Publications by authors named "Jyoti Nangalia"

37 Publications

Lineage tracing of human development through somatic mutations.

Nature 2021 07 12;595(7865):85-90. Epub 2021 May 12.

Wellcome Trust Sanger Institute, Hinxton, UK.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.
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http://dx.doi.org/10.1038/s41586-021-03548-6DOI Listing
July 2021

Extensive heterogeneity in somatic mutation and selection in the human bladder.

Science 2020 10;370(6512):75-82

Cancer, Ageing and Somatic Mutation Programme, Wellcome Sanger Institute, Hinxton CB10 1SA, UK.

The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted ( = 1914 microbiopsies), whole-exome ( = 655), and whole-genome ( = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.
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http://dx.doi.org/10.1126/science.aba8347DOI Listing
October 2020

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

Hemasphere 2020 Jun 21;4(3):e371. Epub 2020 May 21.

Wellcome MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge, CB2 0XY, United Kingdom.

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, CD56CD14 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.
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http://dx.doi.org/10.1097/HS9.0000000000000371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306314PMC
June 2020

Genomic heterogeneity in myeloproliferative neoplasms and applications to clinical practice.

Blood Rev 2020 07 19;42:100708. Epub 2020 May 19.

Wellcome Sanger Institute, Hinxton, Cambridgeshire, UK; Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Puddicombe Way, Cambridge, UK; Department of Haematology, University of Cambridge, Cambridge, UK; Haematopathology and Oncology Diagnostics Service/ Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Hills Rd, Cambridge CB2 0QQ, UK. Electronic address:

The myeloproliferative neoplasms (MPN) polycythaemia vera, essential thrombocythaemia and primary myelofibrosis are chronic myeloid disorders associated most often with mutations in JAK2, MPL and CALR, and in some patients with additional acquired genomic lesions. Whilst the molecular mechanisms downstream of these mutations are now clearer, it is apparent that clinical phenotype in MPN is a product of complex interactions, acting between individual mutations, between disease subclones, and between the tumour and background host factors. In this review we first discuss MPN phenotypic driver mutations and the factors that interact with them to influence phenotype. We consider the importance of ongoing studies of clonal haematopoiesis, which may inform a better understanding of why MPN develop in specific individuals. We then consider how best to deploy genomic testing in a clinical environment and the challenges as well as opportunities that may arise from more routine, comprehensive genomic analysis of patients with MPN.
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http://dx.doi.org/10.1016/j.blre.2020.100708DOI Listing
July 2020

The mutational landscape of normal human endometrial epithelium.

Nature 2020 04 22;580(7805):640-646. Epub 2020 Apr 22.

Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Cambridge, UK.

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.
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http://dx.doi.org/10.1038/s41586-020-2214-zDOI Listing
April 2020

Genome Sequencing during a Patient's Journey through Cancer.

N Engl J Med 2019 11;381(22):2145-2156

From the Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, and Wellcome-MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, the Department of Haematology, University of Cambridge, and the Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge - all in the United Kingdom.

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http://dx.doi.org/10.1056/NEJMra1910138DOI Listing
November 2019

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

Exp Hematol 2019 11 26;79:26-34. Epub 2019 Sep 26.

Blood Cancer Research Group, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, United Kingdom; Centre for Medical Education, Queen's University Belfast, Belfast, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.exphem.2019.09.025DOI Listing
November 2019

Clonal approaches to understanding the impact of mutations on hematologic disease development.

Blood 2019 03 6;133(13):1436-1445. Epub 2019 Feb 6.

Wellcome-MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom.

Interrogation of hematopoietic tissue at the clonal level has a rich history spanning over 50 years, and has provided critical insights into both normal and malignant hematopoiesis. Characterization of chromosomes identified some of the first genetic links to cancer with the discovery of chromosomal translocations in association with many hematological neoplasms. The unique accessibility of hematopoietic tissue and the ability to clonally expand hematopoietic progenitors in vitro has provided fundamental insights into the cellular hierarchy of normal hematopoiesis, as well as the functional impact of driver mutations in disease. Transplantation assays in murine models have enabled cellular assessment of the functional consequences of somatic mutations in vivo. Most recently, next-generation sequencing-based assays have shown great promise in allowing multi-"omic" characterization of single cells. Here, we review how clonal approaches have advanced our understanding of disease development, focusing on the acquisition of somatic mutations, clonal selection, driver mutation cooperation, and tumor evolution.
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http://dx.doi.org/10.1182/blood-2018-11-835405DOI Listing
March 2019

Classification and Personalized Prognosis in Myeloproliferative Neoplasms.

N Engl J Med 2018 10;379(15):1416-1430

From the Wellcome-MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research (J.G., C.E.M., F.L.N., A.R.G., P.J.C.), the Department of Haematology, University of Cambridge (J.G., E.J.B., C.M., J.C., C.E.M., F.L.N., A.R.G.), and the Department of Haematology, Cambridge University Hospitals NHS Foundation Trust (J.G., E.J.B., A.L.G., C.M., J.C., A.R.G.), Cambridge, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus (J.N., D.C.W., N.A., E.P., G.G., L.O., S.O., J.W.T., A.P.B., N.W., P.J.C.), and the European Molecular Biology Laboratory, European Bioinformatics Institute (R.C., M.G.), Hinxton, Big Data Institute, University of Oxford, Oxford (D.C.W.), the Department of Haematology, Queen's University Belfast, Belfast (M.F.M.), and the Department of Haematology, Guy's and St. Thomas' NHS Foundation Trust, London (C.N.H.) - all in the United Kingdom; the Center for Molecular Oncology and the Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York (E.P., G.G.); the Department of Hematology, Zealand University Hospital, Roskilde, and the University of Copenhagen, Copenhagen (C.L.A., H.C.H.); and the Department of Experimental and Clinical Medicine, Center of Research and Innovation of Myeloproliferative Neoplasms, Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy (P.G., A.M.V.).

Background: Myeloproliferative neoplasms, 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 in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. 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.

Results: A total of 2035 patients were included in the analysis. A total of 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. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical 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. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy.

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. (Funded by the Wellcome Trust and others.).
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http://dx.doi.org/10.1056/NEJMoa1716614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030948PMC
October 2018

Hydroxycarbamide Plus Aspirin Versus Aspirin Alone in Patients With Essential Thrombocythemia Age 40 to 59 Years Without High-Risk Features.

J Clin Oncol 2018 12 28;36(34):3361-3369. Epub 2018 Aug 28.

Anna L. Godfrey, Jacob Grinfeld, and Anthony R. Green, Cambridge University Hospitals National Health Service (NHS) Foundation Trust; Peter J. Campbell and Jyoti Nangalia, Wellcome Trust Sanger Institute, Hinxton; Cathy MacLean, Julia Cook, Julie Temple, and Anthony R. Green, University of Cambridge; Anthony R. Green, Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Cambridge; Georgina Buck, University of Oxford, Oxford; Bridget S. Wilkins and Claire N. Harrison, Guy's and St Thomas' NHS Foundation Trust, London; Keith Wheatley, University of Birmingham, Birmingham; Mary Frances McMullin, Queen's University Belfast, Belfast, United Kingdom; Cecily Forsyth, Gosford Hospital, Gosford, and Australasian Leukaemia and Lymphoma Group, Australia; and Jean-Jacques Kiladjian, Hôpital Saint-Louis, Paris, France.

Purpose: Cytoreductive therapy is beneficial in patients with essential thrombocythemia (ET) at high risk of thrombosis. However, its value in those lacking high-risk features remains unknown. This open-label, randomized trial compared hydroxycarbamide plus aspirin with aspirin alone in patients with ET age 40 to 59 years and without high-risk factors or extreme thrombocytosis.

Patients And Methods: Patients were age 40 to 59 years and lacked a history of ischemia, thrombosis, embolism, hemorrhage, extreme thrombocytosis (platelet count ≥ 1,500 × 10/L), hypertension, or diabetes requiring therapy. In all, 382 patients were randomly assigned 1:1 to hydroxycarbamide plus aspirin or aspirin alone. The composite primary end point was time to arterial or venous thrombosis, serious hemorrhage, or death from vascular causes. Secondary end points were time to first arterial or venous thrombosis, first serious hemorrhage, death, incidence of transformation, and patient-reported quality of life.

Results: After a median follow-up of 73 months and a total follow-up of 2,373 patient-years, there was no significant difference between the arms in the likelihood of patients reaching the primary end point (hazard ratio, 0.98; 95% CI, 0.42 to 2.25; = 1.0). The incidence of significant vascular events was low, at 0.93 per 100 patient-years (95% CI, 0.61 to 1.41). There were also no differences in overall survival; in the composite end point of transformation to myelofibrosis, acute myeloid leukemia, or myelodysplasia; in adverse events; or in patient-reported quality of life.

Conclusion: In patients with ET age 40 to 59 years and lacking high-risk factors for thrombosis or extreme thrombocytosis, preemptive addition of hydroxycarbamide to aspirin did not reduce vascular events, myelofibrotic transformation, or leukemic transformation. Patients age 40 to 59 years without other clinical indications for treatment (such as previous thrombosis or hemorrhage) who have a platelet count < 1,500 × 10/L should not receive cytoreductive therapy.
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http://dx.doi.org/10.1200/JCO.2018.78.8414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269131PMC
December 2018

Myeloproliferative neoplasms: from origins to outcomes.

Hematology Am Soc Hematol Educ Program 2017 12;2017(1):470-479

Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, United Kingdom; and.

Substantial progress has been made in our understanding of the pathogenetic basis of myeloproliferative neoplasms. The discovery of mutations in over a decade ago heralded a new age for patient care as a consequence of improved diagnosis and the development of therapeutic JAK inhibitors. The more recent identification of mutations in calreticulin brought with it a sense of completeness, with most patients with myeloproliferative neoplasm now having a biological basis for their excessive myeloproliferation. We are also beginning to understand the processes that lead to acquisition of somatic mutations and the factors that influence subsequent clonal expansion and emergence of disease. Extended genomic profiling has established a multitude of additional acquired mutations, particularly prevalent in myelofibrosis, where their presence carries prognostic implications. A major goal is to integrate genetic, clinical, and laboratory features to identify patients who share disease biology and clinical outcome, such that therapies, both existing and novel, can be better targeted.
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http://dx.doi.org/10.1182/asheducation-2017.1.470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142568PMC
December 2017

Myeloproliferative neoplasms: from origins to outcomes.

Blood 2017 12;130(23):2475-2483

Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, United Kingdom; and.

Substantial progress has been made in our understanding of the pathogenetic basis of myeloproliferative neoplasms. The discovery of mutations in over a decade ago heralded a new age for patient care as a consequence of improved diagnosis and the development of therapeutic JAK inhibitors. The more recent identification of mutations in calreticulin brought with it a sense of completeness, with most patients with myeloproliferative neoplasm now having a biological basis for their excessive myeloproliferation. We are also beginning to understand the processes that lead to acquisition of somatic mutations and the factors that influence subsequent clonal expansion and emergence of disease. Extended genomic profiling has established a multitude of additional acquired mutations, particularly prevalent in myelofibrosis, where their presence carries prognostic implications. A major goal is to integrate genetic, clinical, and laboratory features to identify patients who share disease biology and clinical outcome, such that therapies, both existing and novel, can be better targeted.
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http://dx.doi.org/10.1182/blood-2017-06-782037DOI Listing
December 2017

Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial.

Lancet Haematol 2017 May 20;4(5):e225-e236. Epub 2017 Mar 20.

Department of Haematology, Guy's and St Thomas' NHS Foundation Trust, London, UK.

Background: Available therapies for myelofibrosis can exacerbate cytopenias and are not indicated for patients with severe thrombocytopenia. Pacritinib, which inhibits both JAK2 and FLT3, induced spleen responses with limited myelosuppression in phase 1/2 trials. We aimed to assess the efficacy and safety of pacritinib versus best available therapy in patients with myelofibrosis irrespective of baseline cytopenias.

Methods: This international, multicentre, randomised, phase 3 trial (PERSIST-1) was done at 67 sites in 12 countries. Patients with higher-risk myelofibrosis (with no exclusions for baseline anaemia or thrombocytopenia) were randomly assigned (2:1) to receive oral pacritinib 400 mg once daily or best available therapy (BAT) excluding JAK2 inhibitors until disease progression or unacceptable toxicity. Randomisation was stratified by risk category, platelet count, and region. Treatment assignments were known to investigators, site personnel, patients, clinical monitors, and pharmacovigilance personnel. The primary endpoint was spleen volume reduction (SVR) of 35% or more from baseline to week 24 in the intention-to-treat population as assessed by blinded, centrally reviewed MRI or CT. We did safety analyses in all randomised patients who received either treatment. Here we present the final data. This trial is registered with ClinicalTrials.gov, number NCT01773187.

Findings: Between Jan 8, 2013, and Aug 1, 2014, 327 patients were randomly assigned to pacritinib (n=220) or BAT (n=107). Median follow-up was 23·2 months (IQR 14·8-28·7). At week 24, the primary endpoint of SVR of 35% or more was achieved by 42 (19%) patients in the pacritinib group versus five (5%) patients in the BAT group (p=0·0003). 90 patients in the BAT group crossed over to receive pacritinib at a median of 6·3 months (IQR 5·8-6·7). The most common grade 3-4 adverse events through week 24 were anaemia (n=37 [17%]), thrombocytopenia (n=26 [12%]), and diarrhoea (n=11 [5%]) in the pacritinib group, and anaemia (n=16 [15%]), thrombocytopenia (n=12 [11%]), dyspnoea (n=3 [3%]), and hypotension (n=3 [3%]) in the BAT group. The most common serious adverse events that occurred through week 24 were anaemia (10 [5%]), cardiac failure (5 [2%]), pyrexia (4 [2%]), and pneumonia (4 [2%]) with pacritinib, and anaemia (5 [5%]), sepsis (2 [2%]), and dyspnoea (2 [2%]) with BAT. Deaths due to adverse events were observed in 27 (12%) patients in the pacritinib group and 14 (13%) patients in the BAT group throughout the duration of the study.

Interpretation: Pacritinib therapy was well tolerated and induced significant and sustained SVR and symptom reduction, even in patients with severe baseline cytopenias. Pacritinib could be a treatment option for patients with myelofibrosis, including those with baseline cytopenias for whom options are particularly limited.

Funding: CTI BioPharma Corp.
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http://dx.doi.org/10.1016/S2352-3026(17)30027-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209752PMC
May 2017

Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms.

Haematologica 2017 01 1;102(1):7-17. Epub 2016 Dec 1.

Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK

The myeloproliferative neoplasms are a heterogeneous group of clonal disorders characterized by the overproduction of mature cells in the peripheral blood, together with an increased risk of thrombosis and progression to acute myeloid leukemia. The majority of patients with Philadelphia-chromosome negative myeloproliferative neoplasms harbor somatic mutations in Janus kinase 2, leading to constitutive activation. Acquired mutations in calreticulin or myeloproliferative leukemia virus oncogene are found in a significant number of patients with essential thrombocythemia or myelofibrosis, and mutations in numerous epigenetic regulators and spliceosome components are also seen. Although the cellular and molecular consequences of many of these mutations remain unclear, it seems likely that they interact with germline and microenvironmental factors to influence disease pathogenesis. This review will focus on the determinants of specific myeloproliferative neoplasm phenotypes as well as on how an improved understanding of molecular mechanisms can inform our understanding of the disease entities themselves.
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http://dx.doi.org/10.3324/haematol.2014.113845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210228PMC
January 2017

Pathogenesis of Myeloproliferative Disorders.

Annu Rev Pathol 2016 05;11:101-26

Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, United Kingdom; email:

Myeloproliferative neoplasms (MPNs) are a set of chronic hematopoietic neoplasms with overlapping clinical and molecular features. Recent years have witnessed considerable advances in our understanding of their pathogenetic basis. Due to their protracted clinical course, the evolution to advanced hematological malignancies, and the accessibility of neoplastic tissue, the study of MPNs has provided a window into the earliest stages of tumorigenesis. With the discovery of mutations in CALR, the majority of MPN patients now bear an identifiable marker of clonal disease; however, the mechanism by which mutated CALR perturbs megakaryopoiesis is currently unresolved. We are beginning to understand better the role of JAK2(V617F) homozygosity, the function of comutations in epigenetic regulators and spliceosome components, and how these mutations cooperate with JAK2(V617F) to modulate MPN phenotype.
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http://dx.doi.org/10.1146/annurev-pathol-012615-044454DOI Listing
May 2016

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability.

Cell Rep 2015 Dec 10;13(11):2345-2352. Epub 2015 Dec 10.

Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Broad Institute of the Massachusetts Institute of Technology and Harvard, 415 Main Street, Cambridge, MA 02142; Department of Medical Oncology, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Electronic address:

JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.
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http://dx.doi.org/10.1016/j.celrep.2015.11.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691544PMC
December 2015

Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms.

J Clin Pathol 2016 Feb 19;69(2):155-63. Epub 2015 Aug 19.

School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia PathWest Laboratory Medicine, Western Australia, Australia.

Aims: Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN.

Methods: Immunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and without JAK2(V617F) and CALR mutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12).

Results: Myeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases with CALR mutations, had significant reductions in pro-apoptotic BNIP-3.

Conclusions: Uncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation with CALR mutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN with CALR mutations may be due to greater dysregulation of megakaryocyte apoptosis.
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http://dx.doi.org/10.1136/jclinpath-2015-203177DOI Listing
February 2016

DNMT3A mutations occur early or late in patients with myeloproliferative neoplasms and mutation order influences phenotype.

Haematologica 2015 Nov 6;100(11):e438-42. Epub 2015 Aug 6.

Department of Haematology, Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK Department of Haematology, Addenbrooke's Hospital, Cambridge, UK

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http://dx.doi.org/10.3324/haematol.2015.129510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825297PMC
November 2015

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

Genome Res 2015 06 11;25(6):814-24. Epub 2015 May 11.

Section of Oncology, Department of Clinical Science, University of Bergen, N-5020 Bergen, Norway; Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway;

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells.
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http://dx.doi.org/10.1101/gr.190470.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448678PMC
June 2015

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms.

Nat Commun 2015 Apr 7;6:6691. Epub 2015 Apr 7.

Department of Haematology, UCL Cancer Institute, London WC1 E6BT, UK.

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2(V617F)-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10(-10)) and rs2201862 (MECOM; meta-analysis P=1.96 × 10(-9)). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2(V617F)-positive cases. rs9376092 has a stronger effect in JAK2(V617F)-negative cases with CALR and/or MPL mutations (Breslow-Day P=4.5 × 10(-7)), whereas in JAK2(V617F)-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ(2) P=7.3 × 10(-7)). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype.
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http://dx.doi.org/10.1038/ncomms7691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396373PMC
April 2015

The evolving genomic landscape of myeloproliferative neoplasms.

Hematology Am Soc Hematol Educ Program 2014 Dec 18;2014(1):287-96. Epub 2014 Nov 18.

Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, Department of Haematology, University of Cambridge, Cambridge, United Kingdom; and Department of Haematology, Addenbrooke's Hospital, Cambridge, United Kingdom.

Our understanding of the genetic basis of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) has moved forward at a staggering pace over the last decade. With the discoveries of underlying mutations in JAK2, MPL, and, most recently, calreticulin (CALR), that together account for ∼90% of patients with MPNs, these conditions are now among the best characterized of hematological malignancies. While JAK-STAT pathway activation has been shown to be central to the pathogenesis of the MPN phenotype, the mechanism by which mutant CALR alters cellular function to result in myeloid proliferation remains unclear. Other mutations in several epigenetic modifiers, such as ASXL1, DNMT3a, TET2, EZH2, IDH1, and IDH2, as well as in genes involved in mRNA splicing, such as SF3B1 and U2AF2, have also been described in recent years in patients with MPNs, and evidence is emerging as to how these may be contributing to disease biology. From a therapeutic perspective, the discovery of aberrations in JAK2 has rapidly translated into the successful clinical use of JAK inhibitors in MPNs. Mutant calreticulin has the potential to be a tumor-specific therapeutic target because the mutations generate a novel protein C-terminus. In this chapter, we detail the genomic alterations that underlie MPNs, with a focus on the recent discovery of mutations in CALR, and explore the clinical and biological relevance of the altered genomic landscape in MPNs.
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http://dx.doi.org/10.1182/asheducation-2014.1.287DOI Listing
December 2014

Effect of mutation order on myeloproliferative neoplasms.

N Engl J Med 2015 Feb;372(7):601-612

Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Stem Cell Institute (C.A.O., D.G.K., J.N., Y.S., J.G., E.J.B., C.E.M., A.L.G., D.D., A.R.G.) and Department of Hematology (C.A.O., D.G.K., J.N., Y.S., J.G., E.J.B., C.E.M., A.L.G., D.D., G.S.V., P.J.C., A.R.G.), University of Cambridge, Department of Haematology, Addenbrooke's Hospital (C.A.O., J.N., J.G., E.J.B., A.L.G., G.S.V., P.J.C., A.R.G.), Wellcome Trust Sanger Institute (D.C.W., E.P., G.S.V., P.J.C.), and Cancer Research U.K. Cambridge Institute, Li Ka Shing Centre (S.M.), Cambridge, and Guy's and St. Thomas' National Health Service Foundation Trust, Guy's Hospital, London (C.N.H.) - all in the United Kingdom; Dipartimento di Medicina Sperimentale e Clinica, University of Florence, Florence, Italy (P.G., A.V.); Departments of Pathology (B.B.) and Hematology (C.B.), Hospital del Mar, Barcelona; and the Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany (K.D.).

Background: Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired.

Methods: We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells.

Results: The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as "TET2-first patients"), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first ("JAK2-first patients") had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2-TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation.

Conclusions: The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.).
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http://dx.doi.org/10.1056/NEJMoa1412098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660033PMC
February 2015

Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms.

Leuk Res 2015 Jan 29;39(1):82-7. Epub 2014 Nov 29.

Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK. Electronic address:

The recent discovery of somatically acquired CALR mutations in a substantial proportion of patients with myeloproliferative neoplasms has provided a new marker of clonal disease, advancing both diagnosis and prognosis in these previously difficult to characterise disorders. The mutations, which can be challenging to detect on a routine basis, are heterogeneous insertions/deletions (indels) in exon 9 with mutant allele burden that vary substantially between patients. We evaluated four genetic screening methods for their ability to detect a series of different CALR mutations; Sanger sequencing, fragment analysis PCR, high resolution melt (HRM) and targeted next generation sequencing (NGS). The limit of detection (LoD) of each assay was tested using serial dilution series made with DNA from CALR positive sample DNA and a cell line, MARIMO, found to carry a heterozygous 61 nucleotide CALR deletion. All methods were capable of detecting each mutation; HRM and fragment analysis PCR were better at detecting low mutation levels compared to Sanger sequencing but targeted NGS had the lowest LoD at a 1% mutation burden.
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http://dx.doi.org/10.1016/j.leukres.2014.11.019DOI Listing
January 2015

Nongenetic stochastic expansion of JAK2V617F-homozygous subclones in polycythemia vera?

Blood 2014 Nov;124(22):3332-4

Cambridge Institute for Medical Research and Wellcome Trust/Medical Research Council, Stem Cell Institute and Department of Haematology, University of Cambridge, Cambridge, United Kingdom Department of Haematology, Addenbrooke's Hospital, Cambridge, United Kingdom.

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http://dx.doi.org/10.1182/blood-2014-09-603043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239339PMC
November 2014

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.

Proc Natl Acad Sci U S A 2014 Oct 6;111(42):15190-5. Epub 2014 Oct 6.

Cambridge Institute for Medical Research, Medical Research Council/Wellcome Trust Cambridge Stem Cell Institute, and Department of Haematology, University of Cambridge, Cambridge CB2 0XY, United Kingdom; Department of Haematology, Addenbrooke's Hospital, Cambridge CB2 0XY, United Kingdom; and

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.
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http://dx.doi.org/10.1073/pnas.1401873111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210350PMC
October 2014
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