in the pathogenesis of myeloid leukemias or the stage of hematopoiesis at which CD47 is expressed. In order to identify the stage of hematopoiesis at which alterations in CD47 arise and its role in the pathogenesis of myeloid leukemias, we analyzed CD47 expression in hematopoietic stem cells (HSC), progenitors, and lineage-positive cells derived from three mouse transgenic models of myeloid leukemia including: 1) mice homozygous for overexpression of human bcl-2 in the myeloid lineage (hMRP8 bcl-2 x bcl-2); 2) mice deficient in Fas together with myeloid targeted overexpression of bcl-2 (Faslpr/lpr hMRP8 bcl-2); and 3) mice with both human bcl-2 and BCR-ABL targeted to the myeloid lineage (hMRP8-BCR-ABLxhMRP8-bcl-2). Quantitative RT-PCR analysis demonstrated a 3.01+/− 1.54 fold increase in CD47 transcripts in leukemic compared with control (bcl2+) bone marrow (normalized to b-actin) while FACS analysis revealed approximately a 10-fold increase in CD47 protein expression, as measured by mean fluorescence intensity (MFI), in leukemic GMP compared with wild type GMP. Moreover, transplantation experiments revealed that all mice with both primary (n=14 mice) and secondary (n=19 mice) leukemic transplantation potential had an expansion of granulocyte-macrophage progenitors (GMP) with high level CD47 expression. Human CD47 expression analysis was performed via FACS on human normal, pre-leukemic myeloproliferative disorder (MPD) or AML HSC, progenitors, and lineage positive cells derived from marrow or peripheral blood. MPD samples (n=63) included polycythemia vera (PV; n=15), post-polycythemic myeloid metaplasia/myelofibrosis (PPMM/MF; n=5), essential thrombocythemia (ET; n=8), atypical chronic myelogenous leukemia (aCML; n=2), CML (n=7), chronic eosinophilic leukemia (CEL; n=1), chronic myelomonocytic leukemia (CMML; n= 13) and acute myelogenous leukemia (AML; n=12). As noted with the transgenic leukemic mouse models, progression of human myeloproliferative disorders to AML (n=12) was associated with an expansion of the GMP pool (70.6% +/− S.D. 2.15) compared with normal bone marrow (14.7% +/− S.D. 2.3). Furthermore, FACS analysis revealed that CD47 expression first increased 1.7 fold in AML compared with normal HSC and then increased to 2.2 fold greater than normal with commitment of AML progenitors to the myeloid lineage. CD47 was over-expressed by AML primitive progenitors and their progeny but not by the majority of MPD (MFI 2.3+/−S.D. 0.43) compared with normal bone marrow (MFI 1.9 +/−S.D. 0.07). Thus, increased CD47 expression may serve as a useful diagnostic marker for progression to AML and may represent a novel therapeutic target.

Atypical BCR-ABL Transcripts Chronic Myelogenous Leukemias Show Particular Features, and Their Classical Worse Prognosis Seems Abrogated by Imatinib Mesylate. A Retrospective Analysis of 22 Patients, on the Behalf of the French Intergroup of CML (Fi(ϕ)-LMC Group).

Chronic Myelogenous Leukemia (CML) originates in the chromosome (Ph1), a reciprocal translocation, corresponding to the BCR-ABL fusion oncogene. A small proportion (1–2%) of CML patients show breakpoints falling outside of the M-BCR gene on chromosome 22, leading to the synthesis of a variety of atypical BCR-ABL transcripts [either shortened: e1a2 (m-BCR), e6a2, e8a2, b2a3 (e13a3), b3a3 (e14a3), or elongated transcripts: e19a2 (m-BCR)] and to the synthesis of different BCR-ABL proteins. In this study, we retrospectively analysed the clinical characteristics and outcomes of cohorts of CML patients harbouring atypical transcripts in and treated with imatinib (IM). Twenty-two patients were analysed: 9 e1a2 [Group 1 (G1)], 4 e6a2 [Group 2 (G2)], 5 e19a2 [Group 3 (G3)], and 4 e8a2 [Group 4 (G4)] BCR-ABL transcripts. Two patients were in myeloid blastic phase at onset (1 in G1 and 1 in G2) and others in chronic phase. Age at diagnosis was significantly younger for e19a2 patients (39.5 years versus 64 for G1, 58.5 for G2, 72 for G3, p=0.005). Female patients were predominant for G1 (5F/3M), but not for other groups. All patients presented a classical Ph1 at karyotyping analysis at diagnosis, but 1 had a -7 (G1), 1 an additional t(11;16) with the Ph1 (G2), 1 a +8 (G3) and 1 a -Y (G3). The majority of patients presented typical CML features at diagnosis, however number of differences could be found: WBC counts were higher for e1a2 and e8a2 patients (74.2 109/l and 62.7 respectively vs 20.9 for G2, and 37.8 for G3, p<0.03). A significant relative monocytosis was present for e1a2 patients (10% vs 4 (G2), 2.5 (G3), 5.5 (G4), p<0.05), and a marked basophilia was present for e6a2 patients vs others (p<0.0008). There was a trend for higher platelet counts in G3 vs others. Hasford and Sokal scores were somewhat comparable in all groups. Median follow-up since diagnosis was 24 months for G1, 10 for G2, 17 for G3 and 31 for G4. Only one patient received interferon for 7 months before IM (G1), all other patients did not receive any other treatment than hydroxyurea before IM. All patients were treated with IM alone initially at 400–600 mg/day. Median duration of IM was 18 months for G1, 9 for G2, 12 for G3, and 30 for G4. At time of analysis 1 pt in G2 and 1 patient in G3 died of progression (blastic phase), and the overall survival (OS) since IM start was better for e19a2 and e8a2 patients, but patients remain very few. However, these OS do not seem different from what has been observed for M-BCR transcripts (IRIS study). In conclusion, atypical BCR-ABL transcripts CMLs show particular diagnosis features, but their poor prognosis reputation seems abrogated by IM.

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia

AbstractActivating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.