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

Abstract

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. IntroductionIn 1951, Dr William Dameshek classified polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia/myelofibrosis (MMM), and chronic myelogenous leukemia (CML) as phenotypically related myeloproliferative disorders (MPDs). 1 The molecular pathogenesis of BCR-ABL-negative MPDs was poorly understood until the recent identification of the JAK2V617F activating mutation. 2-8 JAK2V617F confers factor-independent growth and erythropoietin hypersensitivity to hematopoietic cells, 2,3 and expression of JAK2V617F in a murine bone marrow transplantation model results in erythrocytosis. 3 These data suggest that acquisition of JAK2V617F is an important pathogenetic event in JAK2V617F-positive MPDs. Nonetheless, some PV, ET, and MMM patients are JAK2V617F negative, as assessed by DNA resequencing. There are several potential explanations for this observation, including mutations in other genes that phenocopy JAK2V617F. Granulocytes from some patients with PV and ET are polyclonal, 9,10 and thus sequence analysis of granulocyte DNA may underestimate the true frequency of the JAK2V617F allele. Although granulocyte clonality and JAK2V617F mutational status were reported in ET, 11 no study has investigated the relationship between the acquisition of JAK2V617F and the emergence of clonal hematopoiesis in PV, ET, and MMM. We therefore investigated the relationship between clonality and JAK2V617F allelic ratio in MPDs. Study design Harvard Myeloproliferative Disorders StudyThe Harvard Myeloproliferative Disorders Study enrolled patients with PV, ET, and MMM to collect clinical information and biologic samples. 2 All subjects provided informed consent. Approval for the studies was obtained from the Dana-Farber Cancer Institutional Review Board. HUMARA clonality assay and X-inactivation ratio determinationPolymerase chain reaction (PCR) amplification of the polymorphic CAG repeat at the HUMARA locus 12-14 was performed in tandem on undigested (6-FAM-labeled primer) and on HpaII-digested (HEX-labeled primer) DNA. PCR produc...

Genetic complexity of myeloproliferative neoplasms

“…When the abundance of the mutant JAK2 allele was compared with clonality in MPN patients in granulocytes and platelets, a number of ET, PMF and PV patients with monoclonal myeloid cells displayed significantly low abundance of the mutant JAK2 allele, suggesting that only a small proportion of clonal cells carried the JAK2-V617F mutation. 44,54 These studies provided evidence that somatic mutations precede the acquisition of JAK2-V617F and thus experimentally proved that the 'multi-hit' model of MPN pathogenesis is in place at least in a subset of MPN patients ( Figure 1b). Moreover, deletions on chromosome 20q were proposed to be one type of 'pre-JAK2' somatic mutation.…”

“…Before the discovery of JAK2 mutations, polyclonal ET was suspected to represent a reactive condition mimicking ET. The presence of the JAK2-V617F mutation in polyclonal ET patients 44,54,58 not only proved this concept wrong, but at the same time revealed an interesting fact about the ET progenitor pool. In ET, the cells acquiring somatic mutation(s) such as JAK2-V617F do not necessarily undergo selection to full clonality.…”

“…33 (I, j) Clonal structures of patients with a multi-hit pathogenesis, with JAK2-V617F occurring on the background of clonal cells established either by del20q or by unknown mutations. 44,54 (k) Two possible clonal structures in patients carrying both JAK2-V617F and MPL-W515L mutations. 33 Genetic complexity of MPN R Kralovics cytotoxicity but it may also impose a selective pressure on clonal cells negative for JAK2-V617F and increase the chance of acquisition of leukemic mutations such as FLT3-ITD or other mutations facilitating survival in the presence of the drug.…”

JAK2V617F monitoring in polycythemia vera and essential thrombocythemia: Clinical usefulness for predicting myelofibrotic transformation and thrombotic events

“…In patients diagnosed before 2005 the JAK2V617F allele burden was first determined at diagnosis in those cases with available cryopreserved samples or some time after diagnosis in the remaining patients. JAK2V617F allele burden was assessed in DNA from purified granulocytes by real-time allele-specific polymerase chain reaction with probes specific for the mutated and the wild type forms, as previously described [18], using 200 ng in duplicate. In our hands, this technique reached a sensitivity of 2%.…”

Kinase drug discovery approaches in chronic myeloproliferative disorders

“…In this regard, it will be important to develop conditional JAK2-V617F knock-in animals and to determine the phenotypic consequences of the homozygous versus heterozygous JAK2-V617F burden. In addition, there is evidence for an inherited germline allele that precedes and predisposes patients to acquire JAK2-V617F (Goerttler et al, 2005;Levine et al, 2006) as well as loss of chromosomal region 20q in some MPD patients. Although MPD conversion to AML is observed clinically at moderate levels and activating JAK chromosomal translocations are observed in leukemia, epidemiological data suggest that it is questionable that JAK2-V617F is a genetic driver in this context, suggesting that additional genetic alterations are required for full leukemic transformation (Theocharides et al, 2007).…”