Title: An update of molecular pathogenesis and diagnosis of myeloproliferative disorders in the JAK2 era
Abstract: Myeloproliferative disorders (MPD) are clonal haematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased numbers of mature and immature cells in the peripheral blood. MPDs are classified into five categories: polycythemia vera (PV), essential thrombocythaemia (ET), idiopathic myelofibrosis (IMF), chronic myelogenous leukaemia (CML) and atypical MPD. The atypical MPD includes chronic myelomonocytic leukaemia, juvenile myelomonocytic leukaemia, chronic neutrophilic leukaemia, chronic eosinophilic leukaemia, chronic basophilic leukaemia, hypereosinophilic syndrome, systemic mastocytosis, atypical CML and unclassifiable cases.1-3 Apart from the Philadelphia chromosome and BCR-ABL fusion gene as the characteristic genetic abnormality of CML, the molecular pathogenesis of most MPDs such as PV, ET and IMF has not been described. Nonspecific cytogenetic abnormalities were found at diagnosis in numerous patients including deletions of the long arms of chromosome 20 and chromosome 13, trisomies of chromosome 8 and 9 and duplication of part of the long arm of chromosome 1.4-7 Balanced translocations are rare in MPD. Molecular cytogenetic techniques suggested that abnormalities of chromosome 9 may be relatively common in MPD patients.8,9 Also, in early 2005, several independent groups in Boston,10 Paris,11 Switzerland, Italy, 12 Cambridge,13 and Vanderbilt14 identified a gain of function point mutation of Janus associated kinase 2 (JAK2) — JAK2V617F in BCR-ABL negative MPD patients. Subsequent research confirmed this novel finding.15,16 STRUCTURE AND FUNCTION OF JAK2 JAK2, a member of Janus group of cytoplasmic nonreceptor tyrosine kinases (JAKs 1-3 and TYK2), are structurally characterized by seven domains from the amino (N)-terminal to carboxyl (C)-terminal: FERM, SH2, JH1 and JH2. FERM (Band 4.1, Ezrin, Radixin, Moesin) homology domain is responsible for JAK2 binding to the box 1/2 sequences present in the juxtamembrane cytoplasmic region of type I cytokine receptors. SH2 domain contains binding sites. JH2 is pseudokinase domain and JH1 is functional domain.17,18 JAK2 is located on chromosome 9p24.1. JAK2 is widely expressed and plays a central role in transduction of signals from multiple growth factor receptors; erythropoietin (EPO), interleukin-3, granulocyte macrophage colony stimulating factor, stem cell factor, insulin like growth factor 1 and thrombopoietin. Upon ligand binding, receptor dimerization initiates JAK activation by reciprocal transphosphorylation of the JAKs. Once activated, the JAKs phosphorylate multiple sites on cytokine receptors, generating docking sites for SH2 domain containing signalling molecules including the signal transducers and activators of transcription (STATs), protein phosphatases and other adaptor proteins such as Shc, Grb2, Cbl and p85 PI3 kinase. Next, activated STATs separate from the receptor complex, form homodimers or heterodimers and translocate to the nucleus to initiate transcription of diverse target genes.19-21 IDENTIFICATION OF JAK2V617F POINT MUTATION The key role of BCR-ABL fusion protein in the molecular pathogenesis of CML and other protein tyrosine kinases (PTKs) in rare forms of MPD, led to the hypothesis that kinase activation may be a common pathogenic event in MPD.22 The groups in Boston10 and Cambridge13 probed this hypothesis and introduced a high throughput sequencing of kinase genes in the genomic DNA from PV patients. They screened both activation loop and autoinhibitory domains of 85 PTKs in PV patients and identified a commonly occurring JAK2V617F point mutation. Subsequently, the mutation was confirmed in a larger number of MPD patients (PV, ET and IMF), but was not detected in healthy individuals. Matrix assisted laser desorption/ionization, time of flight mass spectrometry genotyping and allele specific polymerase chain reaction detected and confirmed this mutation. Fluorescent in situ hybridization (FISH) and quantitative genomic PCR analyses indicated that homozygosity for the mutant allele was a result of mitotic recombination rather than loss of heterozygosity (LOH). Following observations that inhibition of JAK2 protein with AG490 impaired spontaneous erythroid terminal differentiation in cells from PV patients, the Parisian group found that a short interfering RNA decreased levels of JAK2 protein and markedly inhibited formation of endogenous erythroid colonies. Accordingly, they screened the JAK2 gene and revealed JAK2V617F mutation. FISH assay also demonstrated that homozygosity was due to LOH rather than duplication of the mutant allele.11 Given that molecular cytogenetic techniques such as FISH and CGH indicated abnormalities of chromosome 9 may be more common in MPD patients, the Swiss and Italian group mapped on chromosome 9p the minimal LOH, which contained the JAK2 gene and found the JAK2V617F mutation by sequencing DNA from these patients.12 Considering the general importance of PTKs, protein tyrosine phosphatases and the feature of PV haematopoietic progenitor cells, the Vanderbilt group in collaboration with Chinese researches at Jilin University used sequence analysis of the entire coding regions of PTKs and protein tyrosine phosphatases in cDNA samples from PV patients. The results suggested that cDNA analysis was probably more sensitive than genomic DNA mutation analysis, because the mutant allele has expression advantage.14 JAK2V617F is a G to T substitution that generates valine instead of phenylalanine at position 617 within the JAK2 pseudokinase domain. JAK2V617F is a somatically acquired mutation, which can activate multiple downstream signalling pathways and modifies the growth of haematopoietic cells to be cytokine independent.10-14 In addition, in vivo studies using murine bone marrow transplant (BMT) models indicated that it can cause a short latency, fully penetrating MPD phenotype such as erythrocytosis, splenomegaly, variable leukocytosis or myelofibrosis.23-26 Hitherto, some studies demonstrated that approximately 95% of PV patients have the JAK2V617F mutation, as do approximately 50% of ET and IMF patients.10-16 We next discuss the three groups of MPD individually: PV, ET and PMF. JAK2V617F and other genetic abnormalities of MPD are summarized in Table 1.Table 1: Modern classification of myeloproliferative disordersPOLYCYTHEMIA VERA PV is characterized by an increased haematocrit in the peripheral blood and a hypercellular marrow with increased numbers of trilineage, haematopoietic precursor cells. The clinical manifestation includes thrombosis, bleeding, leukaemic transformation and development of post PV myelofibrosis. The majority of studies using sensitive detection methods such as allele specific PCR indicate that JAK2V617F is found in most PV patients. Moreover, homozygous JAK2V617F mutation, which develops from mitotic recombination and duplication of the mutant, occurs in most of PV patients, but is unusual in either ET or IMF.10,27 JAK2V617F is a sensitive, but not specific, marker and diagnostic utility for PV because this mutation is absent in healthy individuals and in those with lymphoid disorders, solid tumour or reactive erythrocytosis.28 However, not all the PV patients harbour JAK2V617F point mutation. Scott et al29 found that the majority of JAK2V617F negative PV patients carry JAK2 exon 12 mutations, and identified four previously unrecognised somatic mutations in this exon. One of these mutations was the substitution of lysine for leucine at codon 539 (K539L) and the three other mutant alleles were insertions or deletions involving codons 538-543. The JAK2 exon 12 mutations were only observed in JAK2V617F-negative PV patients presenting with an isolated erythrocytosis without concomitant leukocytosis or thrombocytosis. In vitro studies demonstrated that these alleles are constitutively active and can transform BaF3-EPO receptor (EPOR) cells to cytokine independent growth. In addition, expression of JAK2K539L in the murine BMT assay resulted in an MPD phenotype with marked erythrocytosis. These data indicated that JAK2 exon 12 mutations are implicated in the pathogenesis of JAK2V617F-negative PV and suggested that additional activating JAK2 alleles may be identified in the rare JAK2V617F or JAK2 exon 12-negative subset of patients with PV. According to these data, an international expert panel submitted proposals for the revision of the WHO criteria: JAK2V617F or JAK2 exon 12 mutations have been listed as a major criterion for PV diagnosis and the platelet count threshold lowered from 600×109/L to 450×109/ L.30 ESSENTIAL THROMBOCYTHAEMIA Among the MPD, ET is characterized by longer median survival and lower transformation rates into acute leukaemia or post ET myelofibrosis. The clinical course is marked by thrombotic and haemorrhagic episodes that occur more frequently in older patients and those with previous vascular events. Several studies revealed that JAK2V617F-positive ET had higher haemoglobin counts, leukocyte counts and lower platelet counts, suggesting JAK2V617F-positive ET is a distinct clinical variant more similar to PV.31-33 We also found this trend in a sample of Chinese patients with ET.34 However, a significant proportion of patients with ET are JAK2V617F negative. Because cytokine receptor is indispensable for activation of JAK2V617F signalling pathway and mutation of EPOR and thrombopoietin receptor (MPL) are associated with familial erythrocytosis and thrombocytosis, mutational screening of EPOR, MPL and granulocyte colony stimulating factor was performed in a few JAK2V617F-negative MPD patients resulting in the identification of somatic mutations at codon 515 at the transmembrane/ juxtamembrane junction of MPL (MPLW515L or MPLW515K) in some JAK2V617F-negative ET and IMF patients.35,36 Similar to JAK2V617F, in vitro studies demonstrated that MPLW515L can transform haematopoietic cells to factor independent growth and activate multiple downstream signalling pathways such as STAT, MAPK and PI3K-AKT. However, expression of MPLW515L in a murine BMT assay resulted in marked thrombocytosis and myelofibrosis which was different from JAK2V617F.35 PRIMARY MYELOFIBROSIS Myelofibrosis (MF), also known as myelofibrosis with myeloid metaplasia or agnogenic myeloid metaplasia, is characterized by bone marrow fibrosis, extramedullary haemopoiesis with splenomegaly, anaemia with dacryocytes and a leukoerythroblastic blood picture.37 This disease can be divided into primary MF (PMF) or secondary MF evolved from previous PV or ET.38 Although the marrow fibrosis is the distinguishing feature of MF, it actually represents a reactive phenomenon of clonal proliferation of a pluripotent haematopoietic stem cell that acquires a growth advantage due to a somatic mutation. The resulting abnormal cell population releases several cytokines and growth factor from which subsequently develops secondary fibrosis of bone marrow. Moreover, MF can colonize through the blood stream in extramedullary organs such as the spleen and the liver.37 The recently identified JAK2V617F mutation in half of the PMF patients12 and the MPL mutation in a proportion of those without the JAK2 mutation has allowed a better understanding of the pathogenesis and a better diagnostic criteria of this disease.35,36 UNANSWERED QUESTIONS Although the identification of JAK2V617F mutation and the subsequent identification of JAK2 exon 12 mutations and MPLW515L/K mutation have provided crucial clues for the pathogenesis of PV, ET and MF: three unanswered questions arise. First, why the same point mutation causes three different phenotypes remains unclear. JAK2V617F can be detected in myeloid, lymphoid lineages and purified haematopoietic stem cells. Hitherto, no evidence for differences in lineage distribution has been found between patients with PV, ET or PMF.39,40 Other molecular aberrations maybe coexist with JAK2V617F in haematopoietic stem cells and cause different phenotypes. Second, there are several pieces of evidence that JAK2V617F is neither the initiating event nor the only genetic event implicated in the pathogenesis of BCR-ABL-negative MPD phenotype. Besides JAK2 exon 12 mutations and MPLW515L/K mutations underlying the basis of a proportion of JAK2V617F-negative MPD phenotypes; chromosomal translocations that modify fusing JAK2 to another protein such as BCR, ETV6 and PCM1 can modify the myeloproliferative phenotype.41,42 Therefore, knowledge of the set of structural molecular aberrations located in JAK2 and MPL signal pathway is indispensable. In addition, the acute myeloid leukaemia (AML) which was developed from former JAK2V617F-positive MPD was identified as a JAK2V617F-negative clone.43,44 There may be other molecular aberrations, which coexist with JAK2V617F and underlie the transformation of AML from MPD. Third, some studies have demonstrated that JAK2V617F-positive ET patients have higher haematocrit and haemoglobin levels and lower platelet counts than JAK2V617F-negative ET patients and proposed the hypothesis that ET and PV are actually a continuum of phenotypic variation. However, it is not clear why these patients with ET almost never display homozygous progenitor colonies in methylcellulose assays, whereas practically all patients with PV do, irrespective of the allelic ratio of JAK2V617F in peripheral blood granulocytes.45 JAK2 INHIBITORS Given the great achievement of Gleevec with CML patients and identification of specific target in BCR-ABL-negative MPD patients of JAK2 mutation, physicians and patients are eagerly awaiting the results of the first clinical trials of JAK2 inhibitors. Several groups are actively developing orally bioavailable, small molecule, inhibitors of JAK2 kinase. JAK2 inhibitors may be classified as “JAK2 selective” (Class I) or “nonJAK2 selective” (Class II) depending on whether the particular inhibitor was developed to primarily target JAK2 kinase (including JAK2V617F). Class II inhibitors were primarily developed for nonMPD indications but are still considered to have therapeutic potential in MPD given their significant “off-target” JAK2 kinase, inhibitory activity.46 Clinical trials with small molecule, JAK2 inhibitors that are developing are summarized in Table 2.Table 2: Clinical trials of JAK2 inhibitorsOUTLOOK Although our knowledge of the pathogenesis of BCR-ABL negative MPD has been greatly improved by the discovery of JAK2V617F mutation, several studies should be performed consecutively to help us better understand molecular pathogenesis of these MPDs. Current and future research will include screening of JAK signalling molecule to identify mutant alleles in JAK2 and MPL-negative MPD, as well as inherited and/or acquired events that affect JAK2V617F. Moreover, a number of compounds specific for JAK2 are under development. Since all of these compounds also inhibit the wild type JAK2 protein, it may cause serious adverse effects including anaemia, neutropenia and thrombocytopenia. However, the available data demonstrate that cells harbouring the mutant JAK2V617F protein are more sensitive to the inhibitors than wild type cells and it will be interesting to see whether the inhibitors specific for the mutant JAK2V617F protein can be found. The 2001 World Health Organization (WHO) classification and diagnostic criteria of MPD was revised recently for the upcoming 4th edition, again according to the progress of molecular pathogenesis. The term “clonal myeloproliferative disorders”, is replaced by “myeloproliferative neoplasms”, and that category now includes mast cell disease. Myeloid neoplasms with molecularly characterized clonal eosinophilia, previously classified under chronic eosinophilic leukemial hypereosinophilic syndrome, are now removed from the myeloproliferative neoplasm section and assembled into a new category of their own.47