Determinants of clinical phenotype in myeloproliferative neoplasms

Background: Myeloproliferative neoplasms, (MPNs) such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment. Methods: We sequenced coding exons from 69 myeloid cancer genes, common to two separate bait sets, in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes or copy. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort. Cytokine profiles of over 400 patients were also analysed to determine the contribution of the inflammatory microenvironment to phenotype and progression risk. Results: A total of 2035 patients were included in the analysis. 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. In particular a set of mutations that included ASXL1, SRSF2, U2AF1 and EZH2 was enriched in myelofibrosis and associated with poor outcomes. The JAK2 46/1 haplotype strongly correlated with the presence of 9pUPD and independently with a PV phenotype, demonstrating that the underlying germline background can play a role in determining somatic events and can affect the patient’s phenotype in its own right. We defined eight subgroups based solely on clustering of genomic data that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and overall survival. These included a sub-group defined by mutations the same set of chromatin and splicesome component genes described above, and a subgroup enriched for TP53 mutations and chromosomal changes, which carried a significant risk of AML transformation. Patients with no detectable mutations had very low rates of progression or death. By integrating 63 clinical, demographic and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. The prognostic model performed as well as or better than a number of existing risk scores including the high molecular risk genetic score and international prognostic scoring systems and even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy. Cytokine profiles varied significantly across MPN subtypes, with high levels of TNFalpha and IP-10 seen in myelofibrosis, and to a lesser extent in polycythemia vera. Patients with essential thrombocytosis however, were found to have high levels of GROalpha and EGF, and levels of these at single time points or when measured longitudinally were predictive for the risk of progression to myelofibrosis. Conclusions: Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients’ outcomes and may support the treatment of patients with myeloproliferative neoplasms.This research was funded by grants from Bloodwise and Kay Kendall Foundatio

Methylation age as a correlate for allele burden, disease status, and clinical response in myeloproliferative neoplasm patients treated with vorinostat.

The myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal neoplastic disorders. Driver mutations in JAK2, CALR, and MPL genes have been identified in the majority of cases. Alongside these, an increasing number of genes are repeatedly identified as mutated in MPN. These, including ASXL1, TET2, DMNT3A, and EZH2, have key roles in epigenetic regulation. Dysregulation of epigenetic processes is therefore a key feature of MPN. Vorinostat is a pan histone deacetylase inhibitor (HDACi) that has been investigated in MPN. DNA methylation (DNAm) is a well-defined epigenetic mechanism of transcription modification. It is known to be affected by ageing, lifestyle, and disease. Epigenetic ageing signatures have been previously described allowing calculation of a methylation age (MA). In this study we examined the effect of vorinostat on MA in MPN cell lines and in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) treated with vorinostat as part of a clinical trial. An older MA was observed in patients with a higher JAK2 V617F allele burden and those with a longer duration of disease. PV patients had a MA older than that predicted whilst MA was younger than predicted in ET. Treatment with vorinostat resulted in a younger MA in PV patients and older MA in ET patients, in both cases a trend towards the normal chronological age. When MA change was compared against response, nonresponse was associated with a younger than predicted MA in ET patients and a higher than predicted MA in PV patients. The link between MA and JAK2 mutant allele burden implies that allele burden has a role not only in clinical phenotype and disease evolution in MPN patients, but also in the overall methylation landscape of the mutated cells

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF

Longitudinal cytokine profiling identifies GRO-α and EGF as potential biomarkers of disease progression in Essential Thrombocythemia

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.The serum cytokine studies were supported by a research grant from the Rosetrees Trust. NFØ was supported by grants from the Danish Lundbeck Foundation and Danish Cancer Society, J.G. was supported by fellowships from Bloodwise and the Kay Kendall Leukaemia Fund; and M.S.S. is the recipient of a Biotechnology and Biological Sciences Research Council Industrial Collaborative Awards in Science and Engineering PhD Studentship. Work in the R.C.S. laboratory was supported by grants from the Stiftung Blutspendezentrum SRK beider Basel, the Swiss National Science Foundation (31003A-147016/1 and 31003A_166613), and the Swiss Cancer League (KLS-2950-02-2012 and KFS-3655-02-2015). A.K. was supported by the Else Kröner-Fresenius Foundation. Work in the A.R.G. laboratory is supported by the Wellcome Trust, Bloodwise, Cancer Research UK, the Kay Kendall Leukaemia Fund, and the Leukemia and Lymphoma Society of America. Work in the D.G.K. laboratory is supported by a Bloodwise Bennett Fellowship (15008), a European Hematology Association Non-Clinical Advanced Research Fellowship, and an ERC Starting Grant (ERC-2016-STG–715371). D.G.K. and A.R.G. are supported by a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome MRC Cambridge Stem Cell Institute, the National Institute for Health Research Cambridge Biomedical Research Centre, and the CRUK Cambridge Cancer Centre

Longitudinal Cytokine Profiling Identifies GRO-α and EGF as Potential Biomarkers of Disease Progression in Essential Thrombocythemia.

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.The serum cytokine studies were supported by a research grant from the Rosetrees Trust. NFØ was supported by grants from the Danish Lundbeck Foundation and Danish Cancer Society, J.G. was supported by fellowships from Bloodwise and the Kay Kendall Leukaemia Fund; and M.S.S. is the recipient of a Biotechnology and Biological Sciences Research Council Industrial Collaborative Awards in Science and Engineering PhD Studentship. Work in the R.C.S. laboratory was supported by grants from the Stiftung Blutspendezentrum SRK beider Basel, the Swiss National Science Foundation (31003A-147016/1 and 31003A_166613), and the Swiss Cancer League (KLS-2950-02-2012 and KFS-3655-02-2015). A.K. was supported by the Else Kröner-Fresenius Foundation. Work in the A.R.G. laboratory is supported by the Wellcome Trust, Bloodwise, Cancer Research UK, the Kay Kendall Leukaemia Fund, and the Leukemia and Lymphoma Society of America. Work in the D.G.K. laboratory is supported by a Bloodwise Bennett Fellowship (15008), a European Hematology Association Non-Clinical Advanced Research Fellowship, and an ERC Starting Grant (ERC-2016-STG–715371). D.G.K. and A.R.G. are supported by a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome MRC Cambridge Stem Cell Institute, the National Institute for Health Research Cambridge Biomedical Research Centre, and the CRUK Cambridge Cancer Centre

Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2 V617F/V617F TET2 -/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALR del52 or JAK2 +/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10 -/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10 -/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

<p>Abstract</p> <p>Background</p> <p>A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.</p> <p>Methods</p> <p>In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.</p> <p>Results</p> <p>No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (D<smcaps>L</smcaps>_CO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (D<smcaps>L</smcaps>_CO< 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in D<smcaps>L</smcaps>_COat 12 months after baseline (0.48 vs. 0.33; p = 0.023).</p> <p>Conclusions</p> <p>These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.</p

MicroRNA-101 expression is associated with JAK2V617F activity and regulates JAK2/STAT5 signaling.

Philadelphia negative myeloproliferative neopl 28 asms (MPNs) are clonal haematological diseases characterized by excessive production of mature blood cells. Exome sequencing of patient samples have showed a relatively low degree genomic complexity for these diseases1. The majority of MPN patients carry somatic mutations in the JAK2 gene, with the JAK2V617F missense mutation being the most common in poly33 cythemia vera (PV, 95%) and essential thrombocythemia (ET, 60%) 2.FP was supported by Fondazione Umberto Veronesi, and Institute Pasteur - Fondazione Cenci Bolognetti