A Critical Role of YB-1 In NPMc-Induced Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3149-3149
Author(s):  
Yoko Ogawara ◽  
Takuo Katsumoto ◽  
Takeshi Uchiumi ◽  
Kimitoshi Kohno ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Embryonic Lethal ◽  
Hoxa Genes ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract Abstract 3149 Frameshift mutations in Nucleophosmin gene (NPM) are the most frequent abnormality in acute myeloid leukemia (AML), found in approximately 30% of all cases and 50% of patients with normal karyotype (NK) AML. NPM mutations result in an aberrant cytoplasmic localization of NPM protein (NPMc) through a loss of nucleolar localization signal accompanied by acquisition of new nuclear export signal. NPM mutations are heterozygous, so the other wild-type allele is consistently retained. NPMc binds to wild-type NPM through oligomerization domain and impairs its activity by delocalizing to the cytoplasm. It was reported that the NPM-null mice are early embryonic lethal and defective in primary hematopoiesis, suggesting important roles of NPM in early hematopoiesis. However, the molecular mechanism by which NPMc exerts its leukemogenic potential has never been established. Here we show that ectopic expression of NPMc, but not wild type (WT) NPM, in mouse bone marrow (BM) cells enhanced their colony formation activity in methylcellulose media. Increased expression of HoxA7, 9 and 10 genes were observed in cells expressing NPMc but not in those expressing WT NPM. It has been reported that the expression levels of HOXA genes are upregulated in various types of AML including NPMc+ AML. Since overexpression of HoxA9 immortalizes hematopoietic progenitor cells, our findings suggest that up-regulation of HoxA genes are involved in NPMc-mediated leukemogenesis. To clarify roles of NPMc in leukemogenesis, we purified the NPM protein complex and identified Y box-binding protein 1 (YB-1) as a binding partner for NPM. YB-1 belongs to the cold shock family and functions in gene transcription and RNA processing. YB-1 strongly bound to WT NPM but not to NPMc. In addition, interaction between YB-1 and NPM was impaired in the presence of NPMc. YB-1-deficient mice were embryonic lethal and their fetal liver were small. YB-1-deficient yolk sac cells showed decreased colony-forming activity, and decreased number of hematopoietic cells were observed when AGM region of YB-1-deficeint embryo were cultured on OP9 cells. Furthermore, expression of Hoxa9 was decreased in fetal liver cells derived from YB-1 knockout mice. To investigate the roles of YB-1 in NPMc-associated leukemogenesis, WT and YB-1-null E14.5 fetal liver cells were infected with retrovirus expressing NPMc. Analyses of colony-forming activity and mRNA expression showed that YB-1 was essential for NPMc-induced increases in colony formation activity as well as in expression of HoxA genes. However, YB-1 was not necessary for colony formation activity induced by other AML-associated fusion genes, such as AML1-MTG8 and MLL-AF10. These data indicate that YB-1 is specifically required for NPMc-induced leukemogenic transformation of hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Roles of MOZ in Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2269-2269
Author(s):  
Takuo Katsumoto ◽  
Yukiko Aikawa ◽  
Takahiro Ochiya ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Liver Cells ◽  
Recipient Mouse ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Homozygous Mutant ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.

scholarly journals SETDB1 Represses Hox Gene Expression and Suppresses Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1320-1320
Author(s):  
James Ropa ◽  
Nirmalya SAHA ◽  
Andrew G. Muntean
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
H3k9 Methylation ◽  
Acute Myeloid

Abstract Epigenetic regulators play an important role in normal and malignant hematopoiesis. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), including those harboring MLL rearrangements and NPM mutations, as well as others. Expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow into a lethal AML in mouse models. We previously demonstrated that the pro-leukemic genes Hoxa9 and Meis1 are critically regulated by the histone H3 Lysine 9 (H3K9) methyltransferase SETDB1. Recent studies show that SETDB1 is required for normal hematopoiesis and MLL-AF9 mediated leukemia (Koide, et al. Blood 2016). Our lab recently demonstrated that SETDB1 negatively regulates the expression of HoxA9 and Meis1 through deposition of promoter H3K9 methylation in MLL-AF9 AML cells (Ropa et al. Oncotarget 2018). Consistent with these data, HOXA9 and MEIS1 expression negatively correlates with SETDB1 expression in AML patient samples. Therefore, we investigated the biological impact of SETDB1 on AML. We first noted that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlated with a significantly better overall survival (p=0.003) and lower expected hazard (HR=0.9/100RSEM; p=0.009) in AML patients compared with lower SETDB1 expression. These data are consistent with SETDB1 negatively regulating pro-leukemic genes and suggests that SETDB1 expression may be correlated with AML patient prognosis. We tested this directly by expressing high levels of SETDB1 in AML cells. Ex vivo assays show that retroviral overexpression of SETDB1 in MLL-AF9 AML cells leads to cell differentiation, decreased leukemia colony formation, and decreased cell proliferation. Consistent with the AML patient data, overexpression of SETDB1 significantly delays MLL-AF9 mediated leukemogenesis in vivo (p=0.01). Further, we observed a strong selective pressure against exogenous SETDB1 expression in moribund mice. Transcriptome analyses demonstrate that SETDB1 globally represses Hox and pluripotency gene programs. Strikingly, we found that SETDB1 represses many of the same genes that exhibit reduced promoter H3K9me3 in AML patient samples relative to CD34+ cells. These data point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. We also explored how chemical and genetic inhibition of H3K9 methylation and Setdb1 affects AML initiation and maintenance. We first confirmed the previously reported requirement for Setdb1 in AML cell lines by genetically deleting both alleles of Setdb1 in MLL-AF9 cells, which resulted in a complete arrest of proliferation (Koide, et al. Blood 2016). Combined with our data presented above, these results suggest a narrow window of SETDB1 expression is maintained in AML cells. To achieve reduced (but not complete loss of) activity, we investigated how small molecule inhibition of H3K9 methylation (UNC0638) or shRNA mediated knock down of Setdb1 affects AML initiation. We observed increased ex vivo colony formation of normal ckit+ bone marrow cells upon shRNA mediated knockdown of Setdb1 or upon UNC0638 treatment. We hypothesized that this expansion of colony forming unit potential of hematopoietic cells may translate to increased transformation potential by leukemic oncogenes. Indeed, cells pretreated with UNC0638 followed by retroviral transduction with MLL-AF9 exhibit significantly higher capacity for leukemic colony formation than vehicle treated cells. These data are consistent with H3K9 methylation repressing genes required for AML transformation. Our data identified a narrow window of expression of SETDB1 in AML patient samples. SETDB1 expression is reduced in AML patients relative to normal cells and chemical inhibition of H3K9 methylation expands the pool of cells amenable to MLL-AF9 mediated transformation ex vivo. While inhibition of SETDB1 and other H3K9 methyltransferases has been suggested as a possible therapeutic strategy, our data suggests this may also prime bone marrow cells for transformation by inhibiting epigenetic processes that repress pro-leukemic target genes. Further investigation of the roles of SETDB1 and H3K9 methylation levels is necessary to determine the value of these epigenetic modifiers as therapeutic targets in AML and is currently ongoing. Disclosures No relevant conflicts of interest to declare.

Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3907-3914 ◽  
Author(s):  
Masao Mizuki ◽  
Regina Fenski ◽  
Hartmut Halfter ◽  
Itaru Matsumura ◽  
Rainer Schmidt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Map Kinases ◽  
Rapid Development ◽  
Dominant Negative ◽  
Wild Type ◽  
Flt3 Mutations ◽  
Induced Apoptosis ◽  
Acute Myeloid

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3907-3914 ◽  
Author(s):  
Masao Mizuki ◽  
Regina Fenski ◽  
Hartmut Halfter ◽  
Itaru Matsumura ◽  
Rainer Schmidt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Map Kinases ◽  
Rapid Development ◽  
Dominant Negative ◽  
Wild Type ◽  
Flt3 Mutations ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.

scholarly journals Caspase-3 Can Promote Acute Myeloid Leukemia Development By Regulation of Autophagy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2714-2714
Author(s):  
Na Man ◽  
Yurong Tan ◽  
Fan Liu ◽  
Guoyan Cheng ◽  
Sarah Merin Greenblatt ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Fetal Liver ◽  
Liver Cells ◽  
Self Renewal ◽  
Leukemia Development ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract Background AML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML. Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis. Methods We developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection. Results We found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML. Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays. Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells. Conclusion Autophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment. Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phase I/II Study of Combination Therapy With Sorafenib, Idarubicin, and Cytarabine in Younger Patients With Acute Myeloid Leukemia

2010 ◽  
Vol 28 (11) ◽  
pp. 1856-1862 ◽  
Author(s):  
Farhad Ravandi ◽  
Jorge E. Cortes ◽  
Daniel Jones ◽  
Stefan Faderl ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Phase I ◽  
Myeloid Leukemia ◽  
Wild Type ◽  
Platelet Recovery ◽  
Probability Of Survival ◽  
Target Effect ◽  
Acute Myeloid

Purpose To determine the efficacy and toxicity of the combination of sorafenib, cytarabine, and idarubicin in patients with acute myeloid leukemia (AML) younger than age 65 years. Patients and Methods In the phase I part of the study, 10 patients with relapsed AML were treated with escalating doses of sorafenib with chemotherapy to establish the feasibility of the combination. We then treated 51 patients (median age, 53 years; range, 18 to 65 years) who had previously untreated AML with cytarabine at 1.5 g/m2 by continuous intravenous (IV) infusion daily for 4 days (3 days if > 60 years of age), idarubicin at 12 mg/m2 IV daily for 3 days, and sorafenib at 400 mg orally twice daily for 7 days. Results Overall, 38 (75%) patients have achieved a complete remission (CR), including 14 (93%) of 15 patients with mutated FMS-like tyrosine kinase-3 (FLT3; the 15th patient had complete remission with incomplete platelet recovery [CRp]) and 24 (66%) of 36 patients with FLT3 wild-type (WT) disease (three additional FLT3-WT patients had CRp). FLT3-mutated patients were more likely to achieve a CR than FLT3-WT patients (P = .033). With a median follow-up of 54 weeks (range, 8 to 87 weeks), the probability of survival at 1 year is 74%. Among the FLT3-mutated patients, 10 have relapsed and five remain in CR with a median follow-up of 62 weeks (range, 10 to 76 weeks). Plasma inhibitory assay demonstrated an on-target effect on FLT3 kinase activity. Conclusion Sorafenib can be safely combined with chemotherapy, produces a high CR rate in FLT3-mutated patients, and inhibits FLT3 signaling.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

Detection of the JAK2V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System

2006 ◽  
Vol 130 (7) ◽  
pp. 997-1003
Author(s):  
Randall J. Olsen ◽  
Zhouwen Tang ◽  
Daniel H. Farkas ◽  
David W. Bernard ◽  
Youli Zu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Clinical Laboratory ◽  
Melting Curve ◽  
Myeloproliferative Disorder ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Wild Type ◽  
Acute Myeloid

Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.

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