Guide to  

                          the Genetics of

                  Polycythaemia Vera (Rubra)


                  Essential Thrombocythaemia

                              'PV & ET'

A selection of some of the scientific papers that deal
with PV/ET and related subjects

CLICK ON TOPIC:		Polycythaemia Vera
			JAK2 V617F
			Essential Thrombocythaemia
			Risk of thrombosis,
			Maternal Age Effect
			Transmissible Cancer
			Animal models

Papers dealing with PV --------------------------------------------------------------------------------------------------------- Grinfeld J, Nangalia J, Baxter EJ, Wedge DC, Angelopoulos N, Cantrill R, Godfrey AL, Papaemmanuil E, Gundem G, MacLean C, Cook J, O'Neil L, O'Meara S, Teague JW, Butler AP, Massie CE, Williams N, Nice FL, Andersen CL, Hasselbalch HC, Guglielmelli P, McMullin MF, Vannucchi AM, Harrison CN, Gerstung M, Green AR, Campbell PJ. 2018. 'Classification and Personalized Prognosis in Myeloproliferative Neoplasms.' New England Journal of Medicine. 379(15):1416-1430. Free download from: https://www.nejm.org/doi/10.1056/NEJMoa1716614?url_ver= Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dwww.ncbi.nlm.nih.gov Abstract 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. Comment: Important new paper about Driver Mutations. --------------------------------------------------------------------------------------------------------- Mesa, RA. 2018. New 'Refining the management of polycythemia vera' Clinical Advances in Hematology & Oncology. 16(9):587-589 Free download from: http://www.hematologyandoncology.net/files/2018/09/ho0918Mesa-1.pdf Comment: An up-to-date review of therapy for PV. --------------------------------------------------------------------------------------------------------- Verger E, Soret-Dulphy J, Maslah N, Roy L, Rey J, Ghrieb Z, Kralovics R, Gisslinger H, Grohmann-Izay B, Klade C, Chomienne C, Giraudier S, Cassinat B, Kiladjian JJ. 2018. 'Ropeginterferon alpha-2b targets JAK2V617F-positive polycythemia vera cells in vitro and in vivo' Blood Cancer Journal. 8(10):94. Free download from: https://www.nature.com/articles/s41408-018-0133-0 Comment: Using a new interferon in PV. --------------------------------------------------------------------------------------------------------- Grunwald MR, Stein BL, Boccia RV, Oh ST, Paranagama D, Parasuraman S, Colucci P, Mesa R. 2018. New 'Clinical and Disease Characteristics From REVEAL at Time of Enrollment (Baseline): Prospective Observational Study of Patients With Polycythemia Vera in the United States' Clinical lymphoma, myeloma & leukemia. pii: S2152-2650(18)30593-7. Free download from: https://www.clinical-lymphoma-myeloma-leukemia.com/article/S2152-2650(18)30593-7/pdf Abstract BACKGROUND: Polycythemia vera (PV) has a prevalence of 44 to 57 per 100,000 people in the United States. Prospective data concerning the demographics, clinical characteristics, and treatment patterns of patients with PV in the United States are lacking. PATIENTS AND METHODS: The ongoing, prospective, observational REVEAL study evaluates demographics, disease burden, clinical management, patient-reported outcomes, and health care resource utilization of adult patients with PV in the United States. This report summarizes the demographics and clinical characteristics of patients at enrollment (baseline). RESULTS: Patients (n = 2510) were a median age of 67.0 years, 54.2% were male, and 89.1% were white. The median time from PV diagnosis to study enrollment was 4.0 (range, 0-56.3) years. Most patients (89.7%) were diagnosed after an abnormal blood test. Less than half (49.2%) underwent JAK2 mutation analysis, of whom 95.8% were JAK2 V617F mutation positive; < 1% were positive for JAK2 exon 12 mutations. At enrollment, 47.7% of patients had elevated hematocrit (> 45%), 35.8% had elevated platelets (> 400 × 109/L), and 37.0% had elevated leukocytes (> 10 × 109/L). Most patients (94.5%) were receiving active PV treatment, predominantly therapeutic phlebotomy alone (33.6%), hydroxyurea monotherapy (29.0%), or hydroxyurea plus phlebotomy (23.7%). Thrombotic events occurred in 11.9% of patients before PV diagnosis (venous, 6.7%; arterial, 5.7%), and 8.3% between diagnosis and enrollment. Hypertension (70.6%) was the most common previous medical condition. CONCLUSION: REVEAL enrollment data inform our understanding of the baseline demographics, diagnostic approach, disease characteristics, and treatment patterns of patients with PV in the United States. Longitudinal real-world data collected in this study will complement information collected during randomized controlled clinical trials. Comment: An important report that looked at 2,510 patients with PV. Mainly concerned with the therapy given to the patients. --------------------------------------------------------------------------------------------------------- Tefferi A, Barbui T. 2018 New 'Polycythemia vera and essential thrombocythemia: 2019 update on diagnosis, risk-stratification and management' American Journal of Hematology. 2018 Oct 3. ABSTRACT DISEASE OVERVIEW: Polycythemia Vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) respectively characterized by erythrocytosis and thrombocytosis; other disease features include leukocytosis, splenomegaly, thrombosis, bleeding, microcirculatory symptoms, pruritus and risk of leukemic or fibrotic transformation. DIAGNOSIS: Bone marrow morphology remains the cornerstone of diagnosis. In addition, the presence of JAK2 mutation is expected in PV while approximately 90% of patients with ET express mutually exclusive JAK2, CALR or MPL mutations. In ET, it is most important to exclude the possibility of prefibrotic myelofibrosis. SURVIVAL: Median survivals are 14 years for PV and 20 years for ET; the corresponding values for younger patients are 24 and 33 years. Certain mutations (mostly spliceosome) and abnormal karyotype might compromise survival in PV and ET. Life-expectancy in ET is inferior to the control population. Driver mutations have not been shown to affect survival in ET. Risk of thrombosis is higher in JAK2 mutated ET. Leukemic transformation rates at 10 years are estimated at <1% for ET and 3% for PV. THROMBOSIS RISK: In PV, two risk categories are considered: high (age >60 years or thrombosis history present) and low (absence of both risk factors); in ET, four risk categories are considered: very low (age ?60 years, no thrombosis history, JAK2 wild-type), low (same as very low but JAK2 mutation present), intermediate (age >60 years, no thrombosis history, JAK2 wild-type) and high (thrombosis history present or age >60 years with JAK2 mutation). RISK-ADAPTED THERAPY: The main goal of therapy in both PV and ET is to prevent thrombohemorrhagic complications. All patients with PV require phlebotomy to keep hematocrit below 45% and once- or twice-daily aspirin (81 mg), in the absence of contraindications. Very low risk ET might not require therapy while aspirin therapy is advised for low risk disease. Cytoreductive therapy is recommended for high-risk ET and PV but it is not mandatory for intermediate-risk ET. First-line drug of choice for cytoreductive therapy, in both ET and PV, is hydroxyurea and second-line drugs of choice are interferon-? and busulfan. We do not recommend treatment with ruxolutinib in PV, unless in the presence of severe and protracted pruritus or marked splenomegaly that is not responding to the aforementioned drugs. Comment: Up-to-date review. --------------------------------------------------------------------------------------------------------- Hanna MZ, Kalev-Zylinska ML, Jackson SR, Royle G, Blacklock HA. 2018. New 'Distinctive features of polycythaemia vera in New Zealand Polynesians' The New Zealand medical journal. 131(1482):38-45. Abstract AIM: The aim of this study was to examine a potential ethnic disparity in the phenotype of polycythaemia vera (PV) between New Zealand European and Polynesian patients. METHOD: A retrospective review of medical records was conducted at Middlemore Hospital to identify adult patients with PV diagnosed between 1987 and 2007. Data extracted included diagnostic criteria, ethnicity, age, complications and survival. RESULTS: Eighty-eight adult patients with PV were identified during 1987-2007, 49 (55.7%) were Europeans and 36 (40.9%) Polynesians. The most striking finding was that Polynesian patients presented almost 14 years younger than Europeans (mean age of 54 years versus [vs] 68, respectively; P<.001). The white cell and platelet counts were higher in Polynesians compared with Europeans (mean white cell count of 22x109/L vs 13x109/L; mean platelet count of 648x109/L vs 512x109/L, respectively; P<.05 for both). The rate of JAK2 V617F mutation in Polynesians was 96%, equivalent to other large cohorts of European patients. The rates of long-term complications were comparable between Polynesians and Europeans, but the predicted impact on life expectancy was more severe for Polynesians. CONCLUSION: New Zealand Polynesian patients present with a distinctive PV phenotype. Their younger age at presentation suggests a different risk factor profile or a higher genetic susceptibility. We hope our observations initiate larger epidemiological and genetic studies to help elucidate the cause. Free download from: http://www.nzma.org.nz/journal/read-the-journal/all-issues/ 2010-2019/2018/vol-131-no-1482-21-september-2018/7690 Comment: A very interesting paper looking at PV in New Zealanders and Polynesians. --------------------------------------------------------------------------------------------------------- Bertozzi I, Rumi E, Cavalloni C, Cazzola M, Fabris F, Randi ML. 2018 'Pregnancy outcome and management of 25 pregnancies in women with polycythemia vera' American Journal of Hematology. 93(9):E234-E235. Free download from: https://onlinelibrary.wiley.com/doi/epdf/10.1002/ajh.25210 Comment: The risk of pregnancy. --------------------------------------------------------------------------------------------------------- Parasuraman SV, Shi N, Paranagama DC, Bonafede M. 2018. 'Health Care Costs and Thromboembolic Events in Hydroxyurea-Treated Patients with Polycythemia Vera' Journal of Managed Care & Specialty Pharmacy. 24(1):47-55. Free download from: https://www.jmcp.org/doi/10.18553/jmcp.2018.24.1.47 Comment: An interesting summary of the costs of PV. --------------------------------------------------------------------------------------------------------- Paranagama D, Colucci P, Evans KA, Bonafede M, Parasuraman S. 2018. 'Are patients with high-risk polycythemia vera receiving cytoreductive medications? A retrospective analysis of real-world data' Experimental Hematology & Oncology. 7:16. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038180/ Comment: A survey of therapy. --------------------------------------------------------------------------------------------------------- Tefferi A, Vannucchi AM, Barbui T. 2018. 'Polycythemia vera treatment algorithm 2018' Blood Cancer Journal. 8(1):3. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802495/ Comment: An important report that advises how PV should be treated. --------------------------------------------------------------------------------------------------------- Rumi E, Cazzola, M. 2017 Advances in understanding the pathogenesis of familial myeloproliferative neoplasms. British Journal of Haematology. 178(5):689-698. Comment: Even in 2017, using the term 'somatic mutations'. --------------------------------------------------------------------------------------------------------- Pacilli A, Fanelli T, Mannarelli C, Rotunno G, Pancrazzi A, Vannucchi AM, Guglielmelli P. 2017 'Clonal architecture of JAK2V617F mutated cells during treatment with ruxolitinib' Wiley online Library Free download from: https://onlinelibrary.wiley.com/doi/full/10.1002/hon.2424 Comment: The action of ruxolitinib on homozygous and heterozygous cells. --------------------------------------------------------------------------------------------------------- Hinds DA, Barnholt KE, Mesa RA, Kiefer AK, Do CB, Eriksson N, Mountain JL, Francke U, Tung JY, Nguyen HM, Zhang H, Gojenola L, Zehnder JL, Gotlib J. 2016. 'Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms' Blood. 128(8):1121-8. Comment: The full report of the large 23andMe survey. --------------------------------------------------------------------------------------------------------- Arber DA, et al. 2016. 'The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia' Blood. 19;127(20):2391-405. Comment: The 2016 classification includes PV & ET in the list of myeloproliferative neoplasms. --------------------------------------------------------------------------------------------------------- Callum, J. 2008. 'Polycythemia Vera. New diagnostic criteria' Downloaded August 2018: https://sunnybrook.ca/uploads/Polycythemia_Vera.pdf Comment: An interesting review from 2008. --------------------------------------------------------------------------------------------------------- Kelly K, et al. 2008 Blood. 112(10):4356-7. 'Congenital JAK2V617F polycythemia vera: where does the genotype-phenotype diversity end?' Blood. 112(10):4356-7. No abstract; but a free download at: http://www.bloodjournal.org/content/112/10/4356.long?sso-checked=true Comment: The researchers suggested the JAK2 V617F mutation occurred in utero. --------------------------------------------------------------------------------------------------------- Landgren O, Goldin LR, Kristinsson SY, Helgadottir EA, Samuelsson J, Björkholm M. 2008. 'Increased risks of polycythemia vera, essential thrombocythemia, and myelofibrosis among 24,577 first-degree relatives of 11,039 patients with myeloproliferative neoplasms in Sweden' Blood. 112(6):2199-204. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532797/ Abstract Previous small studies have reported familial clustering of myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We identified 6217 PV, 2838 ET, 1172 MF, and 812 MPN unclassifiable (NOS) patients diagnosed in Sweden, 43 550 controls, and first-degree relatives of cases (n = 24 577) and controls (n = 99 542). Using a marginal survival model, we calculated relative risks (RRs) and 95% confidence intervals as measures of familial aggregation. Relatives of MPN patients had significantly increased risks of PV (RR = 5.7; 3.5-9.1), ET (RR = 7.4; 3.7-14.8), and MPN NOS (RR = 7.5; 2.7-20.8). Analyses stratified by type of first-degree relative revealed consistently higher risks for siblings, compatible with a model of recessive genetic inheritance, which can be confirmed only by identifying the susceptibility gene(s). Mean age at MPN diagnosis was not different (P = .20) for affected relatives of cases (57.5 years) versus controls (60.6 years), and risk of MPN by age was not different for parents versus offspring of MPN cases (P = .10), providing no support for anticipation. Relatives of MPN patients had a borderline increased risk of chronic myeloid leukemia (CML; RR = 1.9; 0.9-3.8; P = .09). Our findings of 5- to 7-fold elevated risk of MPNs among first-degree relatives of MPN patients support the hypothesis that common, strong, shared susceptibility genes predispose to PV, ET, MF, and possibly CML. Comment: Clustering of PV and ET in Sweden. --------------------------------------------------------------------------------------------------------- Kralovics R, et al. 2003. 'Clonal hematopoiesis in familial polycythemia vera suggests the involvement of multiple mutational events in the early pathogenesis of the disease' Blood, 15;102(10):3793-6. Comment: An important paper just prior to the discovery of the JAK2 V617F mutation. --------------------------------------------------------------------------------------------------------- Spivak JL. 2002. 'Polycythemia vera: myths, mechanisms, and management' Blood. 100(13):4272-90. Comment: A good review from a few years ago. --------------------------------------------------------------------------------------------------------- Streiff MB, et al. 2002. 'The diagnosis and management of polycythemia vera in the era since the Polycythemia Vera Study Group: a survey of American Society of Hematology members' practice patterns' Blood. 99(4):1144-9. Comment: An important paper in 2002. --------------------------------------------------------------------------------------------------------- Remacha AF, Montserrat I, Santamaria A, Oliver A, Barceló MJ, Parellada M. 1997. 'Serum erythropoietin in the diagnosis of polycythemia vera. A follow-up study' Haematologica. 82(4):406-10. Free download from: http://www.haematologica.org/content/82/4/406.long Comment: An important paper at the time discussing the role of Erythropoietin. --------------------------------------------------------------------------------------------------------- Wasserman LR. 1968. 'The treatment of polycythemia. A panel discussion' Blood. 32(3):483-7. Commment: A very significant presentation given in 1968. --------------------------------------------------------------------------------------------------------- Papers dealing with the JAK2 V617F mutation --------------------------------------------------------------------------------------------------------- Maslah N, Verger E, Schlageter MH, Miclea JM, Kiladjian JJ, Giraudier S, Chomienne C, Cassinat B. 2018 New 'Next-generation sequencing for JAK2 mutation testing: advantages and pitfalls' Annals of hematology. Sep 26 Abstract The JAK2V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R2?=?0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution. Comment: A paper looking at modern JAK2 V617F testing. --------------------------------------------------------------------------------------------------------- Azevedo AP, Silva SN, Reichert A, Lima F, Júnior E, Rueff J. 2017. 'Prevalence of the Janus kinase 2 V617F mutation in Philadelphia-negative myeloproliferative neoplasms in a Portuguese population' Biomedical Reports. 7(4):370-376. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5649586/ Comment: Frequency of JAK2 V617F in Portugal. --------------------------------------------------------------------------------------------------------- Nielsen C, Bojesen SE, Nordestgaard BG, Kofoed KF, Birgens HS. 2014. 'JAK2V617F somatic mutation in the general population: myeloproliferative neoplasm development and progression rate' Haematologica. 99(9): 1448–1455. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562533/ Comment: The incidence of JAK2 V617F in Denmark was 0.1%. --------------------------------------------------------------------------------------------------------- Teofili L, Foa R, Giona F, Larocca LM. 2008. 'Childhood polycythemia vera and essential thrombocythemia: does their pathogenesis overlap with that of adult patients?' Haematologica. 93(2):169-72. Free download from: http://www.haematologica.org/content/93/2/169.long Comment: Children can be JAK2 V617F positive too. --------------------------------------------------------------------------------------------------------- Xu X, Zhang Q, Luo J, Xing S, Li Q, Krantz SB, Fu X, Zhao ZJ. 2007. 'JAK2(V617F): Prevalence in a large Chinese hospital population' Blood. 109(1):339-42. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785080/ Abstract Recently, the JAK2(V617F) mutation was found in patients with myeloproliferative disorders (MPDs), including most with polycythemia vera (PV). The mutant JAK2 has increased kinase activity, and it was shown to be pathogenic in mouse models. Herein, we analyzed blood samples randomly collected from a clinical laboratory. Surprisingly, as many as 37 samples from a total of 3935 were found positive for the JAK2 mutation. However, only one of these samples had blood test results indicative for probable PV, but several had nonhematologic diseases. On average, samples with the mutation had normal red cell counts but significantly higher white blood cell and platelet counts, although most were within the normal range. The data suggest that the JAK2(V617F) mutation is apparently much more common than MPDs. Its occurrence may be a prelude to full blood cell abnormalities and other diseases, but it cannot by itself diagnose MPDs. Comment: An interesting survey looking at JAK2 V617F in China. The frequency was also 0.1%. --------------------------------------------------------------------------------------------------------- Campbell PJ, Baxter EJ, Beer PA, Scott LM, Bench AJ, Huntly BJ, Erber WN, Kusec R, Larsen TS, Giraudier S, Le Bousse-Kerdilès MC, Griesshammer M, Reilly JT, Cheung BY, Harrison CN, Green AR. 2006 'Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation' Blood. 108(10):3548-55. Abstract The identification of an acquired mutation of JAK2 in patients with myeloproliferative disorders has raised questions about the relationship between mutation-positive and mutation-negative subtypes, timing of the JAK2 mutation, and molecular mechanisms of disease progression. Here we demonstrate that patients with V617F(-) essential thrombocythemia do not commonly progress to become V617F(+). Consistent with the concept of distinct pathogenetic mechanisms, we show that patients with and without the JAK2 mutation have different patterns of cytogenetic abnormality, with virtually all patients carrying the 20q deletion or trisomy 9 being V617F(+). We also investigated the existence of a "pre-JAK2" phase by comparing the proportion of clonally derived granulocytes, estimated from X-chromosome inactivation patterns (XCIPs), with the proportion of V617F(+) granulocytes. Our results demonstrate that inherent XCIP variability between granulocytes and T cells produces a systematically biased pattern of results that may be misinterpreted as evidence for an excess of clonally derived granulocytes, an observation that limits the utility of XCIP analysis in this context. Lastly, we studied 4 patients with V617F(+) myeloproliferative disorders who subsequently developed acute myeloid leukemia. In 3 patients the leukemic cells were V617F(-), suggesting that in these patients the leukemia arose in a V617F(-) cell. Free download from: http://www.bloodjournal.org/content/108/10/3548.long?sso-checked=true Comment: A early study of JAK2 V617F behaviour. --------------------------------------------------------------------------------------------------------- McLornan D, Percy M, McMullin MF. 2006. 'JAK2 V617F: a single mutation in the myeloproliferative group of disorders' The Ulster medical journal. 75(2):112-9. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891745/ No abstract. Comment: A good review from 2006. --------------------------------------------------------------------------------------------------------- Kaushansky K. 2005. 'On the molecular origins of the chronic myeloproliferative disorders: it all makes sense' Blood. 105:4187-4190. Comment: An optimistic outlook after the finding of the JAK2 V617F mutation. --------------------------------------------------------------------------------------------------------- Vainchenker W, Constantinescu SN. 2005. 'A unique activating mutation in JAK2 (V617F) is at the origin of polycythemia vera and allows a new classification of myeloproliferative diseases' American Society of Hematology Education Program 2005. Comment: The first of the papers from 2005 that looked at the JAK2 V617F mutation. --------------------------------------------------------------------------------------------------------- Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC. 2005 'A gain-of-function mutation of JAK2 in myeloproliferative disorders' New England Journal of Medicine, 352(17):1779-90. Comment: Another of the papers from 2005 that first linked the mutation JAK2 V617F to Polycythaemia Vera. --------------------------------------------------------------------------------------------------------- Papers dealing with Essential Thrombocythaemia --------------------------------------------------------------------------------------------------------- Masarova L, Verstovsek S. 2018. 'Therapeutic Approach to Young Patients With Low-Risk Essential Thrombocythemia: Primum Non Nocere' Journal of Clinical Oncology. JCO2018793497. Free download from: http://ascopubs.org/doi/10.1200/JCO.2018.79.3497 Abstract The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors' suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice. A 51-year-old woman was diagnosed with essential thrombocythemia (ET) the previous year (April 2016) when she was incidentally found to have increased platelets (747 × 109/L) during a yearly physical examination. Her past medical history was significant only for mild hypertension, which was well controlled with a low dose of a ?-blocker. There was no history of thromboembolic events. A JAK2V617F mutation was detected in her peripheral blood. A repeated platelet count 1 month later showed increased platelets of 871 × 109/L and she began hydroxyurea. One year later, she presented to our clinic with a white cell count of 8.9 × 109/L, hemoglobin 14 g/dL, and platelets 846 ×109/L while receiving hydroxyurea 500 mg one day alternating with 1000 mg the next day and aspirin 81 mg once per day. The differential as well as other laboratory findings were within normal limits. She had chronic mild to moderate itching, but otherwise denied symptoms referable to ET. Her physical examination was notable for the absence of palpable hepatosplenomegaly. Bone marrow aspiration and biopsy revealed normocellular marrow with hyperplastic megakaryocytes in clusters, no reticulin fibrosis, and 2% blasts, compatible with ET. Molecular testing confirmed JAK2V617F mutations at a variant allele frequency of 12% without any other mutations (81-gene panel), and her karyotype was diploid. She visited the clinic to discuss the next steps in her treatment. Comment: An interesting study of a case of ET. --------------------------------------------------------------------------------------------------------- Haunstrup LM, Ebbesen LH, Hansen M, Severinsen MT, Aggerholm A. 2018 New 'Skewed ratio between type 1 and type 2 CALR mutations in Essential Thrombocytosis patients with concomitant JAK2 V617F mutation' Experimental hematology. pii: S0301-472X(18)30848-8. Abstract Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPN). Driver mutations in JAK2, CALR and MPL, are generally considered mutually exclusive, but several reports have suggested that they co-exist in a small subgroup of patients. In this study we retrospectively analyzed for CALR mutations in 136 suspected MPN patients with low allelic burden (?5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutation were identified of which ten were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR mutated MPN cases. Type 1 is generally considered the most common mutation, but interestingly, our double mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of co-existing JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F. Comment: Frequency of JAK2 V617F and other mutations in ET. --------------------------------------------------------------------------------------------------------- Tefferi A, et al. 2018 New 'Decreased survival and increased rate of fibrotic progression in essential thrombocythemia chronicled after the FDA approval date of anagrelide' Abstract First-line cytoreductive drug of choice in high risk essential thrombocythemia (ET) is currently hydroxyurea, a practice based on the results of a randomized study; second-line drugs of choice include pegylated interferon-?, busulfan and anagrelide. Anagrelide clinical trials were pioneered by the late Murray N. Silverstein (1928-1998) of the Mayo Clinic whose studies led to FDA approval in March 1997. The current study represents a retrospective examination of the potential impact of anagrelide therapy on survival and disease complications in ET. 1,076 patients with ET were considered (median age 58 years; females 63%); risk distribution, according to the international prognostic score for ET (IPSET), was 28% high, 42% intermediate and 30% low. Overall (OS), myelofibrosis-free (MFFS) and thrombosis-free survival data were compared for ET patients diagnosed before and after the 1997 FDA approval date for anagrelide; a significant difference was apparent in OS (p=0.006; HR 1.4, 95% CI 1.1-1.7) and MFFS (p<0.001; HR 4.2, 95% CI 2.7-6.5), in favor of patients diagnosed prior to 1997; the difference was sustained during multivariable analysis that included IPSET. Similarly stratified survival data in polycythemia vera (n=665) and primary myelofibrosis (n=1,282) showed no similar impact on survival (p=0.3 and 0.17, respectively). The current study represents a retrospective analysis and suggests significantly decreased OS and MFFS in ET patients diagnosed after the FDA approval date of anagrelide. Whether or not anagrelide therapy was to blame for the worsening of OS and MFFS over time cannot be assumed and requires validation in a prospective study. American journal of hematology. 2018 Sep 25. Comment: The drug anagrelide inhibits the maturation of platelets, but does not appear to improve outcomes. --------------------------------------------------------------------------------------------------------- Godfrey AL, et al. 2018 'Hydroxycarbamide Plus Aspirin Versus Aspirin Alone in Patients With Essential Thrombocythemia Age 40 to 59 Years Without High-Risk Features' Godfrey AL, et al. 2018 Journal of clinical oncology. JCO2018788414. Abstract 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 × 109/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; P = 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 × 109/L should not receive cytoreductive therapy. Comment: Cytoreductive therapy does not appear to help in less severe cases. --------------------------------------------------------------------------------------------------------- Kander EM, Moliterno AR, Rademaker A, Streiff MB, Spivak JL, Stein BL. 2016. 'Practice Patterns in the Diagnosis and Treatment of Polycythemia Vera in the Post-JAK2 V617F Discovery Era' Journal of the National Comprehensive Cancer Network. 14(10):1238-1245. Abstract Polycythemia vera (PV) is an acquired clonal hematopoietic stem cell disorder characterized by an overproduction of red blood cells, white blood cells, and platelets; thrombotic and hemorrhagic complications; and an increased risk of transformation to myelofibrosis and acute leukemia. In 1967, the Polycythemia Vera Study Group proposed the optimal approach to diagnosis and treatment of PV, and in 2002, investigators from Johns Hopkins University School of Medicine surveyed the practice patterns of hematologists as they pertained to PV. Since this survey, the JAK2 V617F mutation was discovered, leading to a new era of discovery in the disease pathogenesis, diagnosis, and classification and treatment of PV. Our objective was to survey hematologists in the diagnosis and treatment of PV in the modern, post-JAK2 V617F discovery era. An anonymous 17-question survey was emailed to members of the Myeloproliferative Neoplasm (MPN) Research Foundation database and Aplastic Anemia and MDS International Foundation. A total of 71 surveys were used in the analysis. Diagnostic testing varied according to the respondent's clinical experience and practice type. In addition, there were marked differences in target hematocrit and platelet count among those surveyed. There continue to be variations in diagnosis and treatment of PV despite WHO guidelines and the JAK2 discovery. US-based guidelines for MPNs are needed to create consistency in the management of PV and other MPNs. Comment: A survey of treatment in 2016. --------------------------------------------------------------------------------------------------------- Langabeer SE, Haslam K, McMahon C. 2013. 'A prenatal origin of childhood essential thrombocythaemia' No abstract. British journal of haematology. 163(5):676-8. Free download from: https://onlinelibrary.wiley.com/doi/abs/10.1111/bjh.12533 Comment: The researchers suggested the JAK2 V617F mutation occurred in utero. --------------------------------------------------------------------------------------------------------- Wiemels J, et al. 2009 'Backtracking of leukemic clones to birth' Methods in molecular biology. 538:7-27. Abstract Many of the acquired genetic changes that contribute to the molecular pathogenesis of leukemia are well characterized. The relative simplicity of the tumor genetics of the common subtypes of leukemia and the availability of archived material in the form of archived neonatal blood spots (ANB or Guthrie cards) has permitted the tracing of many genetic events to fetal origins using sensitive amplification methods. We here described methods for cloning translocations and other rearrangements for "backtracking" studies, and methods for sensitive detection of such rearrangements and a point mutation in ANB cards. Comment: An earlier paper --------------------------------------------------------------------------------------------------------- Teofili L, et al. 2009 'The mutant JAK2 allele burden in children with essential thrombocythemia' British journal of haematology. 145(3):430-2. No abstract, but free download from: https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2141.2009.07591.x Comment: Details of children with ET in Italy. --------------------------------------------------------------------------------------------------------- Teofili L, Giona F, Martini M, Cenci T, Guidi F, Torti L, Palumbo G, Amendola A, Foà R, Larocca LM. 2007. 'Markers of myeloproliferative diseases in childhood polycythemia vera and essential thrombocythemia' Journal of Clinical Oncology. 25(9):1048-53. Free download from: http://ascopubs.org/doi/10.1200/JCO.2006.08.6884 Abstract PURPOSE: Polycythemia vera (PV) and essential thrombocythemia (ET) can present in pediatric age as sporadic or familial diseases. To define the biologic profile of childhood PV and ET, we evaluated specific markers in a cohort of pediatric patients affected by PV and ET, including cases with familial occurrence. PATIENTS AND METHODS: Thirty-eight children with PV and ET were investigated. The control group included 58 adults with PV and ET. Endogenous erythroid colonies, qualitative reverse transcriptase polymerase chain reaction for polycythemia rubra vera-1 (PRV-1) RNA expression, human androgen receptor assay and allele specific polymerase chain reaction for JAK2 V617F mutation were undertaken in all patients. Thrombopoietin, thrombopoietin receptor (c-mpl), and erythropoietin receptor mutation analysis was performed by direct sequencing in familial cases. RESULTS: The JAK2 V617F mutation in children with PV was significantly less frequent than in adult PV. The most common myeloproliferative marker found in these patients was PRV-1 RNA overexpression. Children and adults with sporadic ET showed a similar proportion of patients with PRV-1 RNA overexpression, JAK2 V617F mutation, and clonality, while none of the familial ET showed JAK2 V617F mutation and clonality. Also, PRV-1 RNA overexpression was significantly less common. Furthermore, most patients with familial ET exhibited the dominant-positive activating mutation of c-mpl. Finally, children with PV and ET had a significant lower incidence of thrombosis than adults. CONCLUSION: This study demonstrates that familial and sporadic ET recognize different pathogenetic mechanisms. Myeloproliferative markers are specific tests for the diagnosis of ET in children with sporadic forms, while a significant proportion of children with PV can prove negative. Comment: JAK2 V617F in childhood PV & ET --------------------------------------------------------------------------------------------------------- Papers dealing with the Risk of thrombosis --------------------------------------------------------------------------------------------------------- Podoltsev NA, Zhu M, Zeidan AM, Wang R, Wang X, Davidoff AJ, Huntington SF, Giri S, Gore SD, Ma X. 2018. 'The impact of phlebotomy and hydroxyurea on survival and risk of thrombosis among older patients with polycythemia vera' Blood Advances. 2018 Oct 23;2(20):2681-2690. doi: 10.1182/bloodadvances.2018021436. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199652/ Abstract Current guidelines recommend therapeutic phlebotomy for all polycythemia vera (PV) patients and additional cytoreductive therapy (eg, hydroxyurea [HU]) for high-risk PV patients. Little is known about the impact of these therapies in the real-world setting. We conducted a retrospective cohort study of older adults diagnosed with PV from 2007 to 2013 using the linked Surveillance, Epidemiology, and End Results-Medicare database. Multivariable Cox proportional hazards models were used to assess the effect of phlebotomy and HU on overall survival (OS) and the occurrence of thrombotic events. Of 820 PV patients (median age = 77 years), 16.3% received neither phlebotomy nor HU, 23.0% were managed with phlebotomy only, 19.6% with HU only, and 41.1% with both treatments. After a median follow-up of 2.83 years, 37.2% (n = 305) of the patients died. Phlebotomy (yes/no; hazard ratio [HR] = 0.65; 95% confidence interval [CI], 0.51-0.81; P < .01), increasing phlebotomy intensity (HR = 0.71; 95% CI, 0.65-0.79; P < .01), and a higher proportion of days covered (PDC) by HU were all significantly associated with lower mortality. When thrombosis was the outcome of interest, phlebotomy (yes/no; HR = 0.52; 95% CI, 0.42-0.66; P < .01) and increasing phlebotomy intensity (HR = 0.46; 95% CI, 0.29-0.74; P < .01) were significantly associated with a lower risk of thrombotic events, so was a higher HU PDC. In this population-based study of older adults with PV reflecting contemporary clinical practice, phlebotomy and HU were associated with improved OS and decreased risk of thrombosis. However, both treatment modalities were underused in this cohort of older PV patients. Comment: A recent review of therapy. --------------------------------------------------------------------------------------------------------- Horvat I, Boban A, Zadro R, Antolic MR, Serventi-Seiwerth R, Roncevic P, Radman I, Sertic D, Vodanovic M, Pulanic D, Basic-Kinda S, Durakovic N, Zupancic-Salek S, Vrhovac R, Aurer I, Nemet D, Labar B. 2018. New 'Influence of Blood Count, Cardiovascular Risks, Inherited Thrombophilia, and JAK2 V617F Burden Allele on Type of Thrombosis in Patients With Philadelphia Chromosome Negative Myeloproliferative Neoplasms' Clinical Lymphoma Myeloma & Leukemia. 2018 Sep 10. pii: S2152-2650(18)31049-8. Abstract INTRODUCTION: Thrombosis is the most common complication in Philadelphia chromosome negative (Ph-) myeloproliferative neoplasms patients. PATIENTS AND METHODS: In a cohort of 258 Ph- myeloproliferative neoplasm patients, the difference between patients with and without thrombosis was analyzed according to genetic thrombophilia factors, JAK2 V617F status and burden allele, blood count, cardiovascular risk factors and age. Patients were also divided in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) subgroups as well as by the type of thrombosis. RESULTS: Analysis of cardiovascular risk factors regarding arterial thrombosis showed that PV patients with thrombosis had higher incidence of diabetes (P = .030), ET patients more often had hypertension (P = .003) and hyperlipidemia (P = .005), while PMF patients had hyperlipidemia (P = .046) and at least one cardiovascular risk factor (P = .044). Moreover, leukocytes > 18 × 109/L and V617F burden allele > 25.7% were statistically significantly different in PV patients (P = .019 and borderline significant at P = .055, respectively), while in ET patients leukocytes > 9.2 × 109/L (P < .001) and age at diagnosis of > 55 years were statistically significantly different (P = .002). PMF patients with V617F burden allele ? 34.8% were more prone to thrombosis (P = .032). When comparing patients with and without venous thrombosis, cutoff value of V617F burden allele > 90.4% was significant for PV patients with thrombosis (P = .036), as was > 56.7% for PMF patients with thrombosis (P = .046). Platelets ? 536 × 109/L and age at diagnosis > 54 years showed statistically significant difference for ET patients with thrombosis (P = .015 and P = .041, respectively). CONCLUSION: On the basis of our results, a new scoring system for thrombosis risk in PV could be made, while PMF prognostic model may be expanded for better recognition of potential thrombotic risk factors. Comment: Again a paper stressing the increased risk of thrombosis associated with the JAK2 V617F mutation --------------------------------------------------------------------------------------------------------- Rao R, Grosel J. 2018 'Acute portal vein thrombosis in a 59-year-old male with JAK2 V617F mutation' Radiology Case Reports 13 Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148830/ Comment: Thrombosis risk from the JAK2 V617F mutation. --------------------------------------------------------------------------------------------------------- Cerquozzi S, Barraco D, Lasho T, Finke C, Hanson CA, Ketterling RP, Pardanani A, Gangat N, Tefferi A. 2017. 'Risk factors for arterial versus venous thrombosis in polycythemia vera: a single center experience in 587 patients' Blood Cancer Journal. 7(12):662. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802551/ Comment: A recent review. ---------------------------------- Tevet M, Ionescu R, Dragan C, Lupu AR. 2015 'Influence of the JAK2 V617F Mutation and Inherited Thrombophilia on the Thrombotic Risk among Patients with Myeloproliferative Disorders' Maedica 10(1):27-32. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496761/ Comment: Other inherited factors affect the incidence of thromboses --------------------------------------------------------------------------------------------------------- Kroll MH, Michaelis LC, Verstovsek S. 2015. 'Mechanisms of thrombogenesis in polycythemia vera' Blood Reviews. 29(4):215-21. Comment: Thromboses in PV are a major problem. --------------------------------------------------------------------------------------------------------- Elliott MA. Tefferi A. 2005. 'Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia' British journal of haematology 128(3):275-90. Free download from: http://williams.medicine.wisc.edu/mpdhemostasis.pdf Commment: Detailed review from 2005. --------------------------------------------------------------------------------------------------------- Papers dealing with Maternal Age Effect --------------------------------------------------------------------------------------------------------- Greaves M, et al. 2018. 'Cancer cell transmission via the placenta' Evolution, medicine, and public health. 2018(1):106-115. Comment: A paper looking at maternal-foetal tranfer. --------------------------------------------------------------------------------------------------------- Kazemi M, et al. 2017. 'MeDIP Real-Time qPCR has the Potential for Noninvasive Prenatal Screening of Fetal Trisomy 21' International journal of molecular and cellular medicine. 6(1):13-21. Comment: A paper looking at screening for Trisomy 21 (Downs syndrome). --------------------------------------------------------------------------------------------------------- Langabeer SE, Haslam K, McMahon C. 2013. A prenatal origin of childhood essential thrombocythaemia. British Journal of Haematology. 163(5):676-8. Comment: Looked at the possiblitiy of maternal inheritance, but decided the mutation occurred in utero. --------------------------------------------------------------------------------------------------------- Lo YM, et al. 1998. 'Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis' American Journal of Human Genetics. 62(4):768-75. Comment: An early paper on maternal markers. --------------------------------------------------------------------------------------------------------- Papers dealing with Cancer --------------------------------------------------------------------------------------------------------- Thorsson V, et al. 2018. 'The Immune Landscape of Cancer' Immunity. 48(4):812-830 Comment: An important paper looking at the genetics of cancer. --------------------------------------------------------------------------------------------------------- Kent DG, et al. 2017. 'Order Matters: The Order of Somatic Mutations Influences Cancer Evolution' Cold Spring Harbor perspectives in medicine. 7(4). pii: a027060. Comment: A paper looking at Driver-Mutation. --------------------------------------------------------------------------------------------------------- Arber DA, et al. 2016. 'The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia' Blood. 19;127(20):2391-405. Comment: The latest classification myeloid neoplasms; in which they include Polycythaemia and Essential Thombocythaemia. --------------------------------------------------------------------------------------------------------- Tomczak K, et al. 2015. 'The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge' Contemporary Oncolology. 19(1A):A68-77. Comment: An important survey from 2015. --------------------------------------------------------------------------------------------------------- de Boer RJ, et al. 1987. 'Immunological discrimination between self and non-self by precursor depletion and memory accumulation' Journal of theoretical biology, 7;124(3):343-69. Comment: Another famous paper that introduced the idea of 'self and non-self'. --------------------------------------------------------------------------------------------------------- Billingham RE, 1953. 'Actively acquired tolerance of foreign cells' Nature. 172:603–606. Comment: A famous paper that started the field transplant genetics. --------------------------------------------------------------------------------------------------------- Papers dealing with Transmissible cancer --------------------------------------------------------------------------------------------------------- Frampton D, et al. 2018. 'Molecular Signatures of Regression of the Canine Transmissible Venereal Tumor' Cancer Cell, 33, 620–633. Comment: A recent paper looking at transmissible cancers. --------------------------------------------------------------------------------------------------------- Pye RJ, Woods GM, Kreiss A. 2016. 'Devil Facial Tumor Disease' Veterinary Pathology, 53(4):726-36. Comment: Cancer in the Tasmanian Devil. --------------------------------------------------------------------------------------------------------- Murchison EP, Wedge DC, Alexandrov LB, Fu B, Martincorena I, Ning Z, Tubio JMC, Werner EI, Allen J, De Nardi AB, Donelan EM, Marino G, Fassati A, Campbell PJ, Yang F, Burt A, Weiss RA, Stratton MR. 2014. 'Transmissible dog cancer genome reveals the origin and history of an ancient cell lineage' Science, 24;343(6169):437-440. Freedwonload from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918581/ Comment: A very detailed report looking at transmissible cancer. --------------------------------------------------------------------------------------------------------- Ujvari B, Pearse AM, Peck S, Harmsen C, Taylor R, Pyecroft S, Madsen T, Papenfuss AT, Belov K. 2013. 'Evolution of a contagious cancer: epigenetic variation in Devil Facial Tumour Disease' Proceedings of the Royal Society. Biological sciences, 280(1750):20121720. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574417/ Comment: A paper looking at transmissible cancer in the Tasmanian Devil. --------------------------------------------------------------------------------------------------------- Murchison EP. 2008. 'Clonally transmissible cancers in dogs and Tasmanian devils' Oncogene, 27 Suppl 2:S19-30. Free download from: https://www.nature.com/articles/onc2009350 Comment: A detailed look at transmissible cancer in animals. --------------------------------------------------------------------------------------------------------- Papers dealing with Clones --------------------------------------------------------------------------------------------------------- Kaushansky K, Zhan H. 2018. 'The regulation of normal and neoplastic hematopoiesis is dependent on microenvironmental cells' Advances in biological regulation. 69:11-15. Abstract Each day the adult human produces 4x1011 red blood cells, 1x1011 white blood cells and 1x1011 platelets, levels of production which can increase 10-20 fold in times of heightened demand. Hematopoiesis, or the formation of the ten different types of blood and marrow cells, is a complex process involving hematopoietic stem cells (HSCs), cytokine growth factors and cell surface adhesion molecules, and both specific and ubiquitous transcription factors. The marrow micro-environmental niche is defined as the site at which HSCs reside and are nurtured, receiving the signals that lead to their survival, replication and/or differentiation. Using microscopic, biochemical and molecular methods many different cells and the signals responsible for niche function have been identified. Early studies suggested two distinct anatomical sites for the niche, perivascular and periosteal, but the preponderance of evidence now favors the former. Within the "vascular niche" much evidence exists for important contributions by vascular endothelial cells (ECs), CXCL12-abundant reticular (CAR) cells and mesenchymal stromal cells, through their elaboration of chemokines, cytokines and cell surface adhesion molecules. In a series of studies we have found, and will present the evidence that megakaryocytes (MKs), the precursors of blood platelets, must be added to this list. In addition to normal blood cell development, numerous studies have implicated the perivascular niche as contributing to the pathogenesis of a variety of hematological malignancies. Our laboratory focuses on the Ph (Crane et al., 2017) -negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). These diseases are characterized by clonal expansion of HSCs and one or more mature blood cell types, hypermetabolism, a propensity to disorders of hemostasis (thrombosis > bleeding) and in some, evolution to acute leukemia. While a variety of therapies can control the abnormal expansion of the progeny of the malignant HSC, the only curative therapy is myeloablation with conditioning therapy or immunological means, followed by allogeneic stem cell transplantation (SCT), a procedure that is often inadequate due to relapse of the malignant clone. While the three disorders were postulated by Dameshek in the 1950s to be related to one another, proof came in 2005 when an acquired mutation in the signaling kinase Janus kinase 2 (Jak2V617F) was identified in virtually all patients with PV, and ?50% of patients with ET and PMF. Since that time a number of other mutations have been identified that account for the "Jak2V617F negative" MPNs, including the thrombopoietin receptor, c-MPL, other mutations of Jak2, calreticulin and a variety of epigenetic modifier genes (e.g. TET2). Using a cell-specific Cre recombinase and SCT techniques we can introduce Jak2V617F into murine megakaryocytes and platelets, hematopoietic stem cells, and endothelial cells, alone or in combination, in order to probe the role of the mutant kinase in various cells on several aspects of the MPNs. Using these tools we have found that the expression of Jak2V617F in HSCs and ECs drives a MPN characterized by neutrophilia, thrombocytosis and splenomegaly, eventually evolving into myelosclerosis. Somewhat surprisingly, we found that Jak2V617F-bearing ECs were required for many features of the MPN, such as enhancing the growth of Jak2V617F-bearing HSCs over that of wild type HSCs, its characteristic radioresistance, and a hemostatic defect. Altogether, our studies suggest that the malignant vascular niche is a critical element in the pathogenesis of MPNs, and a more thorough understanding of the molecular basis for these findings could lead to improved treatment for patients with these disorders. Comment: Details of clone production. --------------------------------------------------------------------------------------------------------- Dzierzak E, Bigas A. 2018 New 'Blood Development: Hematopoietic Stem Cell Dependence and Independence' Cell Stem Cell. 22(5):639-651 Free downlaod from: https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30177-2?_returnURL= https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1934590918301772%3Fshowall%3Dtrue Abstract Evidence of the diversity and multi-layered organization of the hematopoietic system is leading to new insights that may inform ex vivo production of blood cells. Interestingly, not all long-lived hematopoietic cells derive from hematopoietic stem cells (HSCs). Here we review the current knowledge on HSC-dependent cell lineages and HSC-independent tissue-resident hematopoietic cells and how they arise during embryonic development. Classical embryological and genetic experiments, cell fate tracing data, single-cell imaging, and transcriptomics studies provide information on the molecular/cell trajectories that form the complete hematopoietic system. We also discuss the current developmentally informed efforts toward generating engraftable and multilineage blood cells. Comment: A review that makes interesting reading. --------------------------------------------------------------------------------------------------------- Lee-Six H, Obro NF, Shepherd MS, Grossmann S, Dawson K, Belmonte M, Osborne RJ, Huntly BJP, Martincorena I, Anderson E, O'Neill L, Stratton MR, Laurenti E, Green AR, Kent DG, Campbell PJ. 2018. 'Population dynamics of normal human blood inferred from somatic mutations' Nature. 561(7724):473-478. Abstract Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans. Comment: Estimating the number of stem cells. --------------------------------------------------------------------------------------------------------- Henninger J, Santoso B, Hans S, Durand E, Moore J, Mosimann C, Brand M, Traver D, Zon L. 2017. 'Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development' Nature Cell Biology. 19(1):17-27. Abstract Haematopoietic stem cells (HSCs) arise in the developing aorta during embryogenesis. The number of HSC clones born has been estimated through transplantation, but experimental approaches to assess the absolute number of forming HSCs in a native setting have remained challenging. Here, we applied single-cell and clonal analysis of HSCs in zebrafish to quantify developing HSCs. Targeting creERT2 in developing cd41:eGFP+ HSCs enabled long-term assessment of their blood contribution. We also applied the Brainbow-based multicolour Zebrabow system with drl:creERT2 that is active in early haematopoiesis to induce heritable colour barcoding unique to each HSC and its progeny. Our findings reveal that approximately 21 HSC clones exist prior to HSC emergence and 30 clones are present during peak production from aortic endothelium. Our methods further reveal that stress haematopoiesis, including sublethal irradiation and transplantation, reduces clonal diversity. Our findings provide quantitative insights into the early clonal events that regulate haematopoietic development. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535785/ Comment: There are relatively few clones in early embryogenesis. --------------------------------------------------------------------------------------------------------- Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, Klein A, Hofmann O, Camargo FD. 2014 'Clonal dynamics of native haematopoiesis' Nature. 514(7522):322-7. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408613/ Abstract It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease. Comment: A discussion of how haematopoetic stem cells work. --------------------------------------------------------------------------------------------------------- Saliba J, Hamidi S, Lenglet G, Langlois T, Yin J, Cabagnols X, Secardin L, Legrand C, Galy A, Opolon P, Benyahia B, Solary E, Bernard OA, Chen L, Debili N, Raslova H, Norol F, Vainchenker W, Plo I, Di Stefano A. 2013. 'Heterozygous and homozygous JAK2(V617F) states modeled by induced pluripotent stem cells from myeloproliferative neoplasm patients' PLoS One. 8(9):e74257. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774801/ Comment: Looking at homozygous and heterozygous clones. --------------------------------------------------------------------------------------------------------- Catlin SN, Busque L, Gale RE, Guttorp P, Abkowitz JL. 2011 'The replication rate of human hematopoietic stem cells in vivo' Blood. 117(17):4460-6. Abstract Hematopoietic stem cells (HSCs) replicate (self-renew) to create 2 daughter cells with capabilities equivalent to their parent, as well as differentiate, and thus can both maintain and restore blood cell production. Cell labeling with division-sensitive markers and competitive transplantation studies have been used to estimate the replication rate of murine HSCs in vivo. However, these methods are not feasible in humans and surrogate assays are required. In this report, we analyze the changing ratio with age of maternal/paternal X-chromosome phenotypes in blood cells from females and infer that human HSCs replicate on average once every 40 weeks (range, 25-50 weeks). We then confirm this estimate with 2 independent approaches, use the estimate to simulate human hematopoiesis, and show that the simulations accurately reproduce marrow transplantation data. Our simulations also provide evidence that the number of human HSCs increases from birth until adolescence and then plateaus, and that the ratio of contributing to quiescent HSCs in humans significantly differs from mouse. In addition, they suggest that human marrow failure, such as the marrow failure that occurs after umbilical cord blood transplantation and with aplastic anemia, results from insufficient numbers of early progenitor cells, and not the absence of HSCs. Free download from; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099568/ Comment: Estimating clone number. --------------------------------------------------------------------------------------------------------- Zovein AC, Hofmann JJ, Lynch M, French WJ, Turlo KA, Yang Y, Becker MS, Zanetta L, Dejana E, Gasson JC, Tallquist MD, Iruela-Arispe ML. 2008. 'Fate tracing reveals the endothelial origin of hematopoietic stem cells' Cell Stem Cell. 3(6):625-36. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631552/ Abstract Hematopoietic stem cells (HSCs) originate within the aortic-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, and are capable of expansion, self-renewal, and multilineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs. Comment: Earlier work on tracing clones. --------------------------------------------------------------------------------------------------------- Palis J, Segel GB. 1998. 'Developmental biology of erythropoiesis' Blood Reviews. 12(2):106-14. Abstract A newborn infant represents the culmination of developmental events from conception through organogenesis. Red cells are critically important for survival and growth of the embryo. During development, erythropoiesis occurs in two distinct forms. The first 'primitive' form consists of nucleated erythroblasts that differentiate within the blood vessels of the extraembryonic yolk sac. The second 'definitive' form consists of anucleate erythrocytes that differentiate within the liver and third trimester bone marrow of the fetus. While adult bone marrow and cord blood now serve as sources of stem cells for the treatment by transplantation of genetic and malignant diseases, the developmental origin of hematopoietic stem cells has not been determined. During the third trimester the fetus grows rapidly and the production of red cells is approximately 3-5 times that of adult steady state levels. Birth brings dramatic changes in oxygenation and erythropoietin production that result in a tenfold drop in red cell production and in a transient 'physiologic' anemia. Other causes of fetal and infant anemias have their origins in development processes. These include globin gene switching in alpha and beta thalassemia, the expression of red cell antigens in alloimmune hemolytic disease, and the poorly understood defects in the regulation of erythropoiesis in Diamond Blackfan anemia. Even in the adult, vestiges of fetal erythropoiesis are evident during transient states of accelerated erythroid expansion. A better understanding of the development of erythropoiesis will bring improvements in the treatment of anemia, not only in the newborn, but also in the fetus and the adult. Comment: Early description of red cell production. --------------------------------------------------------------------------------------------------------- Papers dealing with Animal models --------------------------------------------------------------------------------------------------------- Edelmann B, Gupta N, Schnoeder TM, Oelschlegel AM, Shahzad K, Goldschmidt J, Philipsen L, Weinert S, Ghosh A, Saalfeld FC, Nimmagadda SC, Müller P, Braun-Dullaeus R, Mohr J, Wolleschak D, Kliche S, Amthauer H, Heidel FH, Schraven B, Isermann B, Müller AJ, Fischer T. 2018. New 'JAK2-V617F promotes venous thrombosis through ?1/?2 integrin activation' The Journal of clinical investigation. 128(10):4359-4371. Free download from: https://www.jci.org/articles/view/90312/pdf Comment: A mouse study looking at venous thrombosis. --------------------------------------------------------------------------------------------------------- Dunbar A, Nazir A, Levine R. 2017. 'Overview of Transgenic Mouse Models of Myeloproliferative Neoplasms (MPNs)' Current protocols in pharmacology. 77:14.40.1-14.40.19. Abstract Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients. Since that time, murine models of JAK2V617F have greatly enhanced the understanding of the role of aberrant JAK-STAT signaling in MPN pathogenesis and have provided an in vivo pre-clinical platform that can be used to develop novel therapies. From early retroviral transduction models to transgenics, and ultimately conditional knock-ins, murine models have established that JAK2V617F alone can induce an MPN-like syndrome in vivo. However, additional mutations co-occur with JAK2V617F in MPNs, often in proteins involved in epigenetic regulation that can dramatically influence disease outcomes. In vivo modeling of these mutations in the context of JAK2V617F has provided additional insights into the role of epigenetic dysregulation in augmenting MPN hematopoiesis. In this overview, early murine model development of JAK2V617F is described, with an analysis of its effects on the hematopoietic stem/progenitor cell niche and interactions with downstream signaling elements. This is followed by a description of more recent in vivo models developed for evaluating the effect of concomitant mutations in epigenetic modifiers on MPN maintenance and progression. Mouse models of other driver mutations in MPNs, including primarily calreticulin (CALR) and Tpo-receptor (MPL), which occur in a significant percentage of MPN patients with wild-type JAK2, are also briefly reviewed. Comment: Recent review of available mouse models. --------------------------------------------------------------------------------------------------------- Mullally A, Lane SW, Ball B, Megerdichian C, Okabe R, Al-Shahrour F, Paktinat M, Haydu JE, Housman E, Lord AM, Wernig G, Kharas MG, Mercher T, Kutok JL, Gilliland DG, Ebert BL. 2010. 'Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells' Cancer Cell. 15;17(6):584-96. Free download from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909585/ Abstract We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN. Comment: Producing mice with JAK2 V617F in their cells. ---------------------------------------------------------------------------------------------------------