Martha Wadleigh

MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

Background The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). Methods and Findings DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Conclusions Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta-analysis of prospective clinical trials.

CONTEXT The optimal treatment of acute myeloid leukemia (AML) in first complete remission (CR1) is uncertain. Current consensus, based on cytogenetic risk, recommends myeloablative allogeneic stem cell transplantation (SCT) for poor-risk but not for good-risk AML. Allogeneic SCT, autologous transplantation, and consolidation chemotherapy are considered of equivalent benefit for intermediate-risk AML. OBJECTIVE To quantify relapse-free survival (RFS) and overall survival benefit of allogeneic SCT for AML in CR1 overall and also for good-, intermediate-, and poor-risk AML. METHODS Systematic review and meta-analysis of prospective trials evaluating allogeneic SCT vs nonallogeneic SCT therapies for AML in CR1. The search used the combined search terms allogeneic; acut* and leukem*/leukaem*/leucem*/leucaem*/aml; myelo* or nonlympho* in the PubMed, Embase, and Cochrane Registry of Controlled Trials databases in March 2009. The search identified 1712 articles. STUDY SELECTION Prospective trials assigning adult patients with AML in CR1 to undergo allogeneic SCT vs nonallogeneic SCT treatment(s) based on donor availability and trials reporting RFS and/or overall survival outcomes on an intention-to-treat, donor vs no-donor basis were identified. DATA EXTRACTION Two reviewers independently extracted study characteristics, interventions, and outcomes. Hazard ratios (HRs) with 95% confidence intervals (CIs) were determined. DATA SYNTHESIS Overall, 24 trials and 6007 patients were analyzed (5951 patients in RFS analyses and 5606 patients in overall survival analyses); 3638 patients were analyzed by cytogenetic risk (547, 2499, and 592 with good-, intermediate-, and poor-risk AML, respectively). Interstudy heterogeneity was not significant. Fixed-effects meta-analysis was performed. Compared with nonallogeneic SCT, the HR of relapse or death with allogeneic SCT for AML in CR1 was 0.80 (95% CI, 0.74-0.86). Significant RFS benefit of allogeneic SCT was documented for poor-risk (HR, 0.69; 95% CI, 0.57-0.84) and intermediate-risk AML (HR, 0.76; 95% CI, 0.68-0.85) but not for good-risk AML (HR, 1.06; 95% CI, 0.80-1.42). The HR of death with allogeneic SCT for AML in CR1 was 0.90 (95% CI, 0.82-0.97). Significant overall survival benefit with allogeneic SCT was documented for poor-risk (HR, 0.73; 95% CI, 0.59-0.90) and intermediate-risk AML (HR, 0.83; 95% CI, 0.74-0.93) but not for good-risk AML (HR, 1.07; 95% CI, 0.83-1.38). CONCLUSION Compared with nonallogeneic SCT therapies, allogeneic SCT has significant RFS and overall survival benefit for intermediate- and poor-risk AML but not for good-risk AML in first complete remission.

Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

JAK2V617F mutation in essential thrombocythaemia: clinical associations and long-term prognostic relevance

Clinical correlates and long‐term prognostic relevance of the JAK2V617F mutation was studied in 150 patients with essential thrombocythaemia (ET) from a single institution and followed for a median of 11·4 years. During this period, thrombotic complications were documented in 62 patients (41·3%) and transformation into acute myeloid leukaemia (AML), polycythaemia vera (PV), or myelofibrosis with myeloid metaplasia (MMM) occurred in 4 (2·7%), 8 (5·3%), and 15 (10%) patients, respectively. JAK2V617F was detected in either archived bone marrow or blood cells from 73 patients (48·7%) but none were homozygous for the mutant allele. Parameters at diagnosis that were significantly associated with the presence of JAK2V617F included advanced age and higher counts of both haemoglobin and leucocytes. During follow‐up, patients with the mutation were more likely to transform into PV but the incidences of AML, MMM, or thrombotic events were similar between patients with and without the mutation. Multivariate analysis identified advanced age, higher haemoglobin level, and thrombosis history but not the presence of JAK2V617F as independent predictors of inferior survival. Therefore, although the presence of JAK2V617F in ET appears to promote a PV phenotype, it might not carry treatment‐relevant information.

A germline JAK2 SNP is associated with predisposition to the development of JAK2V617F-positive myeloproliferative neoplasms

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2V617F) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2V617F is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition.

The burden of fatigue and quality of life in myeloproliferative disorders (MPDs): An international internet-based survey of 1179 MPD patients

Few objective data exist on the burden of fatigue and other constitutional symptoms in patients with myeloproliferative disorders (MPD).

IDH1 and IDH2 Mutation Studies in 1473 Patients with Chronic-, Fibrotic- or Blast-Phase Essential Thrombocythemia, Polycythemia Vera or Myelofibrosis

In a multi-institutional collaborative project, 1473 patients with myeloproliferative neoplasms (MPN) were screened for isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essential thrombocythemia (ET), 421 polycythemia vera (PV), 312 primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast-phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2-R140 and 1 IDH2-R172) were detected: 5 (0.8%) ET, 8 (1.9%) PV, 13 (4.2%) PMF, 1 (1%) post-PV/ET MF and 11 (21.6%) blast-phase MPN (P<0.01). Mutant IDH was documented in the presence or absence of JAK2, MPL and TET2 mutations, with similar mutational frequencies. However, IDH-mutated patients were more likely to be nullizygous for JAK2 46/1 haplotype, especially in PMF (P=0.04), and less likely to display complex karyotype, in blast-phase disease (P<0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (P<0.01; hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.7–5.2), but not IDH mutational status (P=0.55; HR 1.3, 95% CI 0.5–3.4), had an adverse effect on survival. This was confirmed by multivariable analysis. In contrast, in both blast-phase PMF (P=0.04) and blast-phase MPN (P=0.01), the presence of an IDH mutation predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic relevance in MPN and provides additional evidence for the biological effect of distinct JAK2 haplotypes.

Functional screening identifies CRLF2 in precursor B-cell acute lymphoblastic leukemia

The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL–derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications.

The JAK2V617F tyrosine kinase mutation in myelofibrosis with myeloid metaplasia : lineage specificity and clinical correlates

An association between an activating JAK2 mutation (JAK2V617F) and BCR/ABL‐negative myeloproliferative disorders was recently reported in multiple simultaneous publications. In the current study, mutation analysis for JAK2V617F was performed in peripheral blood mononuclear cells (PBMC) from 157 patients with myelofibrosis with myeloid metaplasia (MMM) including 117 with agnogenic (AMM), 22 with postpolycythaemic (PPMM), and 18 with post‐thrombocythaemic (PTMM) myeloid metaplasia. The detection rate for JAK2V617F was significantly higher in PPMM (91%; homozygous in 18%) compared with either AMM (45·3%; homozygous in 2·6%) or PTMM (38·9%; homozygous in 11·1%). Concomitant analysis in granulocytes (n = 57) and CD34+ cells (n = 25) disclosed a higher incidence of homozygous JAK2V617F mutation but the overall mutation rate was similar to that obtained from PBMC. JAK2V617F was not detected in DNA derived from T cells (n = 19). In AMM, the presence of JAK2V617F was associated with an older age at diagnosis and a history of thrombosis or pruritus. Multivariate analysis identified only age and the Dupriez prognostic score as independent prognostic factors; JAK2V617F had no prognostic significance. In conclusion, JAK2V617F is a myeloid lineage‐specific event, its incidence in MMM is significantly higher with an antecedent history of polycythaemia vera (PV), and its presence in AMM does not affect prognosis but is associated with PV‐characteristic clinical features.

The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera

Several studies have recently reported on the occurrence of a JAK2V617F mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation currently is under study.