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Biomedical subjects

D Gary Gilliland

Publications and source records attributed to D Gary Gilliland.

At least 19 recordsLinked to original sources
PubMed2006

Prospective analysis of TEL/AML1-positive patients treated on Dana-Farber Cancer Institute Consortium Protocol 95-01.

In a retrospective analysis, we previously reported that children whose leukemia cells harbored the TEL/AML1 gene rearrangement have excellent outcomes. From 1996 to 2000, we conducted a prospective study to determine the incidence and outcomes of children with TEL/AML1-positive acute lymphoblastic leukemia (ALL). Children with newly diagnosed ALL were treated on DFCI ALL Consortium Protocol 95-01. Patients were risk stratified primarily by current National Cancer Institute (NCI)-Rome risk criteria. With a median follow-up of 5.2 years, the 5-year event-free survival for TEL/AML1-positive patients was 89% compared with 80% for TEL/AML1-negative B-precursor patients (P = .05). The 5-year overall survival rate was 97% among TEL/AML-positive patients compared with 89% among TEL/AML1-negative patients (P = .03). However, in a multivariable analysis, risk group (age and leukocyte count at diagnosis) and asparaginase treatment group, but not TEL/AML1 status, were found to be independent predictors of outcome. We conclude that TEL/AML1-positive patients have excellent outcomes, confirming our previous findings. However, factors such as age at diagnosis and presenting leukocyte count should be taken into consideration when treating this group of patients.

Age Factors↗
PubMed2006

Expression of Jak2V617F causes a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model.

An acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

Amino Acid Substitution↗
PubMed2006

The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera.

BACKGROUND: Several studies have recently reported on the occurrence of a JAK2(V617F) mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation currently is under study. METHODS: In a single institutional study, mutation screening for JAK2(V617F) was performed in DNA derived from archived blood granulocytes from 63 consecutive patients with PV in whom current diagnostic criteria were strictly applied and the diagnosis confirmed by bone marrow histology. RESULTS: The JAK2(V617F) mutant allele was detected in 58 of the 63 patients (92%) with 21% homozygosity. The clinical phenotype of the five patients with the wild-type allele was otherwise typical for the disease. A statistical comparison between JAK2(V617F) heterozygotes (n=45 patients) and homozygotes (n=13 patients) did not reveal any significant associations with regard to age, gender, leukocyte or platelet count at the time of diagnosis, duration of disease, or the incidences of thrombosis or bleeding. However, compared with their heterozygote counterparts, JAK2(V617F) homozygote patients displayed a significantly higher hemoglobin level at the time of diagnosis (P=0.001), an increased incidence of pruritus (69% vs. 38%; P=0.04), a higher rate of fibrotic transformation (23% vs. 2%; P=0.009), and higher PRV-1 transcript levels in their blood granulocytes (P=0.07). CONCLUSIONS: The results of the current clinical study support previous laboratory observations that link JAK2(V617F) with the PV phenotype by demonstrating a mutant allele dose effect on erythrocytosis and clinical and laboratory features characteristic of PV.

Adolescent↗
PubMed2006

Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations.

Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.

Aged↗
PubMed2006

X-inactivation-based clonality analysis and quantitative JAK2V617F assessment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F-negative ET and MMM patients with clonal hematopoiesis.

The JAK2V617F mutation is present in most patients with polycythemia vera (PV) and in some patients with essential thrombocythemia (ET) and myeloid metaplasia/myelofibrosis (MMM). We sought to investigate the relationship between granulocyte clonality and JAK2V617F allelic ratio. A total of 168 of 190 female patients were informative for a clonality assay at the HUMARA locus; 80% of MMM, 75% of PV, and 67% of ET patients demonstrated clonal granulopoiesis. The JAK2V617F allele was detected by quantitative real-time polymerase chain reaction (PCR) in 99% of PV, 72% of ET, and 39% of MMM. A correlation between clonality and JAK2V617F allelic ratio was demonstrated for PV (P < .001) but not for ET or MMM (both P > .6). These data suggest that acquisition of the JAK2V617F mutation may be sufficient for the development of PV, but additional genetic events are necessary in ET and MMM. In addition, some ET and MMM patients with clonal granulopoiesis have somatic mutations other than JAK2V617F.

Amino Acid Substitution↗
PubMed2006

Detection of the FIP1L1-PDGFRA fusion in idiopathic hypereosinophilic syndrome and chronic eosinophilic leukemia.

Idiopathic hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) are related hematological malignancies characterized by sustained, unexplained hypereosinophilia (>1,500 eosinophils/microL). The term CEL is used when there is evidence that the disease is of clonal origin. We recently identified the FIP1L1-PDGFRA fusion gene in approx 50% of HES/CEL cases. Fusion of FIP1L1 to PDGFRA is the consequence of a deletion on chromosome 4, del(4)(q12q12), with the centromeric breakpoint in FIP1L1 and the telomeric breakpoint in PDGFRA. The breakpoints in FIP1L1 are diverse (introns 7 to 10), but the breakpoints in PDGFRA are always in exon 12 (encoding the juxtamembrane region). Because the chromosomal deletion is only 800 kb in size, it remains undetected with standard cytogenetics. In agreement with this, most patients with HES/CEL present with a normal karyotype. Here we describe three different techniques to detect the presence of the FIP1L1-PDGFRA fusion gene in peripheral blood or bone marrow cells.

Centromere↗
PubMed2005

Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation.

A recurrent somatic activating mutation in the nonreceptor tyrosine kinase JAK2 (JAK2V617F) occurs in the majority of patients with the myeloproliferative disorders polycythemia vera, essential thrombocythemia, myelofibrosis with myeloid metaplasia, and, less commonly, chronic myelomonocytic leukemia. We do not understand the basis for the specificity of the JAK2V617F mutation in clonal disorders of the myeloid, but not lymphoid, lineage, nor has the basis for the pleiotropic phenotype of JAK2V617F-associated myeloproliferative disorders been delineated. However, the presence of the identical mutation in patients with related, but clinicopathologically distinct, myeloid disorders suggests that interactions between the JAK2V617F kinase and other signaling molecules may influence the phenotype of hematopoietic progenitors expressing JAK2V617F. Here, we show that coexpression of the JAK2V617F mutant kinase with a homodimeric Type I cytokine receptor, the erythropoietin receptor (EpoR), the thrombopoietin receptor, or the granulocyte colony-stimulating-factor receptor, is necessary for transformation of hematopoietic cells to growth-factor independence and for hormone-independent activation of JAK-STAT signaling. Furthermore, EpoR mutations that impair erythropoietin-mediated JAK2 or STAT5 activation also impair transformation mediated by the JAK2V617F kinase, indicating that JAK2V617F requires a cytokine receptor scaffold for its transforming and signaling activities. Our results reveal the molecular basis for the prevalence of JAK2V617F in diseases of myeloid lineage cells that express these Type I cytokine receptors but not in lymphoid lineage cells that do not.

Animals↗
PubMed2005

Genetics of myeloid malignancies: pathogenetic and clinical implications.

Myeloid malignancies are clonal disorders that are characterized by acquired somatic mutation in hematopoietic progenitors. Recent advances in our understanding of the genetic basis of myeloid malignancies have provided important insights into the pathogenesis of acute myeloid leukemia (AML) and myeloproliferative diseases (MPD) and have led to the development of novel therapeutic approaches. In this review, we describe our current state of understanding of the genetic basis of AML and MPD, with a specific focus on pathogenetic and therapeutic significance. Specific examples discussed include RAS mutations, KIT mutations, FLT3 mutations, and core binding factor rearrangements in AML, and JAK2 mutations in polycythemia vera, essential thrombocytosis, and chronic idiopathic myelofibrosis.

Female↗
PubMed2005

JAK2 in myeloproliferative disorders is not just another kinase.

Myeloproliferative disorders (MPD) represent a subcategory of hematological malignancies and are characterized by a stem cell-derived clonal proliferation of myeloid cells including erythrocytes, platelets, and leucocytes. Traditionally, the term 'MPD' included chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMM). At present, these four disorders are referred to as 'classic' MPD and are distinguished from a spectrum of other MPD-like clinicopathologic entities that are operationally classified as 'atypical' MPD. The oncogenic mutations(s) in classic MPD are unknown except for CML, which is associated with an activating mutation (Bcr/Abl) of the gene encoding for the Abl cytoplasmic protein kinase (PTK). In the last 3 months, a somatic point mutation of JAK2 (JAK2(V617F)), the gene encoding for another cytoplasmic PTK was reported in the majority of patients with PV and approximately half of those with either ET or MMM. The same mutation was also found in a small number of patients with either atypical MPD or the myelodysplastic syndrome but not in normal controls, germline tissue including T lymphocytes, and patients with secondary erythrocytosis. In vitro, JAK2(V617F) was associated with constitutive phosphorylation of JAK2 and its downstream effectors as well as induction of erythropoietin hypersensitivity in cell lines. In vivo, murine bone marrow transduced with a retrovirus containing JAK2(V617F) induced erythrocytosis in the transplanted mice. Taken together, these observations suggest that JAK2(V617F) is an acquired myeloid lineage-specific mutation that engenders a pathogenetic relevance for the PV phenotype in MPD.

Alleles↗
PubMed2005

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

Activating 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.

Aged↗
PubMed2005

The small molecule tyrosine kinase inhibitor AMN107 inhibits TEL-PDGFRbeta and FIP1L1-PDGFRalpha in vitro and in vivo.

AMN107 is a small molecule tyrosine kinase inhibitor developed, in the first instance, as a potent inhibitor of breakpoint cluster region-abelson (BCR-ABL). We tested its effectiveness against fusion tyrosine kinases TEL-platelet-derived growth factor receptorbeta (TEL-PDGFRbeta) and FIP1-like-1 (FIP1L1)-PDGFRalpha, which cause chronic myelomonocytic leukemia and hypereosinophilic syndrome, respectively. In vitro, AMN107 inhibited proliferation of Ba/F3 cells transformed by both TEL-PDGFRbeta and FIP1L1-PDGFRalpha with IC50 (inhibitory concentration 50%) values less than 25 nM and inhibited phosphorylation of the fusion kinases and their downstream signaling targets. The imatinib mesylate-resistant mutant TEL-PDGFRbeta T681I was sensitive to AMN107, whereas the analogous mutation in FIP1L1-PDGFRalpha, T674I, was resistant. In an in vivo bone marrow transplantation assay, AMN107 effectively treated myeloproliferative disease induced by TEL-PDGFRbeta and FIP1L1-PDGFRalpha, significantly increasing survival and disease latency and reducing disease severity as assessed by histopathology and flow cytometry. In summary, AMN107 can inhibit myeloid proliferation driven by TEL-PDGFRbeta and FIP1L1-PDGFRalpha and may be a useful drug for treatment of patients with myeloproliferative disease who harbor these kinase fusions.

Animals↗
PubMed2005

Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation.

The majority of patients with systemic mast cell disease express the imatinib-resistant Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase. Limited treatment options exist for aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). We evaluated whether PKC412, a small-molecule inhibitor of KIT with a different chemical structure from imatinib, may have therapeutic use in advanced SM with the D816V KIT mutation. We treated a patient with MCL (with an associated myelodysplastic syndrome (MDS)/myeloproliferative disorder [MPD]) based on in vitro studies demonstrating that PKC412 could inhibit D816V KIT-transformed Ba/F3 cell growth with a 50% inhibitory concentration (IC50) of 30 nM to 40 nM. The patient exhibited a partial response with significant resolution of liver function abnormalities. In addition, PKC412 treatment resulted in a significant decline in the percentage of peripheral blood mast cells and serum histamine level and was associated with a decrease in KIT phosphorylation and D816V KIT mutation frequency. The patient died after 3 months of therapy due to progression of her MDS/MPD to acute myeloid leukemia (AML). This case indicates that KIT tyrosine kinase inhibition is a feasible approach in SM, but single-agent clinical efficacy may be limited by clonal evolution in the advanced leukemic phase of this disease.

Aspartic Acid↗
PubMed2005

Drug therapy for acute myeloid leukemia.

Although improvement in outcomes has occurred in younger adults with acute myeloid leukemia (AML) during the past 4 decades, progress in older adults has been much less conspicuous, if at all. Approximately 50% to 75% of adults with AML achieve complete remission (CR) with cytarabine and an anthracycline such as daunorubicin or idarubicin or the anthracenedione mitoxantrone. However, only approximately 20% to 30% of the patients enjoy long-term disease survival. Various postremission strategies have been explored to eliminate minimal residual disease. The optimal dose, schedule, and number of cycles of postremission chemotherapy for most patients are not known. A variety of prognostic factors can predict outcome and include the karyotype of the leukemic cells and the presence of transmembrane transporter proteins, which extrude certain chemotherapy agents from the cell and confer multidrug resistance and mutations in or over expressions of specific genes such as WT1, CEBPA, BAX and the ratio of BCL2 to BAX, BAALC, EVI1, KIT, and FLT3. Most recently, insights into the molecular pathogenesis of AML have led to the development of more specific targeted agents and have ushered in an exciting new era of antileukemia therapy. Such agents include the immunoconjugate gemtuzumab ozogamicin, multidrug resistance inhibitors, farnesyl transferase inhibitors, histone deacetylase and proteosome inhibitors, antiangiogenesis agents, Fms-like tyrosine kinase 3 (FLT3) inhibitors, and apoptosis inhibitors.

Acute Disease↗
PubMed2005

The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both "atypical" myeloproliferative disorders and myelodysplastic syndromes.

A somatic mutation in the JH2 autoinhibitory domain of the Janus kinase 2 (JAK2) tyrosine kinase was recently described in polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. The prevalence of this mutation in either "atypical" myeloproliferative disorders (MPDs) or the myelodysplastic syndromes (MDSs) is unknown. Bone marrow-derived genomic DNA from 245 patients--119 with chronic myelomonocytic leukemia (CMML), 101 with MDS, 11 with hypereosinophilic syndrome (HES), 8 with systemic mastocytosis (SM), and 6 with chronic neutrophilic leukemia (CNL)--was screened for the JAK2 V617F mutation. A mutant allele was detected in 11 patients: 3 with CMML (3%), 5 with MDS (5%), 2 with SM, and 1 with CNL. Interestingly, one of the patients with SM and the patient with CNL with JAK2 V617F had a history of lymphoma, and this patient with SM also had associated myelofibrosis and CMML. The current observation strengthens the specific association between JAK2 V617F and classic MPD, but also suggests an infrequent occurrence in other myeloid disorders.

Adult↗
PubMed2005

Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase inhibitor PKC412.

Constitutively activated forms of the transmembrane receptor tyrosine kinase c-KIT have been associated with systemic mast cell disease, acute myeloid leukemia, and gastrointestinal stromal tumors. Reports of the resistance of the kinase domain mutation D816V to the adenosine triphosphate (ATP)-competitive kinase inhibitor imatinib mesylate prompted us to characterize 14 c-KIT mutations reported in association with human hematologic malignancies for transforming activity in the murine hematopoietic cell line Ba/F3 and for sensitivity to the tyrosine kinase inhibitor PKC412. Ten of 14 c-KIT mutations conferred interleukin 3 (IL-3)-independent growth. c-KIT D816Y and D816V transformed cells were sensitive to PKC412 despite resistance to imatinib mesylate. In these cells, PKC412, but not imatinib mesylate, inhibited autophosphorylation of c-KIT and activation of downstream effectors signal transducer and transcriptional activator 5 (Stat5) and Stat3. Variable sensitivities to PKC412 or imatinib mesylate were observed among other mutants. These findings suggest that PKC412 may be a useful therapeutic agent for c-KIT-positive malignancies harboring the imatinib mesylate-resistant D816V or D816Y activation mutations.

Animals↗
PubMed2005

Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype.

Homozygous loss of function of Runx1 (Runt-related transcription factor 1 gene) during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we used a conditional gene-targeting strategy to effect the loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, though a number of significant hematopoietic abnormalities were observed. Runx1 excision had lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1-deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and they provide insight into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies.

Animals↗