Negative Chronic Myeloid Leukemia Research Articles

Kinetic of liver enzymes and serum electrolytes in HCV, HBV, and HIV negative chronic phase chronic myeloid leukemia patients treated with imatinib or nilotinib

The outcome for chronic phase (CP) chronic myeloid leukemia (CML) patients was changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. This study intended to evaluate side effects of TK Imatinib or Nilotinib on liver enzymes and serum electrolytes in relation to hematologic and molecular response in HCV-, HBV-, and HIV-, CP-CML patients. The study was a quasi-experimental pre-post single group design, included 38 HCV-, HBV-, and HIV-newly diagnosed Philadelphia positive CP-CML patients with normal hepatic and renal function. They were divided equally into two groups, 19 received Nilotinib, and 19 received Imatinib. Hematologic, BCR-ABL gene expression by RT-PCR, electrolytes and liver enzymes were measured at baseline and after 6 months of treatment. Patients age ranged between 20 and 62 years. Anemic manifestations represented the highest rate (n=23, 60.5%). The mean WBCs count was significantly reduced after treatment (p < 0.001). The WBCs count was significantly reduced in the Nilotinib group than the Imatinib group (97% and 94%, respectively, p=0.049). The mean hemoglobin level was significantly increased after treatment (p=0.010). The mean platelet level did not change over the treatment period. The mean AST, ALT, and ALP levels were significantly increased after treatment, (p=0.014, p=0.002, and p=0.047, respectively). The ALP level was significantly increased in both groups (p=0.001). The mean sodium potassium, phosphorous, and calcium level was not changed over the treatment period. The mean BCR-ABL gene expression was sharply decreased after treatment (p < 0.001). A higher reduction was observed in the Nilotinib group (99%) than the Imatinib group (91.5%) (p=0.025). Imatinib resulted in rise of AST and ALP levels than Nilotinib, while both had the same effect on the ALT level. Higher reduction in BCR-ABL gene expression was achieved by Nilotinib. Nilotinib and Imatinib did not affect serum levels of sodium, potassium, phosphorous, or calcium.

Impact of GSTT1 and GSTM1 Polymorphisms in the Susceptibility to Philadelphia Negative Chronic Myeloid Leukaemia.

Our research aimed to clarify the role of genetic polymorphisms in GST (T1 and M1) in the development of Ph-ve CML. We report on a case-control study with 126 participants, divided into 26 patients with Ph-ve CML (57.7% male, 42.3% female) and 100 healthy volunteers (51% male, 49% female) with no medical history of cancer as a control population. All Ph-ve CML patients were diagnosed according to standard hematologic and cytogenetic criteria based on CBC, confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to determine the presence or absence of the BCRABL gene, followed by bone marrow (BM) examination. Of the 26 studied cases, 50% had the GSTT1 null genotype against 21% of the control group, a statistically significant difference (CI= 1.519 - 9.317; p-value= 0.004). The GSTM1 null genotype was detected in 23.1% of cases and 35% of controls, a difference not statistically significant (OR= 0.557; CI= 0.205-1.515; p-value= 0.252). The distribution of GSTT1 and GSTM1 polymorphisms was also examined according to gender, age and ethnic grouping; these findings revealed no statistically significant differences. Our study reveals a strong correlation between GSTT1 polymorphism and Ph-ve CML, whereas the data for GSTM1 polymorphisms indicates no role in the initial development of the disease. More studies are required to further clarify these and other genes' roles in disease development.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential.

Simple SummaryIn chronic myeloid leukemia (CML), a neoplasm brilliantly taken care of by a molecularly targeted therapeutic approach, the achievement of cure is nevertheless prevented by the maintenance of a small subset of treatment-resistant leukemia stem cells (LSCs), sustaining the so-called minimal residual disease of CML. The phenotypical and functional characterization of this LSC subset is, therefore, crucial to aim at the eradication of disease. Such a characterization includes the acquisition of information relative to the metabolic profile of treatment-resistant LSCs, which is functional to their maintenance in bone marrow. A number of metabolic features of LSCs were shown to determine their sensitivity or resistance to therapy. Glutamine metabolism emerged from this study as a potential target to overcome the persistence of therapy-resistant LSCs.This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

APRVにより致命的な肺胞出血から救命し得た&lt;i&gt;BCR-ABL1&lt;/i&gt;融合遺伝子陰性非定型慢性骨髄性白血病の1例

APRVにより致命的な肺胞出血から救命し得た&lt;i&gt;BCR-ABL1&lt;/i&gt;融合遺伝子陰性非定型慢性骨髄性白血病の1例

Myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes: a focused review.

Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndromes are unique myeloid neoplasms, with overlapping features of MDS and MPN. They consist of four adult onset entities including chronic myelomonocytic leukemia (CMML), MDS/MPN-ring sideroblasts-thrombocytosis (MDS/MPN-RS-T), BCR-ABL1 negative atypical chronic myeloid leukemia (aCML) and MDS/MPN-unclassifiable (MDS/MPN-U); with juvenile myelomonocytic leukemia (JMML) being the only pediatric onset entity. Among these overlap neoplasms, CMML is the most frequent and is hallmarked by the presence of sustained peripheral blood monocytosis with recurrent mutations involving TET2 (60%), SRSF2 (50%) and ASXL1 (40%); with RAS pathway mutations and JAK2V617F being relatively enriched in proliferative CMML subtypes (WBC ≥13 × 109/L). CMML usually presents in the 7th decade of life, with a male preponderance and is associated with a median overall survival of <36 months. Adverse prognosticators in CMML include increasing age, high WBC, presence of circulating immature myeloid cells, anemia, thrombocytopenia and truncating ASXL1 mutations. While allogeneic stem cell transplantation remains the only curative option, given the late onset of this neoplasm and high frequency of comorbidities, most patients remain ineligible. Hypomethylating agents such as azacitidine, decitabine and oral decitabine/cedazuridine have been US FDA approved for the management of CMML, with overall response rates of 40-50% and complete remission rates of <20%. While these agents epigenetically restore hematopoiesis in a subset of responding patients, they do not impact mutational allele burdens and eventual disease progression to AML remains inevitable. Newer treatment modalities exploiting epigenetic, signaling and splicing abnormalities commonly seen in CMML are much needed.

Update on CML-Like Disorders

Chronic myeloid leukemia (CML) is defined for many years as BCR-ABL1 positive disease, but older publications refer to a poor prognosis, clinically heterogeneous entity termed ‘BCR-ABL1 negative CML’ constituting about 5% of CML cases. Apart from very rare CML cases with cytogenetically cryptic, atypical variant BCR-ABL1 fusions that had been inadvertently missed during the diagnostic work up, most of these cases would now be classified as a subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN), such as atypical CML (aCML), chronic myelomonocytic leukemia (CMML), or chronic neutrophilic leukemia (CNL). A minority would be classified as systemic mastocytosis with associated hematological neoplasm (SM-AHN), myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1 or with PCM1-JAK2 (MLN-eo), or chronic eosinophilic leukemia not otherwise specified (CEL-NOS).1

BCR-ABL Negative CML-Like Disorder

Chronic myeloid leukemia (CML) has been defined for many years as BCR-ABL positive disease only, but older publications refer to a poor prognosis, clinically heterogeneous entity termed ‘BCR-ABL negative CML’ constituting about 5% of CML cases. Apart from very rare CML cases with cytogenetically cryptic, atypical variant BCR-ABL fusions that had been inadvertently missed during the diagnostic work up, most of these cases would now be classified as a subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) such as atypical CML (aCML), chronic myelomonocytic leukemia (CMML) or chronic neutrophilic leukemia (CNL). A minority would be classified as systemic mastocytosis with an associated hematological neoplasm (SM-AHN) or myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2 (MLN-eo).1

Molecular Response with Nilotinib in Patients with Philadelphia Negative (Ph-) Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Molecular Response with Nilotinib in Patients with Philadelphia Negative (Ph-) Chronic Myeloid Leukemia in Chronic Phase (CML-CP): ENEST1st Sub-Analysis

Characteristics and survival of BCR/ABL negative chronic myeloid leukemia: a retrospective analysis of the Surveillance, Epidemiology and End Results database.

BCR/ABL negative or atypical chronic myeloid leukemia (CML) is a rare hematologic malignancy with an estimated incidence of 1–2% of BCR/ABL positive CML. Clinical features of BCR/ABL negative CML resembles those of BCR/ABL positive CML but does not have BCR/ABL fusion gene; rearrangement of platelet-derived growth factor receptor (PDGFR)-A, PDGFR-B or FGFR1 is also absent [Vardiman et al. 2008]. BCR/ABL negative CML, however, is associated with mutations in CSF3R (up to 40%), SETBP1 (~10%) and JAK2V617F (~5%) [Gotlib et al. 2013]. KRAS or NRAS mutations may also be common in BCR/ABL negative CML (35% in a multicenter study) [Wang et al. 2014]. In addition, a study utilizing whole-exome sequencing has revealed the presence of ETNK1 mutations in 8–13% of patients with BCR/ABL negative CML [Gambacorti-Passerini et al. 2015]. World Health Organization (WHO) criteria for the diagnosis of BCR/ABL negative CML additionally include the presence of leukocytosis ⩾13 × 109/l with circulating neutrophil precursors ⩾10%, monocytes <10% and minimal basophils (usually <2%); bone marrow hypercellularity with granulocytic proliferation and dysplasia; and blood and bone marrow blast count of <20% [Vardiman et al. 2008]. The rarity of the disease has largely precluded conduction of any prospective study to optimize treatment strategy of BCR/ABL negative CML. Similarly, the mutations associated with the disease were hitherto undiscovered, which prevented identification of therapeutic targets and drug development. Consequently, BCR/ABL negative CML has been managed with palliative therapy such as hydroxyurea, low-dose cytarabine and interferon, which results in a median overall survival (OS) of approximately 2 years in small retrospective studies [Onida et al. 2002; Breccia et al. 2006]. A recent multicenter study demonstrated that BCR/ABL negative CML is distinct from unclassifiable myelodysplastic/myeloproliferative neoplasms and has a worse OS (12 versus 22 months) and acute myeloid leukemia-free survival (11 versus 19 months) [Wang et al. 2014]. A population-based study on the outcomes of BCR/ABL negative CML is lacking. In this population-based study, we utilized the Surveillance, Epidemiology and End Results (SEER) database to analyze the characteristics and outcomes of BCR/ABL negative CML. Such information is useful for patient education and may provide background information for clinical trials in the future.

Survival of BCR-ABL positive and negative chronic myeloid leukemia: A propensity matched analysis of the Surveillance, Epidemiology, and End Results database.

e18053 Background: BCR-ABL negative chronic myeloid leukemia (CML) is uncommon without any previous largescale studies. Because of the genetic differences between BCR/ABL negative and positive CML,...

Abstract 4763: The tyrosine kinase inhibitor axitinib targets T315I gatekeeper-mutant Philadelphia chromosome-positive leukemias in vitro and in vivo

Abstract The use of ABL1 kinase inhibitors has dramatically improved the outcome for chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. However, resistance after treatment still poses a major clinical challenge. The most common resistance mechanism following treatment with first or second line therapy is the occurrence of a T315I mutation in the kinase domain of BCR-ABL1. Only one clinically available ABL1 inhibitor, ponatinib, has been shown to target this gatekeeper mutation, but has recently been associated with significant adverse effects. Hence, there is an unmet need for new and improved therapies for patients with T315I BCR-ABL1 leukemias. In this study we set out to functionally and molecularly profile BCR-ABL1 T315I-driven CML/Ph+ALL patient samples to understand the disease pathogenesis and identify novel therapies with a drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology compounds. Mononuclear cells isolated from patient bone marrow were plated with drugs on 384-well plates. Each compound was tested for its effect on cell growth and survival in a 10,000-fold concentration range enabling the generation of dose response curves and by comparing to healthy donor mononuclear cells, selective drug sensitivity scores (sDSS). Ex vivo DSRT results of one CML and two Ph+ALL patient samples with the T315I mutation revealed a marked and specific sensitivity (IC50 30-40nM) to the tyrosine kinase inhibitor axitinib, originally developed as a VEGFR inhibitor. Strikingly, sensitivity to axitinib was higher in these T315I positive patient samples than in T315I negative CML or ALL patient samples or any other leukemic samples. Supporting the notion that axitinib is a direct T315I BCR-ABL1 inhibitor we observed that Ba/F3 cells transformed with T315I BCR-ABL1 were sensitive to axitinib while the same cells transformed with wild type BCR-ABL1 were not. Finally, we discovered that axitinib has been described to have selective binding towards T315I ABL1 compared to the wild type kinase (Kd 1.5 nM vs. 36 nM, respectively, Davis et al. 2011, Nat. Biotechnol. 29:1046-1051). Based on this information, the T315I CML patient mentioned above was compassionately treated with axitinib for 2 weeks resulting in a rapid 4-fold reduction of the mutated transcript levels in blood suggesting targeted in vivo activity of the drug. In summary, we demonstrated that axitinib is a potent BCR-ABL1 T315I inhibitor both in vitro and in vivo. In light to the fact that axitinib is currently approved as a second line therapy for renal cell carcinoma and is well tolerated in patients, there is an opportunity to repurpose axitinib for Ph+ leukemia patients with T315I mutations with significantly shorter clinical development time. Citation Format: Tea Pemovska, Mika Kontro, Gretchen A. Repasky, Kimmo Porkka, Krister Wennerberg. The tyrosine kinase inhibitor axitinib targets T315I gatekeeper-mutant Philadelphia chromosome-positive leukemias in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4763. doi:10.1158/1538-7445.AM2014-4763

Location of the BCR/ABL Fusion Genes on Both Chromosomes 9 in Ph Negative Young CML Patients: An Indian Experience.

The BCR/ABL gene rearrangement is cytogenetically visualized in most chronic myeloid leukemia (CML) cases. About 5-10% of CML patients lack its cytogenetic evidence, however, shows BCR/ABL fusion by molecular methods. We describe two CML patients with Philadelphia (Ph) negative (-ve) and BCR/ABL positive by fluorescence in situ hybridization (FISH). Both the cases were in chronic phase at diagnosis. Conventional cytogenetics and different FISH assays were adopted using BCR/ABL probes. Home-brew FISH assay using bacterial artificial clone (BAC) for BAC-CTA/bk 299D3 for chromosomal region 22q13.31-q13.32 was performed in case 1. Both the patients were Ph-ve. In first case, dual color dual fusion (DCDF)-FISH studies revealed 1 Red (R) 2 Green (G) 1 Fusion (F) signal pattern in 80% of cells indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph and 2G2F signal pattern in 20% of cells indicating two BCR/ABL fusions on both chromosomes 9q34 on presentation. In second case, FISH studies revealed the 1R1G1F signal pattern indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph in 100% of cells at presentation. During follow-up, both the patients exhibited 2G2F signal pattern indicating two BCR/ABL fusions on both chromosomes 9q34, which indicated a clonal evolution in 100% cells. Both the patients did not achieve therapeutic response. Relocation of BCR/ABL fusion sequence on sites other than 22q11 represents a rare type of variant Ph, the present study highlights the hot spots involved in CML pathogenesis and signifies their implications in Ph-ve BCR/ABL positive CML. This study demonstrated the genetic heterogeneity of this subgroup of CML and strongly emphasized the role of metaphase FISH, especially in Ph-ve CML cases, as it detects variations of the classical t(9;22).

Validation of Digital-PCR Analysis through Programmed imatinib Interruption in Q-RT-PCR Negative Chronic Myeloid Leukemia Patients

IntroductionImatinib induces complete cytogenetic response (CCyR) in up to 80% of chronic myeloid leukemia (CML) patients (pts) and major molecular response (MMR) in 33-60% of them. These patients enjoy life expectancy similar to general population. However even undetectable BCR-ABL may not equate to eradication of the disease because the sensitivity of the standard diagnostic method, the Q-RT-PCR, is limited. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, corresponding to a 100 times increased sensitivity as compared to conventional Q-RT-PCR, was developed (Goh HG et al., Leuk Lymphoma 52(5): 896-904. 2011). Therefore dPCR, assessing with more sensitivity the presence of minimal residual disease, could potentially identify pts in whom CML is eradicated. The Imatinib Suspension And Validation (ISAV) study is aimed at assessing the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. MethodsThis study involves 15 sites, 10 in Italy and 5 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. In this study CML patients (Chronic Phase or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months, with a minimum of 3 Q-RT-PCR performed in their own centers. After signing the informed consent, pts were tested for dPCR and discontinued imatinib therapy. They are being monitored by standard Q-RT-PCR for 36 months to assess the maintenance of the molecular remission. At the end of this period, a peripheral blood sample for dPCR analysis will be obtained from those pts who will still have undetectable BCR-ABL transcripts by Q-RT-PCR, to verify CML eradication. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resumed imatinib treatment at the same dosage used before interruption. Patients' quality of life during imatinib discontinuation/resumption is being evaluated trough the EORTC – C30 Quality of Life questionnaire. ResultsThe enrollment in the ISAV study began in November 2011 and ended in June 2013. The study enrolled 112 pts: Italy 69.6%, Berlin 21.4%, Montreal 5.3%, Zaragoza 2.6% and Tel Hashomer 0.9%. Sixty-one percent of the pts were male and 38% were aged 65 or older; median duration of imatinib treatment is 102 months with median duration of CMR of 32 months before imatinib discontinuation. To date, the median follow-up (FUP) time is 4.6 months [95% CI: 4.1-5.8] and 92 pts out of 112 (82%) had at least one Q-RT-PCR performed after imatinib discontinuation. The following analysis is restricted to 48 pts with a minimum of 6 months of FUP. Of these 48 pts, 20 remained Q-RT-PCR negative (42%, 95% CI:29-56%, median duration of negativity after imatinib discontinuation: 10.3 months). Nineteen pts (40%, 95% CI:27-53%) relapsed and resumed imatinib. All relapses occurred in the first 10 months and all but 3 of them in the first 6 months. A loss of CCyR happened in 5 pts out of 19 (26%): 1 pt regained CCyR after 3 months of re-treatment and is now in CMR, 1 pt died shortly after the diagnosis of relapse because of lung adenocarcinoma and 3 pts are now being monitored after imatinib resumption. No case of progression of CML was observed. After the resumption of imatinib the median time to either MMR or CMR, whichever came first, was 2.1 [95% CI: 0.9-5.8] months. Finally, nine pts (18%, 95% CI:10-31%) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity in this group of pts was 2.92 months (range 1-5 months), and the range of duration of Q-RT-PCR positivity (below 0.1%) is between 2 and 14 months. No significant correlation between relapse and previous duration of imatinib treatment, time to CCyR or duration of CMR was present. Patients previously treated with interferon showed a trend toward lower risk of relapse which is not significant so far. Finally, 19% of pts complained of musculoskeletal/articular pain after imatinib discontinuation. ConclusionsAfter 21 months from the beginning of the study with a median follow-up of 4.6 months, 40% of pts relapsed; the majority of relapses happened in the first 6 months after imatinib discontinuation. The correlation of dPCR results with clinical outcomes will be presented at the meeting. Disclosures:le Coutre:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Research Funding. Gozzini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Myeloproliferative neoplasm with ETV6-ABL1 fusion: a case report and literature review

ETV6-ABL1 is a rare gene fusion with oncogenic properties, reported so far in 28 patients presenting a variety of haematological malignancies associated with clinical outcome, including chronic myeloid leukaemia (CML), acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and chronic myeloproliferative neoplasm (cMPN). Here we report on a 46-year-old female who presented with Philadelphia negative CML, positive for the ETV6-ABL1 fusion. Whole genome screening carried out with oligonucleotide arrays showed a subtle loss at 12p13 and cryptic imbalances within the 9q34.3 region in a highly unstable genome. FISH mapping with custom BAC probes identified two breakpoints 5 Mb apart within the 9q34 region, together with a break at 12p13. While FISH with commercial BCR-ABL1 probes failed to detect any ABL1 changes, the ETV6 break-apart probe conclusively identified the ETV6-ABL1 fusion thus determining the probe’s role as the primary diagnostic FISH test for this chimeric oncogene. In addition, we confirm the association of the ETV6-ABL1 fusion with imatinib resistance reported so far in three other patients, while recording excellent response to the 2nd generation tyrosine kinase inhibitor (TKI) nilotinib. In summary, we highlight the value of ETV6 FISH as a diagnostic test and the therapy resistance of ETV6-ABL1 positive disorders to imatinib.

Acquired Mutation of the Tyrosine Kinase JAK2V617F in Egyptian Patients with Myeloid Disorders

Janus Kinase 2 (JAK2) is a member of a family of four Janus Kinases, 2, and 3 and tyrosine kinase 2. Mutated JAK2 (V617F) has the ability to activate downstream signal transducer and activator of transcription (STAT)-mediated transcription in the absence of the ligand erythropoietin. The autoinhibitory activity of JAK2 is disrupted by the presence of the V617F mutation. Somatic mutation in JAK2 (V617F) gene has been reported in myeloid disorders. This study reports the prevalence of JAK2V617F using amplification refractory mutation system (ARMS)-polymerase chain reaction in 246 Egyptian patients with different myeloid disorders and studied the relationship between the JAK2V617F mutation and parameters in peripheral blood. The mutation was detected among 88 patients (35.8%) with different myeloid disorders. JAK2V617F was found among 81.4% of polycythemia vera (PV), 50% of essential thrombocythemia, 46.1% of primary myelofibrosis (PMF), 33.3% of philadelphia (Ph)-negative chronic myeloid leukemia, 33.3% of myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN), and 50% of refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T) patients. Hemoglobin and white blood cells were significantly higher in the mutated group of MPN including PV, essential thrombocythemia, and PMF, whereas platelet counts were higher among the mutated PV, PMF, RARS-T, and MDS/MPN group. The identification of JAK2V617F mutations has raised the prospect of developing specific JAK2V617F inhibitors to treat mutated patients.

Numerous aggregates of “tiny” micromegakaryocytes in the bone marrow

A 70-year old male presented with leukocytosis (24.9 9 10/L) and thrombocytosis (727 9 10/L). Hb was 14.9 g/dL, neutrophils 81%, basophils 1%, eosinophils 4%, and myelocytes 1%. He had no splenomegaly. Serum LDH was 477 U/L, B12755 pg/m, and ferritin 104 ng/dL. Neutrophil alkaline phosphatase activity was normal. The marrow was moderately cellular with many aggregates of micromegakaryocytes, each aggregate comprising up to 20 ‘‘tiny’’ micromegakaryocytes (Fig. 1). Small cells were close to the size of lymphocytes. Although usual-sized micromegakaryocytes were occasionally seen, most ‘‘tiny’’ micromegakaryocytes were found to form aggregates accompanying many platelets (Fig. 2a, b) and such aggregates were scattered in the entire length of marrow film. Immature granulocytes showed hypogranulation (Fig. 3a), and occasional mature cells showed hypersegmentation (Fig. 3b, c) and cytoplasmic Dohle bodies (arrowheads Fig. 3d). Erythroid series showed mild megaloblastoid changes. These features resembled myelodysplastic syndromes (MDS). Blast cells were few. Normal marrow cytogenetic analysis and the lack of BCR-ABL in mRNA analysis excluded chronic myeloid leukemia (CML). Marrow WT1 mRNA level was 1600 copies/lg RNA. No marrow fibrosis was seen in biopsy. These findings excluded myeloproliferative neoplasm (MPN). Proliferation and marked dysplasia in megakaryocytic and myeloid lineages falls within the scope of MDS/MPN, unclassifiable, according to the current WHO system, rather than atypical BCR-ABL negative CML. The marrow findings remained essentially unchanged in repeat study although blood counts oscillated in response to the intermittent hydroxyurea (1000 mg) treatment. Y. Yoshida (&) Y. Hamakawa S. Oguma T. Katsurada The Center for Hematological Diseases, Takeda General Hospital, 28-1 Ishida-Moriminami, Fushimi-Ku, Kyoto 601-1495, Japan e-mail: yoshida@takedahp.or.jp

Methylation status of DDIT3 gene in Chronic Myeloid Leukemia

BackgroundDNA-damage-inducible transcript 3 (DDIT3), a candidate tumor suppressor gene (TSG), has been found involved in the regulation of cellular growth and differentiation. The epigenetic changes of TSGs are recently recognized as an abnormal mechanism contributing to the development of chronic myeloid leukemia (CML). The aim of this study was to investigate the methylation status of DDIT3 gene in CML patients.MethodsThe methylation status of DDIT3 promoter was detected in the bone marrow mononuclear cells from 53 patients with CML using methylation-specific PCR (MSP). The expression levels of DDIT3 and bcr/abl transcript were determined by real-time quantitative PCR (RQ-PCR). Clinical data of these patients were collected and analyzed.ResultsThe aberrant methylation of DDIT3 gene promoter was found in 35 of 53 (66%) CML cases. Correlation was not found between DDIT3 promoter hypermethylation and the age, sex, hemoglobin concentration, platelet counts, chromosomal abnormalities, bcr/abl transcript, and staging of CML patients (P > 0.05), but found between DDIT3 promoter hypermethylation and WBC counts of CML cases (R = 0.781, P < 0.001). The level of DDIT3 transcript in CML patients was significantly lower than that in controls (median 3.28 vs 19.69, P < 0.001), however, there was no difference in the level of DDIT3 transcript between methylation-positive CML cases (0.05-65.32, median 2.13) and methylation- negative CML cases (0.12-126.04, median 3.92) (P > 0.05).ConclusionOur results demonstrate that aberrant methylation of DDIT3 occurs in CML frequently.

Frequent Inactivating Mutations of TET2 and CBL Are Associated with Acquired Uniparental Disomy in Atypical Chronic Myeloid Leukemia and Related Disorders.

Abstract 3258Poster Board III-1Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) such as atypical BCR-ABL negative chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML) combine dysplastic morphology with features of a CML-like myeloproliferative disorder. The underlying pathomechanism of these related disorders is poorly understood. Recent evidence has shown that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. In this study we sought to investigate if aUPD characterizes MDS/MPN of unknown molecular etiology, and whether it could be used as a tool to help identify novel driver mutations. Genome wide high resolution SNP 6.0 array analysis was performed on total leukocyte DNA from 148 patients with aCML (n=54), CMML (n=69), and unclassified MDS/MPN (n=25). All patients were negative for BCR-ABL, JAK2 V617F, FIP1L1-PDGFRA and cytogenetic indicators of other tyrosine kinase fusion genes. Homozygous copy neutral SNP calls >20 Mb, considered indicative of aUPD, were seen in 39 (26%) patients. In total, 15 different chromosomes were involved with the most common recurrent abnormalities being seen at 4q (n=7), 7q (n=12), and 11q (n=8). Sequencing of candidate genes in the minimally affected regions excluded the involvement of tyrosine kinases and several other genes. Following the identification of TET2 at 4q24 as a putative novel tumor suppressor gene of unknown function in patients with classic MPN and MDS we identified homozygous TET2 variants in 6 cases with 4q aUPD. Analysis of 210 additional patients revealed TET2 variants in aCML (16/53; 30%), CMML (28/70; 40%), unclassified MDS/MPN (5/17, 29%), hypereosinophilic syndrome (4/30; 13%) but none in blast crisis CML (n=40). In total, 70 TET2 variants were identified that were spread throughout the coding region. Of these, 35 were likely causative changes predicted to result in premature chain termination (nonsense, n=18; deletion, n=10; insertion, n=7) and 35 were missense substitutions that have not been reported as SNPs. 20 missense mutations (57%) clustered in two conserved regions. TET2 mutations were not associated with a change of overall or progression-free survival compared to mutation negative cases. Mutations in CBL, encoding a key regulator of tyrosine kinase signaling, were identified in 5 cases with 11q aUPD and analysis of 574 additional MPN and MDS/MPN patients revealed a total of 27 CBL variants in 26 patients with aCML (12/152; 8%), CMML (10/78; 13%), myelofibrosis (n=3/53; 6%) or hypereosinophilic syndrome (n=1/96; 1%). Most variants were missense substitutions in exons 8 or 9 (encoding the linker/RING domain) that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells that overexpressed FLT3. Patients with CBL mutations had a shorter overall survival and progression-free survival compared to mutation negative cases (OS: 33 months vs. 39 months; PFS: 22 months vs 32 months) but the differences were not significant. We conclude that aUPD is common in atypical CML and related disorders and that inactivating mutations of TET2 and CBL are associated with aUPD at 4q and 11q, respectively. Disclosures:Hochhaus:Novartis: Honoraria; BMS: Honoraria.

Ph陰性M-BCR/ABL陽性でt(9;16)を示し，著明なDICを来した慢性骨髄性白血病急性転化例

Ph陰性M-BCR/ABL陽性でt(9;16)を示し，著明なDICを来した慢性骨髄性白血病急性転化例

GATA-2 L359V Mutation Is Solely Associated with Cml progression but Not Other Hematological Malignancies.

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease with distinct biology and clinical features. According to clinical and biology process, CML can be divided as two different phase: the initial chronic phase (CP) and progression phase including accelerated phase (AP) followed by blast crisis (BC). In a previous study, we have identified the transcription factor GATA-2 L359V mutation in CML BC patients but not CP and its pivotal role in CML progression (PNAS 2008 105:2076–2081). Here, we continued to explore the occurrence of GATA-2 L359V mutation in other hematological malignancies using Mass-ARRAY assay and sequence analysis. A total of 652 patient samples were included in our study. These patients were referred to 270 Acute Myeloid Leukemia (AML) including M1–M7, 30 Myelodysplastic Syndrome (MDS), 50 Acute Lymphoblastic Leukemia (ALL), 12 Chronic Lymphocytic Leukemia (CLL), 40 CML CP and 250 BCR/ABL negative Myeloproliferative Disorder (MPD) patients. 5 ml bone marrow sample before therapy was collected with informed consent and genomic DNA was isolated. The diagnosis of AML, ALL, CML, CLL, MDS and MPD was established according to the 2001 WHO diagnostic criteria. In addition, 8 samples of CML BC harboring GATA-2 L359V were supplied into our study as positive control and 100 peripheral blood samples of healthy adult as normal control. As a result, no L359V mutation was detected in AML, ALL, MDS, CLL, BCR/ABL negative MPD and CML CP. Our data strongly suggested that GATA-2 L359V is solely associated with CML progression but not other hematological malignancies. Therefore, we favored this idea, i.e., BCR/ABL underlies the pathogenic basis of CML CP; However, subsequent genomic instabilities contribute to mutation of key transcription factor such as GATA-2 which ultimately trigger the blastic transformation of CML.