Evolution Of Myeloproliferative Neoplasms Research Articles

Superior detection of low-allele burden Janus kinase 2 V617F mutation and monitoring clonal evolution in myeloproliferative neoplasms using chip-based digital PCR

The JAK2 V617F is a prevalent driver mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph−MPNs), significantly affecting disease progression, immunophenotype, and patient outcomes. The World Health Organization (WHO) guidelines highlight the JAK2 V617F mutation as one of the key diagnostic criterions for Ph−MPNs. In this study, we analyzed 283 MPN samples with the JAK2 V617F mutation to assess the effectiveness of three detection technologies: chip-based digital PCR (cdPCR), real-time quantitative PCR (qPCR), and next-generation sequencing (NGS). Additionally, we investigated the relationship between JAK2 V617F mutant allele burden (% JAK2 V617F) and various laboratory characteristics to elucidate potential implications in MPN diagnosis. Our findings demonstrated high conformance of cdPCR with qPCR/NGS for detecting % JAK2 V617F, but the mutant allele burdens detected by qPCR/NGS were lower than those detected by cdPCR. Moreover, the cdPCR exhibited high sensitivity with a limit of detection (LoD) of 0.08% and a limit of quantification (LoQ) of 0.2% for detecting % JAK2 V617F in MPNs. Clinical implications were explored by correlating % JAK2 V617F with various laboratory characteristics in MPN patients, revealing significant associations with white blood cell counts, lactate dehydrogenase levels, and particularly β2-microglobulin (β2-MG) levels. Finally, a case report illustrated the application of cdPCR in detecting low-allele burdens in a de novo chronic myeloid leukemia (CML) patient with a hidden JAK2 V617F subclone, which expanded during tyrosine kinase inhibitor (TKI) treatment. Our findings underscore the superior sensitivity and accuracy of cdPCR, making it a valuable tool for early diagnosis and monitoring clonal evolution.

Clonal hematopoiesis and its evolution of myeloproliferative neoplasms

The mutations of myeloproliferative neoplasma (MPN) mainly include driver mutations and non-driver mutations. The driver mutations mainly include JAK2 mutations, CALR mutations and MPL mutations and non-driver mutations mainly include ASXL1, DNMT3A, TET2, SF3B1, EZH2, TP53, SRSF2, USAF1, etc. Driver and non-driver mutations and their clonal evolution affect the thrombosis and disease transformation of MPN. Clonal hematopoiesis of MPN can occur decades before diagnosis, even in the fetal stage. After the emergence of clonal hematopoiesis, until the emergence and progression of MPN, gene mutation order, inflammation, interferon therapy affect the disease phenotype and clonal hematopoiesis of MPN. Although great progress has been made in the understanding of MPN clonal hematopoiesis and its evolution with the development of next-generation sequencing, there are still many limitations. In this study, we mainly discuss gene mutations of MPN and their influences on the thrombosis, leukemia and fibrosis transformation, and the influencing factors of clonal evolution, aiming to summarize the influence of clonal hematopoiesis and its evolution on the complications, prognosis and survival of MPN.

Signaling Pathway Gene Set Enrichment Analysis in Whole Exome Sequencing Data from Discordant Identical Twins Unveils Relevant Pathways As a Clue to Understand Somatic Mutation Acquisition and Clonal Evolution in Myeloproliferative Neoplasms

Although the role of classic driver mutations (i.e., JAK2, MPL, and CALR) in myeloproliferative neoplasm (MPN) development is well characterized, additional somatic mutations likely modulate the phenotype. Recurrent somatic mutations, such as those in TET2, DNMT3A, ASXL1 genes are commonly identified in MPN using targeted myeloid panels, however identification of somatic mutations using an unbiased approach may unveil novel pathways involved in MPN pathogenesis. We performed whole exome sequencing of blood samples of three sets of discordant identical twins in which one twin has MPN and the other twin is unaffected. The twin sets included all MPN subtypes (1 polycythemia vera, 1 essential thrombocythemia, 1 primary myelofibrosis). We also performed whole exome sequencing on additional family members, including the mother and father of the PV twin and the brother of the ET twin. Sequencing information from the discordant identical twins is a unique opportunity to identify somatic mutations in the affected twin that may contribute to MPN pathogenesis, and also to potentially identify clonal hematopoiesis in the unaffected twin. We mapped all the identified genetic variants to the affected genes, and we filtered all the genes mapped to those variants depending on whether they were present either in the MPN-affected twin or in the unaffected counterpart for further gene set enrichment pathway analysis. The gene set enriched signaling pathways from identified genetic variants for each condition-exclusive (either MPN or unaffected twin) allows us to identify and prioritize key pathways in MPN development in terms of somatic mutations acquisition. Furthermore, besides performing the analysis within each single MPN set, we analyzed the gene sets resulting from combining the different families allowing us not only to identify pathways at each single MPN type level but also among the different MPN diseases (PV, ET, and MF). We divided all the enrichment analyses into coding and silent variants to obtain a higher degree of pathway refinement. In single discordant twin sets we identified several pathways that may play an important role within MPN pathogenesis such as defective mismatch DNA repair, facilitated mitotic progression, extracellular matrix degradation, MAPK signaling, FGFR signaling, O-glycosylation of proteins, collagen metabolism and laminin interactions, neutrophil degranulation, interleukin-33 signaling, Rho GTPases and cGMP regulation, and nuclear pore complex (NPC) assembly. Interestingly, only when we analyze all the MPN-types together all the statistically significant pathways resulting from coding variants correspond only to DNA repair, specifically defective mismatch repair (PMS2, MLH1, MutSα, MutSβ, and TP53). Our findings reveal that DNA repair genes might occupy a central role within MPN acquisition and development, whereas at the same time each single MPN clinical entity might possess its own distinct mechanisms differentiating each one from the others.

Loss of Heterozygosity of Chromosome 9p and Progression in MPNs

Introduction Copy-neutral loss of heterozygosity (LOH) of chromosome 9p, concomitant with mutated JAK2, can occur in myeloproliferative neoplasms (MPNs), and may be the most common chromosomal aberration in patients with polycythemia vera (PV). The prevalence of LOH of 9p varies amongst the different MPNs and is highest in PV where it has been reported to be between 30-70%. The reported prevalence of LOH of 9p is 20-50% and 10-20% in patients with myelofibrosis (MF) and essential thrombocythemia (ET), respectively. A hallmark feature of MPNs is the tendency to progress to more aggressive disease phenotypes (i.e., PV/ET to MF to accelerated- or blast-phase MPN) through the acquisition or loss of cytogenetic and molecular clonal markers. Although loss of LOH of 9p likely contributes to progression in MPNs, its pathologic role is still incompletely defined. Methods We conducted a retrospective review of patients at a single-center, tertiary-care institution who were diagnosed with an MPN, either chronic, accelerated or blast-phase (MPN-AP/BP), from January 1 st, 2017 until July 1 st, 2023. All patients underwent a microarray-based chromosome analysis using the I Scan System with the Global Diversity Array-8 (GDA) v1.0 Array BeadChip. Mutational profiling was done using the NextGENe software (SoftGenetics LLC) and MiSeq Reporter, and variant annotation and filtration was performed using VarSeq (Golden Helix). Results Fifty-two patients with MF were identified. Twenty-eight patients (53.8%) were found to have LOH of 9p, 15 of whom had secondary MF (7 with preceding ET and 8 with preceding PV). Eight of 9 patients (88.9%) with PV-MF harbored LOH of 9p, vs. 6 of 13 (46.2%) with ET-MF. The median cell fraction affected was 70% (range, 5-95%) and the median JAK2 variant allele frequency (VAF) was 75% (range, 16-97%). Eleven of the 24 patients without LOH of 9p harbored JAK2 mutations, at a median VAF of 40%. Nine patients (32.2%) with LOH of 9p had abnormal cytogenetics by conventional karyotyping, compared to 8 (33.3%) without LOH of 9p. Eleven patients (39.3%) with LOH of 9p were MIPSS-70 v2 high or very high risk, compared to 10 (41.7%) patients without LOH of 9p. Nine patients (32.1%) with LOH of 9p were DIPSS-plus intermediate-2 or high-risk, compared with 9 (37.5%) patients without LOH of 9p. Twenty patients with MPN-AP/BP were identified, of whom 11 had MPN-AP and 9 had MPN-BP. Four patients (20%), all with MPN-BP, had LOH of 9p. Median JAK2 VAF in these patients was 50% (range, 38-80%), vs. 4% (range, 3-48%) in the 6 patients with JAK2 without LOH of 9p. Two of 19 patients (10.5%) with ET were found to have LOH of 9p, with a corresponding JAK2 VAF of 70% and 20%. Twelve of the 17 patients (70.6%) who did not have LOH of 9p had a JAK2 mutation, at a median VAF of 27% (range, 4-44%). One patient with LOH of 9p had a thrombotic event, vs. 4 of 17 patients (23.5%) without LOH of 9p. Three of 9 patients (33%) with PV were found to have a LOH of 9p, with a median corresponding JAK2 VAF of 61% (range, 55-81%). Median JAK2 VAF for the other 6 patients (all with a JAK2 mutation) was 34% (range, 26-46%). One patient with LOH of 9p had a thrombotic event vs. 3 of 6 patients (50%) without LOH of 9p. Discussion The highest prevalence of LOH of 9p in patients with an MPN was seen in those with MF (>50%), followed by PV, MPN-AP/BP and ET. These results are generally in line with those previously reported, though the prevalence in PV was lower than other studies (likely due to small sample size). The prevalence of LOH of 9p in ET-MF was more than four times higher than in ET, suggesting that LOH of 9p may be an acquired cytogenetic aberration involved in ET progression. LOH of 9p was seen in almost 90% of PV-MF vs. 33% of PV patients, further supporting that LOH of 9p may be implicated in MPN progression. Notably, LOH-9p was seen in far fewer patients with accelerated- or blast-phase disease. It is likely that clonal evolution of MPNs to MPN-AP/BP is driven more so by the acquisition of high-risk mutations (e.g., TP53, ASXL1, U2AF1) and less by changes such as LOH of 9p and increased JAK2 VAF. In summary, our data largely confirms previous reports regarding the prevalence of LOH of 9p in MPNs while suggesting a role for the gain or loss of LOH of 9p in the progression of these hematologic cancers.

Clonal Hematopoiesis in Myeloproliferative Neoplasms Confers a Predisposition to both Thrombosis and Cancer.

This review focuses on vascular complications associated with chronic myeloproliferative neoplasms (MPN) and more specifically aims to discuss the clinical and biological evidence supporting the existence of a link between clonal hematopoiesis, cardiovascular events (CVE), and solid cancer (SC). The MPN natural history is driven by uncontrolled clonal myeloproliferation sustained by acquired somatic mutations in driver (JAK2, CALR, and MPL) and non-driver genes, involving epigenetic (e.g., TET2, DNMT3A) regulators, chromatin regulator genes (e.g., ASXL1, EZH2), and splicing machinery genes (e.g., SF3B1). The genomic alterations and additional thrombosis acquired risk factors are determinants for CVE. There is evidence that clonal hematopoiesis can elicit a chronic and systemic inflammation status that acts as driving force for the development of thrombosis, MPN evolution, and second cancer (SC). This notion may explain the mechanism that links arterial thrombosis in MPN patients and subsequent solid tumors. In the last decade, clonal hematopoiesis of indeterminate potential (CHIP) has been detected in the general population particularly in the elderly and initially found in myocardial infarction and stroke, rising the hypothesis that the inflammatory status CHIP-associated could confer predisposition to both cardiovascular diseases and cancer. In summary, clonal hematopoiesis in MPN and CHIP confer a predisposition to cardiovascular events and cancer through chronic and systemic inflammation. This acquisition could open new avenues for antithrombotic therapy both in MPNs and in general population by targeting both clonal hematopoiesis and inflammation.

Additional Genetic Alterations and Clonal Evolution of MPNs with Double Mutations on the MPL Gene: Two Case Reports

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50–60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3–5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPLV501A-W515R and JAK2V617F mutations and a man with PMF displaying an uncommon double MPLV501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF.

Metabolic profiling of myeloproliferative neoplasms reveals subtype specific metabolic traits

Introduction: Myeloproliferative neoplasms (MPN) are haematopoietic disorders leading to aberrant expansion of myeloid, erythroid, and megakaryocytic lineages, which are classified into three phenotypes namely Polycythaemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). The evolution of MPN mainly originates from three somatic mutations in haematopoietic stem cells (HSCs) including Janus Kinases2 (JAK2), Calreticulin (CALR), Myeloproliferative Leukemia Virus Oncogene (MPL). The JAK2 inhibitor Ruxolitinib, despite being the state-of-the- art therapy is unable to completely eradicate leukemic stem cells (LSCs). . Metabolic alterations are crucial in the development of cancer by creating different anaplerotic pathways supporting cancer cell proliferation. Metabolic vulnerabilities can be utilized to target MPN mutational hematopoietic stem cells. Methods: We preformed Seahorse analysis on three independent biological replicates for Jak2V617F wild type (WT), heterozygous (HET, represents ET) and homozygous (HOM, represent PV) c-kit positive cells and for even more primitive lineage negative, c-kit positive, Sca1 positive (LSK). We additionally used Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) and RNA sequencing to investigate metabolic differences in LSCs. Results: We see increased respiration in Jak2V617F HET c-kit cells compared to WT and Jak2V617F HOM cells. However, mitochondrial numbers as determined my mitotracker green flow cytometry are similar between all genotypes. Moreover, mitochondrial membrane potential, as measured my TRMT staining and flow cytometry, does not differ between genotypes in c-kit positive and in LSK cells. These data indicate that differences in respiration between genotypes is not due to changes in the mitochondrial compartment, but might instead be caused by changes in e.g. gene expression levels of respiratory enzymes or other players involved in cellular respiration. We therefore performed bulk RNA-sequencing on c-Kit positive and LSK cells. We found the glutamate receptor signalling pathway elevated in Jak2V617F Hom, with single cell RNASeq data from highly purified Hom Jak2V617F LSCs showing significant upregulation in glutathione metabolism, feeding into the same metabolic processes. To elucidate changes in metabolism in more detail, we generated data from two independent biological replicates of c-kit positive, murine cells from all three genotypes for LC-MS. We found that Jak2V617F Het cells show increased activity of the TCA cycle compared to Hom. This is in accordance with our Seahorse data showing increased respiration in Het compared to Hom. We therefore added an OxPhos inhibitor (IACS) to Het and Hom c-kit positive cells to characterise a possible vulnerability in the Het cells. Jak2V617F Het c-kit positive cells seem indeed more sensitive to IACS compared to Hom cells, where IACS yields little effect. In contrast, higher glutamate levels were discovered medium of Jak2V617F Hom cells after 24 hours of culture, indicating glutamate evacuation out of the cells. Conclusions Our data demonstrate, for the first time, differences in metabolic needs in subtypes of MPNs, with ET-like murine cells depending on the TCA cycle compared to PV-like murine cells, which deregulate the glutamate/ glutathione metabolism pathway.

Expression of Jak2 R1063H Leads to Altered Epor/Tpor Signaling, Changes in HSC Fitness and Represents Increased Risk of Thrombosis

Non-canonical weakly activating germline JAK2 variants may predispose progenitor cells to acquire JAK2 V617F during the evolution of myeloproliferative neoplasms (MPN) (Lanikova et al. Blood 2016;128(18):2266) and represent risk factors for MPN transformation into AML (Benton et al. Cancer 2019;125:1855). We have previously described and functionally characterized the JAK2 R1063H germline variant, cooperating with the JAK2 E846D variant in a case of hereditary MPN with erythrocytosis and megakaryocytic atypia (Kapralova et al. Blood 2016;128:1418) or with the acquired JAK2 V617F causing increased JAK2 signaling and higher disease severity in patients with essential thrombocythemia (Mambet et al. Blood 2018;132:2695). The mouse model bearing Jak2 R1063H mutation was created using microinjection of synthesized ssODNs containing the R1063H mutation, sgRNA mRNA and Cas9 protein into C57BL6/N-derived zygotes. In order to intensify a possible effect of the mutation, we bred and analyzed the knock-in Jak2 R1063H homozygous mice. The mice are born in normal (expected) frequency and display increased platelets (PLT) counts when compared to wild-type (wt) mice during life-span. Despite increased PLT counts, the plasma thrombopoietin level is normal whereas erythropoietin levels are increased without marked erythrocytosis. The preliminary co-immunoprecipitation experiments with human erythropoietin and thrombopoietin receptors (EPOR/TPOR) and JAK2 variants suggest increased TPOR and decreased EPOR binding with JAK2 R1063H kinase, possibly explaining the hematological phenotype. Also experiments with humanized mouse model of EPOR (Divoky et al. Proc Natl Acad Sci USA 2001;98:986) crossed with Jak2 R1063H mice supported these findings, where anemia due to hypoactivation of wt human EPOR signaling is more severe in Jak2 R1063H mice than in a control group and mild thrombocytosis is still present in the double mutant mice. The Jak2 R1063H model displays increased number of long-term hematopoietic stem cells (HSC) suggesting increased cycling in this population at young age (3 months) and shift to increased short-term HSC and multipotent progenitors with aging (12 months). The immunophenotypically-defined myeloid progenitor cells (LK, Lin-/Sca1-/cKithigh) are statistically increased in Jak2 R1063H mice due to disproportionately increased megakaryocytic/erythroid progenitors (MEP) over other myeloid progenitors at 3 months of age. The LK compartment is compromised in old Jak2 R1063H mice due to substantial myeloid bias by disproportionately increased CMP cells and marked decreased MEP cells, explaining the more pronounced anemia of aging in these mice. Some of these features are reminiscent of aging-associated myeloid and megakaryocytic bias, increased LKs, and relative exhaustion of progenitors with an erythroid lineage-priming program in aged bone marrow (BM) of a mouse model of primary familial and congenital polycythemia (PFCP) due to gain-of-function EPOR (Kralova et al, Am J Hematol 2022; doi: 10.1002/ajh.26658). However, the gain-of-function EPOR-introduced changes into the BM microenvironment are not associated with augmented hypoxia signaling and systemic inflammation and do not represent permanent challenges that would contribute to an increased incidence of myeloid malignancies in PFCP. In this regard, it will be interesting to compare these characteristics with the possible predisposing potential of Jak2 R1063H; further analysis of proliferative history of Jak2 R1063H HSC populations and expression profiling are ongoing. In the cohort of Jak2 R1063H mice, we also observed a relatively high frequency of sudden death in mice with median overall survival of 215 days (5/15) in comparison with the control group (0/15). We therefore measured the levels of D-dimers as a marker of thrombosis and found it statistically increased in the Jak2 R1063H group. Interestingly, in a WES study of patients with venous thromboembolism, the JAK2 R1063H was identified in one case, being considered a probable disease-causing variant (Lee at el. Blood Adv 2017;1:1224). In conclusion, our results confirm a direct effect of germline Jak2 R1063H mutation on EpoR/TpoR signaling but also on HSC biology, suggesting that the variant may represent a risk factor for BM remodeling in the process of myeloid cell transformation. Grant support: Czech Health Research Council NU21/03/00338.

Clinical Presentation, Risk Factors and Outcome of Splanchnic Venous Thrombosis: A Report on 305 Cases

Background and Aims: Splanchnic vein thrombosis (SVT) is a manifestation of unusual site venous thromboembolism. Most common predisposing factors for SVT include liver cirrhosis, solid abdominal cancer, myeloproliferative neoplasms (MPN), and abdominal infection or inflammatory diseases. Complex balance between bleeding risk and the risk of thrombus extension or recurrence makes the management of SVT a clinical challenge. In this study, we reported clinical presentation and outcome data of patients with SVT followed in our center from January 2000 to December 2021, with a focus on MPN and cirrhotic patients. Methods: We performed a retrospective review of clinical reports of 305 SVT patients. According to PLT count in patients with no active bleeding, anticoagulation treatment was started as soon as possible with: no treatment for a platelet count <30.000/μl; prophylactic dose of LMWH or related drugs for a platelet count range 30.000 -50.000/μl and therapeutic dose low-molecular-weight heparin (LMWH) followed by early initiation of vitamin-K antagonist (VKA; INR range 1.8-2.5) for a platelet count >50.000/μl. Moreover, cytoreductive therapy was administered in MPN patients and prophylaxis with beta-blockers and endoscopic therapies were applied in cirrhotic SVT. Cox regression analysis was used to identify independent predictors of recanalization and overall survival. The Kaplan-Meier method was performed to estimate the overall survival and the time to reach vessel recanalization. Results: In a median follow up of 16.1 months (range, 1-346), 305 patients experienced a SVT (median age 59 years; 68.2% males). Isolated portal vein thrombosis was the most common site of thrombosis (55.1%), followed by multiple venous involvement (28.9%). Six patients developed a Budd-Chiari syndrome. SVT were associated to chronic liver disease, solid cancer and abdominal infections in 212 (69.5%), 100 (32.8%) and 34 (11.5%) patients, respectively. Ninety-seven patients had splenomegaly. MPN was identified in 38 (12.5%) patients. Among these patients, JAK2 and CALR mutation were detected in 81.6% and 13.2%, respectively, and abnormal karyotype in 10.5%. Ascites and esophageal varices were present in 47.5% and 48.8% of patients, respectively. Therapeutic decisions were taken as a multidisciplinary team, involving the use of VKA and LMWH in 70 and 163 patients, respectively. In addition, trans-jugular intrahepatic portosystemic (TIPS) or other surgical shunting was undertaken in 12% and variceal banding in 11%. In a median duration therapy of 9.6 months, bleeding complications and recanalization were reported in 9.5% and 35.4% of patients, respectively. At univariate analysis, VKA exposure was associated with a significantly lower risk of bleeding events compared to LMWH (p=0.039), while esophageal varices (p=0.049), leukocytosis at time of thrombosis (p=0.048), liver cirrhosis (p=0.010) and solid tumor (p=0.021) were associated with lack of recanalization. Only one death was attributed to fatal bleeding, whereas 76 patients died for causes not related to anticoagulation (cirrhosis, cancer, MPN evolution) with a median OS was not reached (range, 107,89-NR). Factors associated with poor survival included age (p<0.001), MPN (p=0.047), recurrent thrombosis (p=0.041), alkaline phosphatase (p=0.013), gamma-GT (p<0.001), albumin (p=0.021), MELD score (p=0.004), lack of recanalization (p<0.001), bleeding event (p=0.043), ascites (p=0.009), and antithrombotic therapy (p=0.006, Figure 1). Among MPN patients, poor survival outcome was also associated with driver mutations (p=0.034), hemoglobin level at thrombosis (p<0.001), splenomegaly (p=0.019), and cytoreductive therapy (p=0.019). Conclusions: Outcome in SVT patients mostly depends on the presence of an underlying MPN, liver failure, active bleeding, recurrence and recanalization rate. Moreover, the anticoagulant treatment was associated with a low bleeding incidence also in patients with major risk factors for bleeding (i.e. liver cirrhosis, cancer or esophageal varices). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1064: CIRCULATING CYTOKINE LEVELS IN PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS – PROFILE AND CLINICAL CORRELATIONS – A SINGLE CENTER STUDY

Background: Myeloproliferative neoplasms(MPNs) are a group of disorders with heterogeneous features and often progressive clinical course. Over the past few years a remarkable advance has been made in the knowledge on the pathogenesis and therapeutic management of the Philadelphia(Ph)-negative MPNs. Despite the wide variety of studies in this field, there are still unsolved issues regarding their biology, disease course, prognosis and treatment. The improved recognition of molecular markers is not sufficient for a comprehensive explanation of the variable clinical symptoms and complications among these patients. The role of the chronic inflammation has been widely considered as a co-factor, involved in the pathogenesis and progression of MPNs. Aims: To analyze the serum levels of a panel of cytokines, associated with the mechanisms of chronic inflammation, among patients with Ph-negative MPNs and healthy controls and the possible correlations of these cytokines with the disease’s features. Methods: One hundred and fifty adults were included in the study, distributed in comparable groups, as follows: 36 patients with Essential thrombocytemia (ET), 49 patients with Polycythemia vera (PV), 30 patients with Myelofibrosis (MF) and 35 age-matched healthy controls. The patients with MPNs were diagnosed according to the latest WHO criteria. The cytokines’ levels were tested in serum samples with a specific kit Cytokine Human Panel for Luminex™. Assessments of the symptoms burden were performed with MPN-SAF- questionnaire. Results: Among the 11 tested cytokines we detected significantly higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF among the MF patients (p<0.05), in comparison with the healthy controls, as well as significantly higher levels of Interleukin-6 in the MF-group, compared to the PV-patients. Elevated serum Interleukin-6, Interleukin-8, Interleukin-18 and GM-CSF were observed in the ET-group versus the control group levels (p<0.05). The cytokine profile analysis also revealed higher serum levels of Interleukin-2, Interleukin-5, Interleukin-6 and Interleukin-18 in the patients with PV, than in the healthy subjects. In terms of estimation of the clinical impact of the circulating inflammatory cytokines among the analyzed MPN cohort - for the ET-patients a positive correlation was present between GM-CSF and the fatigue severity and between Interleukin-6 levels and the weight loss severity index, both assessed according to the MPN-SAF score. A positive correlation was detected between Interleukin-10 levels and the bone pain burden and the degree of weight loss for the patients with MF. Interleukin-18 values demonstrated a positive correlation with the weight loss among the patients with PV. Summary/Conclusion: Our single center study results can give a ground to assume the potential role of the chronic inflammation and the pathogenetic impact of the circulating inflammatory cytokines on the MPNs clinical features and course. The cytokine profile of the different subgroups of MPNs is a topic of interest and can be monitored as a potential predictor of disease progression. It can motivate future research attempts, aiming to alter the clinical evolution of MPNs by affecting the pathogenetic mechanisms of the inflammatory process. It can create prerequisites for future studies regarding more effective therapeutic targets, related to both – the molecular pathways of the mutations in MPNs and the molecular mechanisms of the chronic inflammation.

OBESITY AND MYELOPROLIFERATIVE NEOPLASMS: A SYSTEMATIC REVIEW

Objective: Obesity is a known trigger for carcinogenesis and its involvement is proven in the development of solid and blood cancers. However, the involvement of obesity in myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), has not been assessed so far. Design and method: Systematic review in PubMed/MEDLINE from its inception to March 13th, 2022. The database was searched using specific keywords/word combinations to identify relevant papers which were further evaluated if they were original articles and investigated the role of obesity in MPNs. Reviews, case reports, meeting abstracts, letters to the editor, in vivo/in vitro studies were excluded. Results: The initial search yielded 88 results of which 62 remained after the exclusion of reviews/case reports (n = 26). After screening of titles/abstracts, 25 full-texts were assessed of which 18 were entered into the qualitative analysis. Most conclusions were drawn from singular studies. Two papers pointed out that obesity affects the evaluation of red cell mass in PV due to the hypovascular nature of the adipose tissue, arguing for new reference ranges in obese subjects. Adults with obesity during adolescence had an increased risk of MPNs development during adulthood. Obesity was particularly linked to the development of ET. Obese MPNs adults had an elevated number of bone marrow adipocytes, higher total symptom burden (U-shaped association with BMI), higher rate of cerebrovascular accidents (ET) and venous thrombosis, lower risk of post-PV myelofibrosis and stroke, higher survival rates. Obesity did not influence anxiety in MPNs or the occurrence of thrombosis (ET). However, lower survival rates and thrombosis-free survival were noted in MPNs patients with multiple cardiovascular risk factors. PMF patients treated with ruxolitinib who had higher BMI values registered better outcomes. Ruxolitinib-treated subjects exhibited an increase in BMI. Obesity did not influence the outcome of allogeneic bone marrow transplantation in PMF, nor did it influence the outcome of this procedure in elderly patients transplanted for myeloid malignancies. Conclusions: Obesity can influence the development and evolution of MPNs and should be carefully monitored. Further studies should assess the obesity paradox in MPNs.

Chronic Exposure to Cytoreductive Treatment Shapes Clonal Evolution in Myeloproliferative Neoplasms

▪Introduction: Myeloproliferative neoplasms (MPN) are a heterogeneous group of chronic myeloid malignancies resulting from the combination of a driver mutated gene (JAK2, MPL or CALR) and a variety of acquired additional somatic mutations. Although next-generation sequencing (NGS) has identified high molecular risk mutations associated with adverse prognosis (Vannucchi et al., Leukemia, 2013 , Guglielmelli et al., Leukemia, 2014 ), the clonal evolution of these mutations remains poorly described. Chronic exposure to cytoreductive treatment, especially genotoxic drugs such as hydroxyurea (HU), could impact clonal evolution. A previous study suggested that Interferon-α (IFN) could limit the accumulation of cytogenetic abnormalities compared to HU (Mondello et al., Blood, 2018). The objective of our study was to describe the long-term evolution of the mutational landscape in the era of NGS in a large cohort of MPN patients.Methods: A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. This study included 1039 of them in whom a NGS molecular analysis targeting 36 myeloid genes with a sensitivity of 1% was performed at diagnosis and/or during follow-up. Patients with only one NGS (n=588), AML/MDS transformation at either the first (n=3) or the second NGS (n=2) were excluded from the analysis. Serial NGS data obtained in chronic MPN phase were thus analyzed for 446 patients. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Mutation rates per year were calculated for each gene as the difference in the number of mutations between first and last NGS divided by the time interval (in years) between both NGS.Results : Median age at MPN diagnosis in our whole cohort was 51 years [IQR 41-60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with Polycythemia Vera (PV), Essential thrombocythemia (ET) and primary myelofibrosis (MF) respectively. 279 patients (63%) had at least one additional mutation at first NGS, and respectively 27 (6%) and 104 (23%) patients had TP53 and high molecular risk mutations. Median interval between MPN diagnosis and the first NGS was 6.5 years [IQR 1.7-13] while median time between the first and the last NGS was 2.5 years [IQR 1.6-4, range 0.3-14.3]. Overall, 178 patients (39.9%) acquired an additional mutation at last NGS evaluation, most frequently involving TET2, DNMT3A, ASXL1, TP53 and NFE2 genes . To study the impact of chronic MPN therapy on clonal evolution, we focused on patients who electively received HU (n=112) or IFN (n=92) as a monotherapy, or did not receive any cytoreductive treatment (n=119) between the first and the last NGS. The remaining patients received ruxolitinib (n=44), anagrelide (n=10), vercyte (n=7) or polytherapy (n=62). At last follow-up, 74 patients receiving IFN (80.4%) and 65 (58%) treated with HU had a complete hematological response. When combining all additional mutations, the global mutation rate per year did not significantly differ between treatment groups. When analyzing individual genes, TP53 mutation rate was higher in patients treated with HU compared to the patients receiving IFN (p=0.014) or not treated (p=0.008) (Figure). MDS/AML evolution occurred in 4 patients (3.6%) treated with HU, 2 (1.7%) without cytoreductive therapy versus none of the 92 patients treated with IFN (ns). In the whole cohort, MDS/AML evolution was significantly increased in patients harboring TP53 mutations (p= 0.004). In contrast, DNMT3A mutation rate was significantly increased in patients receiving IFN compared to patients treated with HU (p=0.045) (Figure). The latest result is in line with previous observations showing that loss of DNMT3A could confer resistance to IFN in a JAK2-V617F mouse model (Stetka et al., Blood, 2020).Conclusion: Our results highlight the impact of chronic cytoreductive therapy on clonal evolution shaping in MPN. IFN limits the emergence of TP53 mutated clones compared to HU, thus potentially reducing the risk of leukemogenesis. Emergence of DNMT3A mutated clones under IFN therapy requires further exploration and could potentially play a role in therapeutic resistance. This study on a large clinically and biologically annotated cohort illustrates how serial NGS analysis may guide therapeutic options for MPN patients. [Display omitted] DisclosuresRaffoux: PFIZER: Consultancy; ASTELLAS: Consultancy; ABBVIE: Consultancy; CELGENE/BMS: Consultancy. Kiladjian: AbbVie: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; PharmaEssentia: Other: Personal fees. Benajiba: Pfizer: Research Funding; Gilead: Research Funding.

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 [Display omitted] DisclosuresGotlib: 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.

Myeloproliferative Neoplasms (MPN) Clonal Evolution Landscape and Its Impact on Patients' Prognosis

▪Introduction:Although myeloproliferative neoplasms (MPN) are driven by three mutually exclusive driver mutations (JAK2, CALR and MPL), targeted deep sequencing studies identified multiple additional somatic mutations potentially impacting MPN evolution. Presence of a high molecular risk (HMR: ASXL1, EZH2, SRSF2 and IDH1/2) or a TP53 mutations has been associated with adverse prognosis. However, to date, the effect of clonal evolution (CEv) on MPN patients' outcome has not been evaluated, as most of the studies assessed mutational-based prognosis stratification from single baseline molecular genotyping. The objective of our study was to describe the clinical and molecular characteristics of patients with CEv in a large cohort of MPN patients and analyze its impact on patients' outcome.Methods:A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. From this large retrospective cohort, we included in this study 446 patients who had at least 2 molecular analyses during the chronic phase of MPN, performed at diagnosis and/or during follow-up using next generation sequencing (NGS), targeting a panel of 36 genes involved in myeloid malignancies. Significant variants were retained with a sensitivity of 1%. CEv was defined as the acquisition of a new additional non-driver mutation between baseline and subsequent NGS evaluation. Statistical analyses were performed using the STATA software (STATA 17.0 for Mac Corporation, College Station, TX).Results:The median age at MPN diagnosis in our whole cohort was 51 years [IQR 41 - 60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with polycythemia vera, essential thrombocythemia and primary myelofibrosis (MF) respectively.With a median interval of 1.6 years [IQR 1.0 - 2.8] between the first and the second NGS analysis in the whole cohort, CEv occurred in 128 patients (29%). Patients with CEv were significantly older compared to patients without CEv (n=318) (p=0.03). MPN diagnosis, the type of driver mutation and complete blood counts at MPN diagnosis did not differ between the 2 groups. Eighty-one (63%) and 198 (62%) patients with or without CEv respectively had at least one additional non-driver mutation at baseline NGS (p=0.59), while the rate of HMR (n=25 (20%) versus n=79 (25%)) or TP53 (n=7 (5%) versus n=20 (6%)) mutations at baseline NGS did not differ between the 2 groups.Thirty six out of 128 (28%) of patients with CEv had more than 1 acquired mutation. Most recurrently acquired mutations involved the epigenetic regulators TET2 and DNMT3A that were mutated in respectively 33% and 25% of patients with CEv (Figure 1A). Moreover, 38% of CEv patients acquired HMR (ASXL1 (14%), EZH2 (6%), SRSF2 (3%), IDH1/2 (2%)) or TP53 (13%) mutations. After a median follow up of 10.8 years [IQR 6.6 - 17.2] in the whole cohort representing a total of 5635 patient years, 32 (7%) patients died, and 11 (2.5%) and 11 (2.5%) patients with at least 2 NGS performed during MPN chronic phase transformed respectively into secondary MF or myelodysplastic syndrome / acute myeloid leukemia (MDS/AML). Interestingly, CEv (HR 11.27, 95%CI [5.09; 24.96], p<0.001) (Figure 1B), age at MPN diagnosis (HR 1.11, 95%CI [1.07; 1.15], p<0.001) and the presence of HMR mutations at baseline NGS (HR 4.48, 95%CI [2.05; 9.77], p <0.001) independently adversely impacted OS in a COX regression multivariate analysis. CEv also independently adversely impacted MDS/AML free survival (HR 13.15, 95%CI [3.88; 44.47], p<0.001) and secondary MF free survival (HR 21.13, 95%CI [6.18; 72.20], p<0.001) in a COX regression multivariate analysis.Conclusion:Our study on a large retrospective clinically and biologically annotated real-life cohort of MPN patients with long-term follow up shows that CEv independently adversely impacts OS, MDS/AML and secondary MF free survivals. CEv occurred in a clinically relevant proportion of MPN patients (28%) and was associated with patients' age. Acquired mutations mainly involved epigenetic regulators, HMR and TP53 genes. These results suggest that serial molecular monitoring using NGS could be routinely implemented in MPN patients follow up, to assess more accurately disease evolution and potentially update therapeutic management. [Display omitted] DisclosuresRaffoux: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ABBVIE: Consultancy; ASTELLAS: Consultancy. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Benajiba: Gilead: Research Funding; Pfizer: Research Funding.

Clonal Analysis of Acute Myeloid Leukemia Secondary to Myeloproliferative Neoplasms

[Background] Prognosis of patients with secondary acute myeloid leukemia (sAML) transformed from myeloproliferative neoplasms (MPNs) is extreamly poor. It has been demonstrated that several additional genetic alterations, which are engaged in epigenetic regulation, cell-growth signaling and RNA splicing machinery, cooperate with JAK2, CALR and MPL mutations for the development of sAML from MPN. The order of mutation acquisition also influences the clonal evolution in MPN. However, little is known about the mechanisms of its leukemic transformation. In previous studies, both JAK2 -mutated and -unmutated AML clones were identified in sAML cases transformed from JAK2 -mutated MPNs, suggesting the diversity of the clonal evolution during the leukemic transformation from MPNs. In this study, we investigated genetic alterations and clonal evolutions in sAML from MPN, especially in JAK2 -unmutated sAML, to elucidate the mechanisms of the leukemic transformation.[Methods] We performed targeted deep sequencing of 54 genes in six paired MPN and sAML samples. Whole-exome sequencing (WES) was also performed in a paired MPN, sAML and normal tissue cells. For a clonal analysis, bone marrow (BM) cells at complete remission (CR) stage after chemotherapy were sorted into hematopoietic stem cell (HSC) and hematopoietic progenitor fractions such as common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocytic/erythroid progenitor (MEP) fractions, and analyzed the mutations detected in targeted deep sequencing or WES. Furthermore, we sorted single cells from the HSC fraction and performed single-cell mutational analysis to clarify the clonal evolutions precisely during the development of sAML. In addition, we intravenously inoculated BM cells at the sAML stage into NOD/SCID/IL-2Rγnull (NOG) mice, and compared mutation status between primary AML cells and engrafted leukemia cells to evaluate the clonal hierarchy of leukemia cells.[Results] Four JAK2 -mutated and two CALR -mutated MPNs were analyzed in this study. Targeted deep sequencing identified TP53 mutations in five of six sAML samples with high variant allele frequencies (VAFs). In two of the four JAK2 -mutated MPNs, VAFs of JAK2 -V617F were significantly decreased in sAML cells compared with those in MPN cells, whereas VAFs of TP53 mutations were emerged or increased in sAML cells, suggesting that sAML clones with TP53 mutations were raised from JAK2-V617F negative hematopoietic cells in both cases. Moreover, we identified the mutations which was common to MPN and AML cells in both cases. These mutations formed common initiating clones prior to MPN and sAML, and additional mutation in JAK2 or TP53 was associated with their MPN or sAML development. The single-cell analysis in CD34+/CD38- HSC fractions revealed that a small part of the cells harbored TP53 -R248W and JAK2 -wild type (WT), half of the cells harbored homozygous JAK2 -V617F and TP53 -WT, and the other half cells harbored JAK2 - and TP53 -WT. Notably, three common mutations detected both in MPN and in sAML with the same VAFs were mainly found in JAK2 -WT cells. Thus, we validated that JAK2 and TP53 mutations were mutually exclusive HSC fractions, and confirmed that sAML clones were raised from JAK2 -unmutated clone. In patient derived xenotransplant models using JAK2 -WT sAML cells following JAK2 -mutated MPN or CALR -mutated sAML, engrafted leukemia cells showed the same mutational pattern as primary sAML cells. These results suggested that sAML clones but not MPN clones were able to engraft and proliferate in mice.[Conclusions] The genetic analysis in paired MPN and sAML samples revealed the diverse clonal evolution patterns during the development of sAMLs from MPNs. TP53 mutation was strongly associated with leukemic transformations both in JAK2 -V617F mutated and CALR -mutated MPNs. However, we demonstrated two cases in whom driver mutations in MPNs were not preserved in sAML clones. In those cases, there might be independent common initiating clones corresponding to MPN or sAML, and each initiating clone might be acquired JAK2 or TP53 mutation before the development of MPN or sAML, respectively. Our data also suggested the importance of single-cell analysis which enable us to clarify the clonal evolutions in leukemic transformation from MPN, that were not identified in targeted deep sequencing as well as WES. DisclosuresKiyoi:Phizer Japan Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding.

Inflammatory Microenvironment and Specific T Cells in Myeloproliferative Neoplasms: Immunopathogenesis and Novel Immunotherapies.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

Neutrophils prothrombotic characteristics during myeloproliferative neoplasms

Arterial and venous thrombosis are the most frequent complications during evolution of myeloproliferative neoplasms (MPN) but the events causing these clotting abnormalities remain unclear. Neutrophils are now recognized as important actors of thrombosis, especially by their capacity to emit neutrophil extracellular traps (NETs). To assess if JAK2V617F neutrophils are more prone to form NETs, thus promoting thrombosis. We studied two different mouse models: PF4-iCre; JAK2V617/WT mice with expression of JAK2V617F in all hematopoietic cells and MRP8-iCre; JAK2V617/WT mice with expression of JAK2V617F in neutrophils only. In these models, we quantified: – NET formation; – plasma levels of circulating DNA; – pulmonary thrombus formation. In PF4-iCre; JAK2V617/WT mice, we analysed thrombus formation after DNAse administration. Finally, we quantified NET formation from JAK2WT or JAK2V617F neutrophils in presence or absence of JAK2WT or JAK2V617F platelets. We observed an increased NETs formation in both mouse models. We observed increased plasma levels of free DNA and increased thrombus formation in PF4-iCre; JAK2V617F/WT mice only. Thrombus formation was abrogated in PF4-iCre; JAK2V617F/WT mice after DNAse administration. We observed an increased NET formation in presence of JAK2V617F neutrophils and JAK2V617F platelets, with a decreased NETs formation after platelet inhibition. Our results show that neutrophils are prone to form NETs in two different mouse models. More, our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis and that the cooperation with platelets is important in NETs formation.

Splicing Anomalies in Myeloproliferative Neoplasms: Paving the Way for New Therapeutic Venues.

Since the discovery of spliceosome mutations in myeloid malignancies, abnormal pre-mRNA splicing, which has been well studied in various cancers, has attracted novel interest in hematology. However, despite the common occurrence of spliceosome mutations in myelo-proliferative neoplasms (MPN), not much is known regarding the characterization and mechanisms of splicing anomalies in MPN. In this article, we review the current scientific literature regarding “splicing and myeloproliferative neoplasms”. We first analyse the clinical series reporting spliceosome mutations in MPN and their clinical correlates. We then present the current knowledge about molecular mechanisms by which these mutations participate in the pathogenesis of MPN or other myeloid malignancies. Beside spliceosome mutations, splicing anomalies have been described in myeloproliferative neoplasms, as well as in acute myeloid leukemias, a dreadful complication of these chronic diseases. Based on splicing anomalies reported in chronic myelogenous leukemia as well as in acute leukemia, and the mechanisms presiding splicing deregulation, we propose that abnormal splicing plays a major role in the evolution of myeloproliferative neoplasms and may be the target of specific therapeutic strategies.

Emerging translational science discoveries, clonal approaches, and treatment trends in chronic myeloproliferative neoplasms.

The 60th American Society of Hematology (ASH) held in San Diego in December 2018 was followed by the 13th Post-ASH chronic myeloproliferative neoplasms (MPNs) workshop on December 4 and 5, 2018. This closed annual workshop, first introduced in 2006 by Goldman and Mughal, was organized in collaboration with Alpine Oncology Foundation and allowed experts in preclinical and clinical research in the chronic MPNs to discuss the current scenario, including relevant presentations at ASH, and address pivotal open questions that impact translational research and clinical management. This review is based on the presentations and deliberations at this workshop, and rather than provide a resume of the proceedings, we have selected some of the important translational science and treatment issues that require clarity. We discuss the experimental and observational evidence to support the intimate interaction between aging, inflammation, and clonal evolution of MPNs, the clinical impact of the unfolding mutational landscape on the emerging targets and treatment of MPNs, new methods to detect clonal heterogeneity, the challenges in managing childhood and adolescent MPN, and reflect on the treatment of systemic mastocytosis (SM) following the licensing of midostaurin.

Cytogenetic evolution in myeloproliferative neoplasms with different molecular abnormalities

We investigated the changes in chromosomal abnormalities in myeloproliferative neoplasm (MPN) patients during long-term follow-up. In total, 28 MPN patients (22 with primary myelofibrosis and 6 with polycythemia vera) were included. Among them, 25 patients underwent serial bone marrow (BM) biopsies during disease progression, and 3 patients had cytogenetic abnormalities at initial diagnosis but lacked follow-up BM biopsies. JAK2, CALR, and MPL mutation analyses were performed. Targeted sequencing analysis was conducted in 11 patients. Among the 28 patients, 21 (75.0%) had cytogenetic abnormalities either at diagnosis (8/26) or during follow-up. The median time from the initial analysis to the appearance of additional cytogenetic abnormalities was 8.4 years. Among the chromosomal abnormalities at initial diagnosis, trisomy 8 (3/26, 11.5%) was the most frequent, followed by gain of 1q, del(20q), and del(9q) (each in 2/26). Among all chromosomal abnormalities, including those that occurred during follow-up, the most frequent was del(20q) and +1q (8/28, 28.6%), followed by del(6p) (14.3%) and trisomy 8 (10.7%). Del(20q) was more frequent in CALR-mutated patients (4/6, 66.7%) than in JAK2-mutated patients (3/19, 15.8%, P = 0.016). The presence of cytogenetic abnormalities at initial diagnosis was associated with poor prognosis. Cytogenetic evolution may provide interesting insights into the disease course.