Kimberly Hickman

A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement

The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway-mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.

Methylation of AR locus does not always reflect X chromosome inactivation state

Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

Quantification of Fibrosis and Osteosclerosis in Myeloproliferative Neoplasms: A Computer-Assisted Image Study

Evaluation of bone marrow fibrosis and osteosclerosis in myeloproliferative neoplasms (MPN) is subject to interobserver inconsistency. Performance data for currently utilized fibrosis grading systems are lacking, and classification scales for osteosclerosis do not exist. Digital imaging can serve as a quantification method for fibrosis and osteosclerosis. We used digital imaging techniques for trabecular area assessment and reticulin fiber quantification. Patients with all Philadelphia negative MPN subtypes had higher trabecular volume than controls (p<or=0.0015). Results suggest that the degree of osteosclerosis helps differentiate primary myelofibrosis from other MPN. Numerical quantification of fibrosis highly correlated with subjective scores, and interobserver correlation was satisfactory. Digital imaging provides accurate quantification for osteosclerosis and fibrosis.

Plasma quantitation of JAK2 mutation is not suitable as a clinical test: an artifact of storage

To the editor: JAK2V617F is a gain-of-function somatic mutation of JAK2 tyrosine kinase present in some but not all BCR/ABL-negative myeloproliferative neoplasms (MPN). High JAK2V617F allelic burdens correspond with homozygosity generated by uniparental disomy[1][1] present in some progenitors and

Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes

Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes

Myelofibrotic transformations of polycythemia vera and essential thrombocythemia are morphologically, biologically, and prognostically indistinguishable from primary myelofibrosis.

A fraction of polycythemia vera (PV) and essential thrombocythemia (ET) cases will, in time, undergo myelofibrotic transformation. In such cases, fibrosis may mask the diagnostic histologic features of the original underlying myeloproliferative neoplasm. Thus, confidently differentiating postfibrotic PV/ET from primary myelofibrosis (PMF) histologically may not be possible. It is controversial whether post-PV/ET myelofibrosis (MF) differs clinicopathologically from PMF, or whether these entities are biologically, clinically, and prognostically indistinguishable. To answer this question, we compared multiple candidate biological, morphologic, and prognostic parameters between 19 postfibrotic ET/PV individuals and 18 PMF individuals. The postfibrotic ET/PV and PMF cases did not differ with regard to clinical outcome, cytogenetic abnormalities, serum lactate dehydrogenase level, peripheral blast count, bone marrow morphology, or grade of reticulin fibrosis. Only JAK2 allele burden, which was higher in the postfibrotic PV/ET population (P=0.011), differed between the 2 groups. Cardinal morphologic features of PMF (ie, marrow cellularity, intrasinusoidal hematopoiesis, osteosclerosis, etc.) were commonly observed in post-PV/ET MF marrow biopsies, and only a minority of post-PV/ET MF marrow biopsies the retained diagnostic features of the primary myeloproliferative neoplasm (panmyelosis in PV and megakaryocytic hyperplasia in ET). Our study indicates that PMF and post-PV/ET MF are clinically and biologically indistinguishable.

Could hypoxia increase the prevalence of thrombotic complications in polycythemia vera

Thromboses represent a major cause of morbidity and mortality in polycythemia vera but the contributing mechanisms are not fully described. To evaluate whether environmental conditions such as altitude/hypoxia could impact thrombosis history, we retrospectively analyzed thrombosis history in 71 polycythemia vera patients living at an elevation of 5000 feet or more in the Salt Lake City (SLC) area and 166 polycythemia vera patients living near sea level in the Baltimore (BLM) area. The SLC cohort was older with a longer disease duration. No significant differences in type of anticoagulation therapy or prothrombotic factors were present between the two cohorts. After adjusting for age, sex and disease duration, SLC patients experienced an estimated 3.9-fold increase in the odds of a history of thrombosis compared with BLM patients (95% confidence interval 1.8–7.6; P = 0.0004). A history of a cardiovascular event was present in 58% of the SLC patients compared with 27% of the BLM patients (P < 0.0001). Before diagnosis, thrombosis occurred in 18 and 4% of the SLC and BLM groups, respectively (P = 0.003). No correlation between the JAK2V617F allele burden and thrombosis was observed in this study. This retrospective study suggests that even moderate hypoxia associated with 5000 feet elevation should be considered as an independent prothrombotic risk factor. This observation needs to be confirmed by prospective studies.

Abstract LB-311: Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by augmented myelopoiesis leading to increased red cell mass with frequent excessive proliferation of other myeloid lineages. Over 95% of PV patients harbor JAK2V617F mutation, which confers proliferative advantage to affected myeloid progenitors. JAK2V617F, which is thought to be necessary but not sufficient for the disease, is often associated with acquired uniparental disomy on chromosome 9p (9pUPD). To better understand the underlying molecular basis of PV, we performed whole-exome sequencing and DNA copy-number analysis of 31 JAK2V617F-positive patients and further investigated the evolution of somatic mutations using longitudinal samples. In addition to JAK2 V617F and 9pUPD, we identified recurrent somatic mutation in an additional 5 genes including ASXL1, TET2, DNMT3A, and SF3B1 and recurrent DNA copy-number alterations at 1q and 18p. Forty-two percent of patients had at least one somatic mutation in an epigenetic modifier gene, and 23% of patients harbored mutation in RTK/RAS, RNA processing pathways, tumor suppressors and protein kinases. In 4 of 31 cases, variant allele abundance suggested somatic mutations of ASXL1, DNMT3A and SF3B1 may have preceded JAK2V617F. Interestingly, in 7 PV patients mutations also present in COSMIC were shared between the patients’ T-cell controls and granulocytes: 4 NF1, 2 ASXL1, and one each in TET2, IDH2, and DNMT3A, suggesting these genes mutated early in hematopoietic development. Absence of JAK2V617F in these T-cells ruled out cross-contamination of cell lineages. In an additional 4 cases, a minor JAK2V617F subclone acquired new mutations in DNMT3A, SF3B1, SMARCA2, and TET2 during PV progression. In summary, seventy percent of patients had mutation of at least one gene important to development of myeloid neoplasms, other than JAK2V617F. Our data reveal that these mutations, in particular NF1, may have arisen in some cases early in hematopoiesis but also in some patients during progression after diagnosis. These results suggest that the order of mutation is not as important as the total mutational burden in the development of PV. Citation Format: David A. Wheeler, Linghua Wang, Sabina Swierczek, Kimberly Hickman, Jennifer A. Drummond, Donna Muzny, Richard A. Gibbs, Joseph Prchal. Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes. [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 LB-311. doi:10.1158/1538-7445.AM2014-LB-311