Ophelia Rogers

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.

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.

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.

The quantitative JAK2 V617F neutrophil allele burden does not correlate with thrombotic risk in essential thrombocytosis

The quantitative JAK2 V617F neutrophil allele burden does not correlate with thrombotic risk in essential thrombocytosis

Disease burden at the progenitor level is a feature of primary myelofibrosis: a multivariable analysis of 164 JAK2 V617F-positive myeloproliferative neoplasm patients

OBJECTIVE Suppression of normal hematopoiesis by the neoplastic clone (clonal dominance) is a feature of the myeloproliferative neoplasms, but the determinants that predict clonal dominance are unknown. The objective of this study was to identify clinical and laboratory variables that associate with the JAK2 V617F CD34(+) progenitor allele burden and clonal dominance, which was defined by congruence of the JAK2 V617F CD34(+) progenitor and neutrophil allele burdens. MATERIALS AND METHODS A cross-sectional analysis was performed on 164 consecutive JAK2 V617F-positive patients: 30 with essential thrombocytosis (ET), 100 with polycythemia vera (PV), and 34 with myelofibrosis (MF), including 8 post-ET MF and 3 post-PV MF. The JAK2 V617F CD34(+) progenitor and neutrophil allele burdens were measured using an allele-specific, quantitative real-time polymerase chain reaction assay. RESULTS After adjusting for genotype, sex, age at diagnosis, and disease duration, disease type was the strongest predictor of clonal dominance, with the odds ratio being nearly 61.9 times higher for MF patients when compared with ET patients (p < 0.001), and 9.7 times higher when compared with PV patients (p = 0.002). Additionally, clonal dominance was associated with a clinical phenotype of an increased spleen size (p = 0.006), increased white blood cell count (p = 0.009), and lower hemoglobin (p < 0.001), even after adjusting for disease type and duration. CONCLUSIONS These data indicate that loss of wild-type clones at the progenitor level is a feature of MF (primary MF, post-ET MF, and post-PV MF), presumably due to expansion of the JAK2 V617F clone and that this characteristic is surprisingly independent of JAK2 V617F homozygosity, suggesting that additional genomic lesions may contribute to this unique molecular process that distinguishes MF from ET and PV.

Two Clinical Phenotypes in Polycythemia Vera

BACKGROUND Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. (Funded by the Department of Defense and the National Institutes of Health.).

Ascites Specific Inhibition of CD1d-Mediated Activation of Natural Killer T Cells

Purpose: Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer–associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells. Experimental Design: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin–based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells. Results: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-galactosylceramide–loaded CD1d-immunoglobulin–based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2–mediated activation of classic CD8+ T cells. Conclusions: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.