Alison R. Moliterno

Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34(+) cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34(+) cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34(+)CD45(+)) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34(+) cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Impaired Expression of the Thrombopoietin Receptor by Platelets from Patients with Polycythemia Vera

BACKGROUND The cause of polycythemia vera, which originates from a multipotent hematopoietic progenitor cell, is unknown. Thrombopoietin is a hematopoietic growth factor that regulates the production of multipotent hematopoietic progenitor cells and platelets. To evaluate the possibility that an abnormality in thrombopoietin-mediated signal transduction might be involved in the pathogenesis of polycythemia vera, we examined thrombopoietin-induced tyrosine phosphorylation of proteins and the expression of the thrombopoietin receptor in platelets from patients with the disease. METHODS Platelets were isolated from the blood of patients with polycythemia vera or other chronic myeloproliferative disorders and control subjects. The platelets were exposed to either thrombopoietin or thrombin and then lysed for analysis of tyrosine phosphorylation of platelet proteins and the expression of the proteins by means of immunoblotting. Expression of the thrombopoietin receptor (Mpl) by platelets and megakaryocytes was also assessed. RESULTS Thrombopoietin-mediated tyrosine phosphorylation of proteins was impaired in platelets from 20 patients with polycythemia vera and 3 with idiopathic myelofibrosis, but not in 4 patients with essential thrombocytosis, 3 with chronic myelogenous leukemia, 6 with secondary erythrocytosis, 2 with iron-deficiency anemia, 4 with hemochromatosis, or 5 normal subjects. Thrombin-mediated tyrosine phosphorylation of proteins was intact in platelets from patients with polycythemia vera, and the tyrosine kinases and substrates involved in the process were present in normal amounts. However, expression of the platelet thrombopoietin receptor MpI was markedly reduced or absent in 34 of 34 patients with polycythemia vera and in 13 of 14 patients with idiopathic myelofibrosis. Impaired thrombopoietin-induced tyrosine phosphorylation of proteins in patients with these two diseases was uniformly associated with markedly reduced expression of MpI or the lack of its expression. In patients with polycythemia vera, reduced expression of MpI by platelets was associated with reduced expression of MpI by megakaryocytes. CONCLUSIONS Reduced expression of the thrombopoietin receptor MpI is characteristic of polycythemia vera and idiopathic myelofibrosis. The abnormality appears to distinguish polycythemia vera from other-forms of erythrocytosis.

Novel homo- and hemizygous mutations in EZH2 in myeloid malignancies

Systematic application of single-nucleotide polymorphism arrays (SNP-As) as a karyotyping tool led to the realization that segmental somatic uniparental disomy (UPD) is a common defect in many cases of myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), MDS/MPN and acute myeloid leukemia (AML).1 Discovery of UPD9p paved the way for identification of the JAK2V617F mutation in MPN. Since then, through application of SNP-A, several new mutations have been identified in the homozygous configuration in the malignant cells of patients with hematological malignancies. These include mutations in CBL2, 3 and TET2,4 for example. Similarly, SNP-A analysis demonstrated that MPL5 or TP536 mutations can occur in homozygous configurations. Assays of JAK2V617F mutant allele burden consequent to UPD are increasingly being utilized clinically because of diagnostic and prognostic relevance. Based on these observations, it can be postulated that areas of somatic UPD may identify regions that harbor mutations in the regions affected by the copy number neutral loss of heterozygosity/UPD.7 If found, somatic UPD most often spans large areas of the affected chromosome, thus making identification of mutated target genes quite challenging.

Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (−7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of −7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

Disruption of the ASXL1 gene is frequent in primary, post-essential thrombocytosis and post-polycythemia vera myelofibrosis, but not essential thrombocytosis or polycythemia vera: analysis of molecular genetics and clinical phenotypes.

Background The myeloproliferative neoplasms, essential thrombocytosis, polycythemia vera and primary myelofibrosis, share the same acquired genetic lesion, but the concept of JAK2 V617F serving as the sole lesion responsible for these neoplasms is under question, and there has been interest in identifying additional mutations that may contribute to disease pathogenesis. Because ASXL1 lesions have been increasingly identified in myeloid neoplasms, we examined the relationships of ASXL1 mutation or deletion to both clinical phenotype and associated molecular features in 166 patients with myeloproliferative neoplasms. Design and Methods Exon 12 of ASXL1 was amplified from neutrophil genomic DNA and bidirectionally sequenced in 77 patients with myelofibrosis (including patients with primary and post-essential thrombocytosis or post-polycythemia myelofibrosis), 42 patients with polycythemia vera, 41 with essential thrombocytosis and 6 with post-myelofibrosis acute myeloid leukemia. Pyrosequencing assays were designed to determine the allele percentages of JAK2 V617F (G5073770T), ASXL1 2475dupA, and ASXL1 2846_2847del in neutrophil genomic DNA samples. Clinical and laboratory characteristics of patients with wild-type and ASXL1 mutations were then compared. Results We identified nonsense mutations or hemizygous deletion of ASXL1 in 36% of the patients with myelofibrosis, but very rarely among those with polycythemia vera or essential thrombocytosis. Among the patients with myelofibrosis, those with ASXL1 lesions were not distinguished from their wild-type counterparts with regard to JAK2 V617F status, exposure to chemotherapy or evolution to leukemia. Myelofibrosis patients with ASXL1 lesions were more likely to have received anemia-directed therapy compared to those without lesions [15/26 (58%) versus 11/39 (23%); P=0.02]. Using serial banked samples and quantitative ASXL1 mutant allele burden assays, we observed the acquisition and accumulation of ASXL1 mutations over time in two patients with post-essential thrombocytosis myelofibrosis. Conclusions ASXL1 haploinsufficiency is associated with a myelofibrosis phenotype in the context of other known and unknown lesions, and disruption of ASXL1 function may contribute to the disease pathogenesis of myelofibrosis.

Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A-isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

Phenotypic variability within the JAK2 V617F-positive MPD: Roles of progenitor cell and neutrophil allele burdens

OBJECTIVE The myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), differ phenotypically, but share the same JAK2(V617F) mutation. We examined the relationship of the quantitative JAK2(V617F) allele burden to MPD disease phenotype among the three MPD classes and within PV. MATERIALS AND METHODS We measured the JAK2(V617F) allele percentage in genomic DNA from neutrophils, CD34(+) cells, and cloned progenitors in 212 JAK2(V617F)-positive MPD patients and correlated the allele burdens to both disease class and disease features. RESULTS In ET and PV, mean CD34(+) cell JAK2(V617F) allele burdens were lower than the corresponding neutrophil allele burdens, but these were equivalent in PMF. JAK2(WT) progenitors were present in ET and PV when the CD34(+) JAK2(V617F) allele burden was lower than the neutrophil allele burden, but not in PV and PMF subjects in whom the CD34(+) cell and neutrophil allele burdens were similar. CD34(+) cell JAK2(V617F) clonal dominance, defined as coherence between the CD34(+) cell and neutrophil JAK2(V617F) allele burdens, was present in 24% of ET, 56% of PV, and 93% of PMF patients, and was independent of the CD34(+) cell JAK2(V617F) genotype. Clonally dominant PV patients had significantly longer disease durations, higher white cell counts, and larger spleens than nondominant PV patients. CONCLUSIONS We conclude that the extent of JAK2(V617F) CD34(+) cell clonal dominance is associated with disease phenotype within the MPD and, in PV, is associated with extramedullary disease, leukocytosis, and disease duration.

Anemia of cancer

Cancer patients frequently develop anemia, due either to the cancer itself or to the effects of cancer-related therapy. Recent years have brought insights into both the pathogenesis of the anemia of cancer and the extent to which erythropoietin regulation participates in this process. Although transfusion therapy was the mainstay of therapy for symptomatic anemia in the past, clinical trials have demonstrated that recombinant human erythropoietin can alleviate both anemia and transfusion requirements in many cancer patients and may prove to have an important role in the treatment of cancer-related anemia in the future.

Sex differences in the JAK2V617F allele burden in chronic myeloproliferative disorders

Background The JAK2V617F allele burden is a variable measure, determined by the frequency of mitotic recombination events and the expansion of JAK2V617F clones. Since variability in the JAK2V617F allele burden is partly responsible for the distinct phenotypes seen in the myeloproliferative disorders, the objective of this study was to identify modifiers of the allele burden. Design and Methods Blood samples were obtained between May 2005 and January 2009 from 272 patients with essential thrombocytosis, polycythemia vera, and myelofibrosis. The JAK2V617F allele burden was measured by an allele-specific quantitative polymerase chain reaction using DNA from purified neutrophils. Repeated measures, on average 2 years apart, were available for 104 patients. Results Sex, age at diagnosis, and disease duration all independently influenced the JAK2V617F allele burden. When considering all patients with myeloproliferative disorders, women had significantly lower allele burdens than men (P=0.04). In those patients with repeated measures, the increase in allele burden per year between the first and second evaluations was significantly less in females than in males. Among those who experienced disease evolution, females were 4.5 times more likely to have evolution from essential thrombocytosis to polycythemia vera, but 0.23 times as likely to have evolution from essential thrombocytosis to myelofibrosis. Conclusions Sex is an independent factor accounting for variability in the JAK2V617F allele burden. We speculate that lower allele burdens in females reflect a lower frequency of mitotic recombination events in females than in males, and should be considered when evaluating the relationship of allele burden to disease phenotype and also in evaluating responses to JAK2V617F-inhibitors. Because sex may influence genotype and/or clonal expansion, underpinning the variability in JAK2V617F allele burden, it will be important to explore factors that determine susceptibility to mitotic recombination events.

Sex differences in the JAK2V617F allele burden in the chronic myeloproliferative disorders

Background The JAK2V617F allele burden is a variable measure, determined by the frequency of mitotic recombination events and the expansion of JAK2V617F clones. Since variability in the JAK2V617F allele burden is partly responsible for the distinct phenotypes seen in the myeloproliferative disorders, the objective of this study was to identify modifiers of the allele burden. Design and Methods Blood samples were obtained between May 2005 and January 2009 from 272 patients with essential thrombocytosis, polycythemia vera, and myelofibrosis. The JAK2V617F allele burden was measured by an allele-specific quantitative polymerase chain reaction using DNA from purified neutrophils. Repeated measures, on average 2 years apart, were available for 104 patients. Results Sex, age at diagnosis, and disease duration all independently influenced the JAK2V617F allele burden. When considering all patients with myeloproliferative disorders, women had significantly lower allele burdens than men (P=0.04). In those patients with repeated measures, the increase in allele burden per year between the first and second evaluations was significantly less in females than in males. Among those who experienced disease evolution, females were 4.5 times more likely to have evolution from essential thrombocytosis to polycythemia vera, but 0.23 times as likely to have evolution from essential thrombocytosis to myelofibrosis. Conclusions Sex is an independent factor accounting for variability in the JAK2V617F allele burden. We speculate that lower allele burdens in females reflect a lower frequency of mitotic recombination events in females than in males, and should be considered when evaluating the relationship of allele burden to disease phenotype and also in evaluating responses to JAK2V617F-inhibitors. Because sex may influence genotype and/or clonal expansion, underpinning the variability in JAK2V617F allele burden, it will be important to explore factors that determine susceptibility to mitotic recombination events.