Combination approach to diagnosis and treatment of an elderly patient with chronic Ph-negative myeloproliferative neoplasm and concomitant surgical pathology. Clinical observation

Chronic myeloproliferative neoplasms (CMPN), Ph-negative, are of clonal nature, develop on the level of hematopoietic stem cell and are characterized by proliferation of one or more hematopoietic pathways. Currently, the group of Ph-negative CMPN includes essential thrombocythemia, primary myelofibrosis, polycythemia vera, myeloproliferative neoplasm unclassifiable.Identification of mutations in the Jak2 (V617F), CALR, and MPL genes extended understanding of biological features of Ph-negative CMPN and improved differential diagnosis of myeloid neoplasms. Nonetheless, clinical practice still encounters difficulties in clear separation between such disorders as primary myelofibrosis, early-stage and transformation of essential thrombocythemia into myelofibrosis with high thrombocytosis. Thrombocytosis is one of the main risk factors for thromboembolic complications, especially in elderly people.A clinical case of an elderly patient with fracture of the left femur developed in the context of Ph-negative CMPN (myelofibrosis) with high level of thrombocytosis is presented which in combination with enforced long-term immobilization and presence of additional risk created danger of thrombosis and hemorrhage during surgery and in the postoperative period.

Platelet Transcriptome Yields Progressive Markers in Chronic Myeloproliferative Neoplasms and Identifies Putative Targets of Therapy

Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs), as a model, we identify the blood platelet transcriptome as a proxy strategy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine learning algorithms. Using RNA sequencing (RNA-seq), we derive disease-relevant gene expression in purified platelets from 120 peripheral blood samples constituting two time-separated cohorts of patients diagnosed with one of three MPN subtypes at sample acquisition - essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), and healthy donors (n=21). The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy and discriminates each clinical phenotype. Differential markers in each of ET, PV and MF also highlight candidate genes as potential mediators of the pro-thrombotic and pro-fibrotic phenotypes in MPNs. In ET and PV, a strong thromboinflammatory profile is revealed by the upregulation of several interferon inducible transmembrane genes (IFITM2, IFITM3, IFITM10, IFIT3, IFI6, IFI27L1, IFI27L2), interleukin receptor accessory kinases/proteins (IRAK1, IL15, IL1RAP, IL17RC) and several solute carrier family genes (SLC16A1, SLC25A1, SLC26A8, SLC2A9) as glucose and other metabolic transport proteins, and coagulation factor V (F5). In MF, fibrosis-specific markers were identified by an additional focused comparison of MF patients versus ET and PV, showing increased expression of several pro-fibrotic growth factors (FGFR1, FGFR3, FGFRL1), matrix metalloproteinases (MMP8, MMP14), vascular endothelial growth factor A (VEGFA), insulin growth factor binding protein (IGFBP7), and cell cycle regulators (CCND1, CCNA2, CCNB2, CCNF). Also, focusing on the JAK-inhibitor ruxolitinib/RUX-specific signatures, we not only confirm previous observations on its anti-inflammatory and immunosuppressive effects (e.g. downregulation in our RUX-treated cohort of IL1RAP, CXCR5, CPNE3, ILF3) but also identify new gene clusters responsive to RUX - e.g. inhibition of type I interferon (e.g. IFIT1, IFIT2, IFI6), chromatin regulation (HIST2H3A/C, HIST1H2BK, H2AFY, SMARCA4, SMARCC2), epigenetic methylation in mitochondrial genes (ATP6, ATP8, ND1-6 and NDUFA5), and other proliferation, and proteostasis-associated markers as putative targets for MPN-directed therapy. Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with a persistent integrated stress response. Preliminary ex vivo data on MPN patient bone-marrow-derived CD34+ cells and cultured megakaryocytes validate a proteostasis-focused subset of our peripheral platelet RNA-seq signatures. Further leveraging this substantive dataset, and in particular a progressive expression gradient across MPN, we develop a machine learning model (Lasso-penalized regression) predictive of the advanced subtype MF at high accuracy and under two conditions of validation: i) temporal Stanford internal (AUC-ROC of 0.96) and ii) geographic external cohorts (AUC-ROC of 0.97, using independently published data of an additional n=25 MF and n=46 healthy donors). Lasso-derived signatures offer a robust core set of < 5 MPN progression markers. Together, our platelet transcriptome snapshot of chronic MPNs demonstrates a methodological avenue for disease risk stratification and progression beyond genetic data alone, with potential utility in a wide range of age-related disorders. Part of the work contributing to this abstract has been posted as a preprint at this link: https://www.biorxiv.org/content/10.1101/2021.03.12.435190v2 Figure 1 Figure 1. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; Allakos: Consultancy.

Genetic Landscape of Myeloproliferative Neoplasms with an Emphasis on Molecular Diagnostic Laboratory Testing

Chronic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell neoplasms with driver events including the BCR-ABL1 translocation leading to a diagnosis of chronic myeloid leukemia (CML), or somatic mutations in JAK2, CALR, or MPL resulting in Philadelphia-chromosome-negative MPNs with constitutive activation of the JAK-STAT signaling pathway. In the Philadelphia-chromosome-negative MPNs, modern sequencing panels have identified a vast molecular landscape including additional mutations in genes involved in splicing, signal transduction, DNA methylation, and chromatin modification such as ASXL1, SF3B1, SRSF2, and U2AF1. These additional mutations often influence prognosis in MPNs and therefore are increasingly important for risk stratification. This review focuses on the molecular alterations within the WHO classification of MPNs and laboratory testing used for diagnosis.

Immune Phenomena in Myeloid Neoplasms: An “Egg or Chicken” Question

Immune phenomena are increasingly reported in myeloid neoplasms, and include autoimmune cytopenias/diseases and immunodeficiency, either preceding or complicating acute myeloid leukemia, myelodysplastic syndromes (MDS), chronic myeloproliferative neoplasms, and bone marrow failure (BMF) syndromes. Autoimmunity and immunodeficiency are the two faces of a dysregulated immune tolerance and surveillance and may result, along with contributing environmental and genetic factors, in an increased incidence of both tumors and infections. The latter may fuel both autoimmunity and immune activation, triggering a vicious circle among infections, tumors and autoimmune phenomena. Additionally, alterations of the microbiota and of mesenchymal stem cells (MSCs) pinpoint to the importance of a permissive or hostile microenvironment for tumor growth. Finally, several therapies of myeloid neoplasms are aimed at increasing host immunity against the tumor, but at the price of increased autoimmune phenomena. In this review we will examine the epidemiological association of myeloid neoplasms with autoimmune diseases and immunodeficiencies, and the pivotal role of autoimmunity in the pathogenesis of MDS and BMF syndromes, including the paroxysmal nocturnal hemoglobinuria conundrum. Furthermore, we will briefly examine autoimmune complications following therapy of myeloid neoplasms, as well as the role of MSCs and microbiota in these settings.

Philadelphia-Negative Chronic Myeloproliferative Neoplasms during the COVID-19 Pandemic: Challenges and Future Scenarios

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) started in December 2019 in China and then become pandemic in February 2020. Several publications investigated the possible increased rate of COVID-19 infection in hematological malignancies. Based on the published data, strategies for the management of chronic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are provided. The risk of severe COVID-19 seems high in MPN, particularly in patients with essential thrombocythemia, but not negligible in myelofibrosis. MPN patients are at high risk of both thrombotic and hemorrhagic complications and this must be accounted in the case of COVID-19 deciding on a case-by-case basis. There are currently no data to suggest that hydroxyurea or interferon may influence the risk or severity of COVID-19 infection. Conversely, while the immunosuppressive activity of ruxolitinib might pose increased risk of infection, its abrupt discontinuation during COVID-19 syndrome is associated with worse outcome. All MPN patients should receive vaccine against COVID-19; reassuring data are available on efficacy of mRNA vaccines in MPNs.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Progressive and predictive markers of disease evolution: platelet transcriptome in chronic myeloproliferative neoplasms

Predicting disease natural history remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs), as a model, we identify the blood platelet transcriptome as a generalizable strategy for highly sensitive progression biomarkers that also enable prediction via machine learning algorithms. Using RNA sequencing (RNA seq), we derive disease relevant gene expression and alternative splicing in purified platelets from 120 peripheral blood samples constituting two independently collected and mutually validating patient cohorts of the three MPN subtypes: essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), as well as healthy donors (n=21). The MPN platelet transcriptome discriminates each clinical phenotype and reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy. Leveraging this dataset, in particular the progressive expression gradient noted across MPN, we develop a machine learning model (Lasso-penalized regression) predictive of the advanced subtype MF at high accuracy (AUC-ROC of 0.95-0.96) with validation under two conditions: i) temporal, with training on the first cohort (n=71) and independent testing on the second (n=49) and ii) 10 fold cross validation on the entire dataset. Lasso-derived signatures offer a robust core set of < 10 MPN progression markers. Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with persistent integrated stress response. We also identify JAK inhibitor-specific signatures and other interferon, proliferation, and proteostasis associated markers as putative targets for MPN-directed therapy. Our platelet transcriptome snapshot of chronic MPNs establishes a methodological foundation for deciphering disease risk stratification and progression beyond genetic data alone, thus presenting a promising avenue toward potential utility in a wide range of age-related disorders.