Search

The current retrospective study involving a total of 1607 patients was designed to identify clinical and molecular variables that were predictive of inferior myelofibrosis-free survival (MFS) in WHO-defined essential thrombocythemia (ET), utilizing three independent patient cohorts: University of Florence, Italy (n = 718); Mayo Clinic, USA (n = 479) and Policlinico Gemelli, Catholic University, Rome, Italy (n = 410). The Florence patient cohort was first examined to identify independent risk factors for MFS, which included age > 60 years (HR 2.5, 95% CI 1.3-4.9), male sex (2.1, 1.2-3.9), palpable splenomegaly (2.1, 1.2-3.9), CALR 1/1-like or MPL mutation (3.4, 1.9-6.1) and JAK2V617F variant allele frequency > 35% (4.2, 1.6-10.8). Subsequently, an operational molecular risk category was developed and validated in the other two cohorts from Mayo Clinic and Rome: "high molecular risk" category included patients with JAK2V617F VAF >35%, CALR type 1/1-like or MPL mutations; all other driver mutation profiles were assigned to "low molecular risk" category. The former, compared to the latter molecular risk category, displayed significantly higher risk of fibrotic transformation: Florence cohort with respective fibrotic transformation risk rates of 8% vs. 1.2% at 10 years and 33% vs. 8% at 20 years (p

Venetoclax (Ven) combined with a hypomethylating agent (HMA) has now emerged as an effective treatment regimen for acute myeloid leukemia, in both de novo and relapsed/refractory setting. The current multicenter study retrospectively examined Ven + HMA treatment outcome among 32 patients (median age 69 years; 59% males) with blast‐phase myeloproliferative neoplasm (MPN‐BP). Pre‐leukemic phenotype included essential thrombocythemia (ET)/post‐ET myelofibrosis (34%), polycythemia vera (PV)/post‐PV myelofibrosis (38%) and primary myelofibrosis (28%). Twenty‐nine study patients were fully annotated cytogenetically and molecularly (NGS): 69% harbored complex karyotype and/or mutations, including TP53 (41%), IDH1/2 (21%), ASXL1 (21%), N/KRAS (14%), SRSF2 (10%), EZH2 (10%) and U2AF1 (7%). All patients received Ven combined with either azacitidine (n = 12) or decitabine (n = 20); either up front (n = 23) or after failing another induction therapy (n = 9). Complete remission with (CR) or without (CRi) count recovery was achieved in 14 (44%) patients and was more likely to occur in the absence of pre‐leukemic PV/post‐PV myelofibrosis phenotype (p

Background: Polycythemia vera (PV) is one of the Philadelphia-negative myeloproliferative neoplasms (MPN), characterized by a pan-myelosis with an erythroid-predominant proliferation mainly driven by somatic JAK2V617F gain-of-function mutation. Hairy cell leukemia (HCL) is a rare B-cell lineage lymphoproliferative disease (LPD) with a typic immunophenotypic profile. BRAFV600E, leading to constitutive activation of the RAF/MEK/ERK signalling pathway and increased cell proliferation, is identified as the driver mutation in almost all cases. Although the risk of developing an LPD is significantly increased in patients with MPN compared with the general population, few cases of co-occurring PV and HCL are reported to date. BRAF is one of the most frequently mutated oncogenes in human cancer and some point mutations were identified in multiple neoplasms in addition to HCL, including follicular and papillary thyroid adenoma and carcinoma. Case presentation: Here we report a molecular diagnostic challenge in a woman with a concomitant diagnosis of JAK2V617F PV, BRAFV600E HCL, and HRASQ61K thyroid follicular adenoma. Conclusion: In the age of molecular and precision medicine, this case underlines the importance of integrating molecular results with clinical, radiologic, cytologic, and histopathologic investigations.

We report the clinical presentation and risk factors for survival in 175 patients with myeloproliferative neoplasms (MPN) and COVID-19, diagnosed between February and June 2020. After a median follow-up of 50 days, mortality was higher than in the general population and reached 48% in myelofibrosis (MF). Univariate analysis, showed a significant relationship between death and age, male gender, decreased lymphocyte counts, need for respiratory support, comorbidities and diagnosis of MF, while no association with essential thrombocythemia (ET), polycythemia vera (PV), and prefibrotic-PMF (pre-PMF) was found. Regarding MPN-directed therapy ongoing at the time of COVID-19 diagnosis, Ruxolitinib (Ruxo) was significantly more frequent in patients who died in comparison with survivors (p=0.006). Conversely, multivariable analysis found no effect of Ruxo alone on mortality, but highlighted an increased risk of death in the 11 out of 45 patients who discontinued treatment. These findings were also confirmed in a propensity score matching analysis. In conclusion, we found a high risk of mortality during COVID-19 infection among MPN patients, especially in MF patients and/or discontinuing Ruxo at COVID-19 diagnosis. These findings call for deeper investigation on the role of Ruxo treatment and its interruption, in affecting mortality in MPN patients with COVID-19.

Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2P95-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.

Philadelphia-chromosome negative myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by an increased risk of thrombosis and progression to acute myeloid leukemia. MPN are associated with driver mutations in JAK2, CALR and MPL which are crucial for the diagnosis and lead to a constitutive activation of the JAK-STAT signaling, independent of cytokine regulation. Moreover, most patients have concomitant mutations in genes involved in DNA methylation, chromatin modification, messenger RNA splicing, transcription regulation and signal transduction. These additional mutations may arise before, in the context of clonal hematopoiesis of indeterminate potential (CHIP), or after the acquisition of the driver mutation. The clinical phenotype of MPN results from complex interactions between mutations and host factors. The increased application of next-generation sequencing (NGS) techniques to a large series of patients with MPN has expanded the knowledge of mutational landscape and contributed to define the clinical significance of mutations. This molecular information is being increasingly used to refine diagnosis, risk stratification, monitoring of residual disease and response to treatment. ASXL1, SRSF2, EZH2, IDH1/IDH2 and U2AF1 mutations are associated with a more advanced disease and reduced overall survival in primary myelofibrosis (PMF), whereas spliceosome mutations in Polycythemia vera (PV) and essential thrombocythemia (ET) adversely affect both overall (SF3B1, SRSF2 in ET and SRSF2 in PV) and myelofibrosis-free (U2AF1, SF3B1 in ET) survival. This review discusses current knowledge of the molecular landscape of MPN, and how the availability of those molecular information may impact patient management.

The dysregulation of the JAK/STAT pathway drives the pathogenesis of myelofibrosis (MF). Recently, several JAK inhibitors (JAKis) have been developed for treating MF. Select mutations (MTs) have been associated with impaired outcomes and are currently incorporated in molecularly annotated prognostic models. Mutations of RAS/MAPK pathway genes are frequently reported in cancer and at low frequencies in MF. In this study, we investigated the phenotypic, prognostic, and therapeutic implications of NRASMTs, KRASMTs, and CBLMTs (RAS/CBLMTs) in 464 consecutive MF patients. A total of 59 (12.7%) patients had RAS/CBLMTs: NRASMTs, n = 25 (5.4%); KRASMTs, n = 13 (2.8%); and CBLMTs, n = 26 (5.6%). Patients with RAS/CBLMTs were more likely to present with high-risk clinical and molecular features. RAS/CBLMTs were associated with inferior overall survival compared with patients without MTs and retained significance in a multivariate model, including the Mutation-Enhanced International Prognostic Score System (MIPSS70) risk factors and cytogenetics; however, inclusion of RAS/CBLMTs in molecularly annotated prognostic models did not improve the predictive power of the latter. The 5-year cumulative incidence of leukemic transformation was notably higher in the RAS/CBLMT cohort. Among 61 patients treated with JAKis and observed for a median time of 30 months, the rate of symptoms and spleen response at 6 months was significantly lower in the RAS/CBLMT cohort. Logistic regression analysis disclosed a significant inverse correlation between RAS/CBLMTs and the probability of achieving a symptom or spleen response that was retained in multivariate analysis. In summary, our study showed that RAS/CBLMTs are associated with adverse phenotypic features and survival outcomes and, more important, may predict reduced response to JAKis.

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the cornerstone of curative approach to myelofibrosis (MF), although it is burdened by not negligible toxicity and mo...

Pre-fibrotic myelofibrosis (pre-PMF) and essential thrombocythemia (ET) are characterized by similarly increased rate of thrombotic events, but no study specifically analyzed risk factors for thrombosis in pre-PMF. In a multicenter cohort of 382 pre-PMF patients collected in this study, the rate of arterial and venous thrombosis after diagnosis was 1.0 and 0.95% patients/year. Factors significantly associated with arterial thrombosis were age, leukocytosis, generic cardiovascular risk factors, JAK2V617F and high molecular risk mutations, while only history of previous thrombosis, particularly prior venous thrombosis, was predictive of venous events. The risk of total thromboses was accurately predicted by the the international prognostic score for thrombosis in essential thrombocythemia (IPSET) score, originally developed for ET, and corresponded to 0.67, 2.05, and 2.95% patients/year in the low-, intermediate-, and high-risk categories. IPSET was superior to both the conventional 2-tiered score and the revised IPSET in this cohort of pre-PMF patients. We conclude that IPSET score can be conveniently used for thrombosis risk stratification in patients with pre-PMF and might represent the basis for individualized management aimed at reducing the increased risk of major cardiovascular events. Further refinement of the IPSET score in pre-PMF might be pursued by additional, prospective studies evaluating the inclusion of leukocytosis and/or adverse mutational profile as novel variables.

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

The management of patients with myelofibrosis (MF) has dramatically changed since the introduction of ruxolitinib as a tailored treatment strategy. However, the perceptions about the use of this drug in clinical practice remain, at times, a matter of discussion. We conducted a survey about the diagnostic evaluation, prognostic assessment, and management of ruxolitinib in real-life clinical practice in 18 Italian hematology centers. At diagnosis, most hematologists do not use genetically or molecularly inspired score systems to assess prognosis, mainly due to scarce availability of next-generation sequencing (NGS) methodology, with NGS conversely reserved only for a subset of lower-risk MF patients with the aim of possibly improving the treatment strategy. Some common points in the management of ruxolitinib were 1) clinical triggers for ruxolitinib therapy, regardless of risk category; 2) evaluation of infectious risk before the starting of the drug; and 3) schedule of monitoring during the first 12 weeks with the need, in some instances, of supportive treatment. Further development of international recommendations and insights will allow the achievement of common criteria for the management of ruxolitinib in MF, before and after treatment, and for the definition of response and failure. Electronic supplementary material The online version of this article (10.1007/s00277-019-03847-z) contains supplementary material, which is available to authorized users.

ROMEI, a prospective, observational study in patients with myelofibrosis receiving the oral JAK1/2 inhibitor ruxolitinib in real-world practice, assesses treatment adherence based on the 8-item Morisky Medication Adherence Scale (MMAS-8). Here, we present MMAS-8 results at week 24. Overall, 101 of 188 evaluable patients completed the questionnaire at every visit (full completers). Mean (±standard deviation) total MMAS-8 scores remained stable from week 4 to week 24 in the overall population (7.54 ± 0.77 and 7.67 ± 0.70, respectively) and full completers (7.53 ± 0.79 and 7.67 ± 0.73, respectively). Rates of low (MMAS-8 ˂6) or medium (MMAS-8 ≥ 6 to ˂8) adherence were 25–40% and 26–36%, respectively. Fifty-five full completers (54%) reported ≥1 change in adherence category (improvement and/or worsening), most of which were associated with unintentional behavior. The data suggest that one-third of patients receiving ruxolitinib may be undertreated due to non-adherence, potentially undermining disease control, and indicate a need for better interventions addressing noncompliance to oral therapies.

TO THE EDITOR: The discovery of prognostically informative mutations in patients with primary myelofibrosis (PMF)[1][1] prompted the development of mutation-enhanced risk scores.[2][2][⇓][3][⇓][4][⇓][5]-[6][6] Among these mutations, those in ASXL1 are invariably associated with adverse

Systemic mastocytosis (SM) is characterized by extreme heterogeneity of manifestations and prognosis. Several disease-related biomarkers, including clinical, hematological and molecular variables, have been correlated with prognosis. Although relevant, the mutation profile closely reflects the WHO classification that has per se prognostic value. High-risk mutations (HRM) are largely confined to advanced forms, and thus fail in providing information regarding progression and outcome in the not-advanced variants. In this work, we studied hematopoietic cells by multi-parameter flow cytometry (MFC) in order to highlight dysplastic traits that might provide insights into outcome. A score previously validated for myelodysplastic syndromes, with high reproducibility in standard diagnostics, was used. The application of an MFC score to a cohort of 71 SM cases, concurrently genotyped for configuring a HRM category, resulted in the identification of two separate patients' categories (MFC+ and MFC-) characterized by significantly different clinical and laboratory features at presentation. The extent of dysplasia by MFC tended to parallel WHO-category and genotype-related stratification. MFC+ patients had shorter survival compared to MFC- ones, for whom the incidence of progression and/or death was virtually null. Of note, MFC score remained prognostically informative in unadvanced subsets. Furthermore, the integration of MFC and HRM was an independent predictor for outcome, also overcoming WHO-categories in multivariate analysis for EFS. Our results support the use of MFC analysis in the evaluation of patients with SM, alone and in combination with HRM, for refinement of prognosis assessment.

Ruxolitinib, a potent Janus kinase 1/2 inhibitor, has demonstrated durable improvements in patients with myelofibrosis. In this analysis of the Phase 3b JUMP study, which included patients aged ≥18 years with a diagnosis of primary or secondary myelofibrosis, we assessed the safety and efficacy of ruxolitinib in patients stratified by Dynamic International Prognostic Scoring System (DIPSS) risk categories. Baseline characteristic data were available to assess DIPSS status for 1844 of the 2233 enrolled patients; 60, 835, 755, and 194 in the low‐, intermediate (Int)‐1‐, Int‐2‐, and high‐risk groups, respectively. Ruxolitinib was generally well tolerated across all risk groups, with an adverse‐event (AE) profile consistent with previous reports. The most common hematologic AEs were thrombocytopenia and anemia, with highest rates of Grade ≥3 events in high‐risk patients. Approximately, 73% of patients experienced ≥50% reductions in palpable spleen length at any point in the ≤24‐month treatment period, with highest rates in lower‐risk categories (low, 82.1%; Int‐1, 79.3%; Int‐2, 67.1%; high risk, 61.6%). Median time to spleen length reduction was 5.1 weeks and was shortest in lower‐risk patients. Across measures, 40%–57% of patients showed clinically meaningful symptom improvements, which were observed from 4 weeks after treatment initiation and maintained throughout the study. Overall survival (OS) was 92% at Week 72 and 75% at Week 240 (4.6 years). Median OS was longer for Int‐2‐risk than high‐risk patients (253.6 vs. 147.3 weeks), but not evaluable in low‐/Int‐1‐risk patients. By Week 240, progression‐free survival (PFS) and leukemia‐free survival (LFS) rates were higher in lower‐risk patients (PFS: low, 90%; Int‐1, 82%; Int‐2, 46%; high risk, 15%; LFS: low, 92%; Int‐1, 86%; Int‐2, 58%; high risk, 19%). Clinical benefit was seen across risk groups, with more rapid improvements in lower risk patients. Overall, this analysis indicates that ruxolitinib benefits lower‐risk DIPSS patients in addition to higher risk.

Myeloproliferative neoplasms (MPN) patients share driver mutations in JAK2, MPL or CALR genes leading to the activation of the thrombopoietin receptor (TPOR) and downstream signaling pathways. JAK2 mutation drives all the three major entities of MPN (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis) through the constitutive activation of TPOR, erythropoietin (EPOR) and colony stimulating factor 3 receptor (CSF3R) signaling. MPL is a proto-oncogene encoding for TPOR, the hematopoietic growth factor receptor of myeloid stem cells. MPL mutants induce the stable dimerization of TPOR that in turn activate JAK2 and the thrombopoietin pathway. The thrombopoietin pathway plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of hematopoietic stem cells. Little wonder therefore that mutations of MPL result in thrombocytosis, leading to an abnormal MPL trafficking or receptor activation. Finally, some extremely rare germline genetic variants in MPL can induce MPN-like hereditary disease. Against this molecular background, TPOR is a key actor in the MPN development and MPL mutations are of major relevance to fully elucidate the molecular mechanisms underlying the clinical manifestations of MPN and to arrange novel therapeutic strategies aiming to disrupt the dysegulated signaling cascade. This chapter will focus on the role MPL in the pathogenesis of MPN and in familial thrombocytosis and will review these different subtypes of somatic and germline genetic variants by dissecting how they impact clinical phenotype.

Data from the large, prospective, multinational, phase 3b JUMP study were analyzed to identify factors predictive of spleen and symptom responses in myelofibrosis patients receiving ruxolitinib. Factors associated with higher spleen response rates included International Prognostic Scoring System (IPSS) low/intermediate-1 risk vs intermediate-2/high risk (43.1% vs 30.6%; adjusted OR [aOR] 0.65 [95% CI 0.44-0.95]), ruxolitinib as first- vs second- or later-line therapy (40.2% vs 31.5%; aOR 0.53 [95% CI 0.38-0.75]), and a ruxolitinib total daily dose at Week 12 of20 mg/day vs ≤20 mg/day (41.3% vs 30.4%; aOR 0.47 [95% CI 0.33-0.68]). No association was seen between baseline characteristics or total daily dose at Week 12 and symptom response. Ruxolitinib led to higher spleen response rates in patients with lower IPSS risk, and when used earlier in treatment. Higher doses of ruxolitinib were associated with higher spleen response rates, but not with symptom improvement.Trial registrationINC424 for patients with primary myelofibrosis, post polycythemia myelofibrosis or post-essential thrombocythemia myelofibrosis (JUMP).2010-024473-39; NCT01493414Date of registration: 16 December 2011https://www.clinicaltrialsregister.eu/ctr-search/search?query=2010-024473-39https://clinicaltrials.gov/ct2/show/NCT01493414.

Among 382 patients with WHO-defined prefibrotic myelofibrosis (pre-PMF) followed for a median of 6.9 years, fibrotic or leukemic transformation or death accounts for 15, 7, and 27% of cases, respectively. A multistate model was applied to analyze survival data taking into account intermediate states that are part of the clinical course of pre-PMF, including overt PMF and acute myeloid leukemia (AML). Within this multistate framework, multivariable models disclosed older age (>65 years) and leukocytosis (>15 × 109/L) as predictors of death and leukemic transformation. The risk factors for fibrotic progression included anemia and grade 1 bone marrow fibrosis. The outcome was further affected by high molecular risk (HMR) but not driver mutations. Direct transition to overt PMF, AML, or death occurred in 15.2, 4.7, and 17.3% of patients, respectively. The risk of AML was the highest in the first 5 years (7%), but leveled off thereafter. Conversely, the probability of death from overt PMF or AML increased more rapidly over time, especially when compared to death in the pre-PMF state without disease progression. The probability of being alive with pre-PMF status decreased to 70 and 30% at 10 and 20 years, respectively. This study highlights the aspects of the clinical course and estimates of disease progression in pre-PMF.

Summary Background There is no evidence that phlebotomy alone is sufficient to steadily maintain haematocrit on target level in low-risk patients with polycythaemia vera. This study aimed to compare the efficacy and safety of ropeginterferon alfa-2b on top of the standard phlebotomy regimen with phlebotomy alone. Methods In 2017, we launched the Low-PV study, a multicentre, open-label, two-arm, parallel-group, investigator-initiated, phase 2 randomised trial with a group-sequential adaptive design. The study involved 21 haematological centres across Italy. Participants were recruited in a consecutive order. Participants enrolled in the study were patients, aged 18–60 years, with a diagnosis of polycythaemia vera according to 2008–16 WHO criteria. Eligible patients were randomly allocated (1:1) to receive either phlebotomy and low-dose aspirin (standard group) or ropeginterferon alfa-2b on top of the standard treatment (experimental group). Randomisation sequence was generated using five blocks of variable sizes proportional to elements of Pascal's triangle. Allocation was stratified by age and time from diagnosis. No masking was done. Patients randomly allocated to the standard group were treated with phlebotomy (300 mL for each phlebotomy to maintain the haematocrit values of lower than 45%) and low-dose aspirin (100 mg daily), if not contraindicated. Patients randomly allocated to the experimental group received ropeginterferon alfa-2b subcutaneously every 2 weeks in a fixed dose of 100 μg on top of the phlebotomy-only regimen. The primary endpoint was treatment response, defined as maintenance of the median haematocrit values of 45% or lower without progressive disease during a 12-month period. Analyses were done by intention-to-treat principle. The study was powered assuming a higher percentage of responders in the experimental group (75%) than in the standard group (50%). Here we report results from the second planned interim analysis when 50 patients had been recruited to each group. The trial is ongoing, and registered with ClinicalTrials.gov , NCT03003325 . Findings Between Feb 2, 2017, and March 13, 2020, 146 patients were screened, and 127 patients were randomly assigned to the standard group (n=63) or the experimental group (n=64). The median follow-up period was 12·1 months (IQR 12·0–12·6). For the second pre-planned interim analysis, a higher response rate in the experimental group was seen (42 [84%] of 50 patients) than in the standard group (30 [60%] of 50 patients; absolute difference 24%, 95% CI 7–41%, p=0·0075). The observed z value (2·6001) crossed the critical bound of efficacy (2·5262), and the stagewise adjusted p value early showed superiority of experimental treatment. Thus, the data safety monitoring board decided to stop patient accrual for overwhelming efficacy and to continue the follow-up, as per protocol, for 2 years. Under the safety profile, no statistically significant difference between groups in frequency of adverse events of grade 3 or higher was observed; the most frequently reported adverse events were neutropenia (four [8%] of 50 patients) in the experimental group and skin symptoms (two [4%] of 50 patients) in the standard group. No grade 4 or 5 adverse events occurred. Interpretation Supplementing phlebotomy with ropeginterferon alfa-2b seems to be safe and effective in steadily maintaining haematocrit values on target in low-risk patients with polycythaemia vera. Findings from the current study might have implications for changing the current management of low-risk patients with polycythaemia vera. Funding AOP Orphan Pharmaceuticals, Associazione Italiana per la Ricerca sul Cancro

Numerous studies have documented ultrastructural abnormalities in malignant megakaryocytes from bone marrow (BM) of myelofibrosis patients but the morphology of these cells in spleen, an important extramedullary site in this disease, was not investigated as yet. By transmission-electron microscopy, we compared the ultrastructural features of megakaryocytes from BM and spleen of myelofibrosis patients and healthy controls. The number of megakaryocytes was markedly increased in both BM and spleen. However, while most of BM megakaryocytes are immature, those from spleen appear mature with well-developed demarcation membrane systems (DMS) and platelet territories and are surrounded by platelets. In BM megakaryocytes, paucity of DMS is associated with plasma (thick with protrusions) and nuclear (dilated with large pores) membrane abnormalities and presence of numerous glycosomes, suggesting a skewed metabolism toward insoluble polyglucosan accumulation. By contrast, the membranes of the megakaryocytes from the spleen were normal but these cells show mitochondria with reduced crests, suggesting deficient aerobic energy-metabolism. These distinctive morphological features suggest that malignant megakaryocytes from BM and spleen express distinctive metabolic impairments that may play different roles in the pathogenesis of myelofibrosis.

Polycythemia vera (PV) is the most common myeloproliferative neoplasm (MPN). PV is characterized by erythrocytosis, leukocytosis, thrombocytosis, increased hematocrit, and hemoglobin in the peripheral blood. Splenomegaly and myelofibrosis often occur in PV patients. Almost all PV patients harbor a mutation in theThis article examines the recentPreclinical models of PV are valuable tools for enabling an understanding of the pathophysiology and the molecular mechanisms of the disease. These models provide new biological insights on the contribution of concomitant mutations and the efficacy of novel drugs in a 'more faithful' setting. This may facilitate an enhanced understanding of pathogenetic mechanisms and targeted therapy.

Since myeloproliferative neoplasms (MPN) pose a significant risk for vascular and thrombotic complications, cytoreductive therapies, such as hydroxyurea (HU), interferon (IFN) inhibitors, and Janus kinase (JAK) inhibitors are recommended for patients at high risk. However, these agents also place patients at increased risk for drug-related cutaneous adverse events. Herein, we review the literature on skin toxicity related to the use of drugs for the treatment of MPN. Overall, the cytoreductive agents used for MPN are generally well tolerated and considered to be safe, except IFN, for which dropout rates as high as 25% have been reported. While IFN is known to give rise to flu syndrome, it rarely leads to hematological alterations. The most common hematological side effects of HU are mild and include granulocytopenia, anemia, and thrombocytopenia. The JAK inhibitor ruxolitinib has been associated with cytopenia and a higher incidence of viral infections, as well as increased risk for basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Based on the present analysis, it can be concluded that cutaneous toxicity is not a negligible complication of commonly used treatments for MPN. While further research is needed, patients on these agents, and especially those with a history of cutaneous malignancies, should undergo thorough skin examination before and during therapy. In addition, detailed history is critical since many patients who develop non-melanoma skin cancer have multiple preexisting risk factors for cutaneous carcinogenesis.

Myelofibrosis (MF), either appearing de novo (primary MF, PMF) or after a previous diagnosis of essential thrombocythemia or of polycythemia vera, is a progressive disease burdened by symptomatic splenomegaly, debilitating systemic symptoms, ineffective hematopoiesis, and overall reduced survival. Patients often present worsening cytopenias, including thrombocytopenia, secondary to progression of the disease as well as to cytoreductive treatment. Patients with MF and thrombocytopenia have few therapeutic options and there is limited information regarding the management of disease in these settings. This article reviews current evidence for the management of patients with MF and thrombocytopenia, in the era of JAK inhibitors.

Myeloproliferative Neoplasms (MPN) course can be complicated by thrombosis involving unusual sites as the splanchnic veins (SVT). Their management is challenging, given their composite vascular risk. We performed a retrospective, cohort study in the framework of the International Working Group for MPN Research and Treatment (IWG-MRT), and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). A total of 518 MPN-SVT cases were collected and compared with 1628 unselected, control MPN population, matched for disease subtype. Those with MPN-SVT were younger (median 44 years) and enriched in females compared to controls; PV (37.1%) and ET (34.4%) were the most frequent diagnoses. JAK2V617F mutation was highly prevalent (90.2%), and 38.6% of cases had an additional hypercoagulable disorder. SVT recurrence rate was 1.6 per 100 patient-years. Vitamin K-antagonists (VKA) halved the incidence of recurrence (OR 0.48), unlike cytoreduction (OR 0.96), and were not associated with overall or gastrointestinal bleeding in multivariable analysis. Esophageal varices were the only independent predictor for major bleeding (OR 17.4). Among MPN-SVT, risk of subsequent vascular events was skewed towards venous thromboses compared to controls. However, MPN-SVT clinical course was overall benign: SVT were enriched in PMF with lower IPSS, resulting in significantly longer survival than controls; survival was not affected in PV and slightly reduced in ET. MPN-U with SVT (n = 55) showed a particularly indolent phenotype, with no signs of disease evolution. In the to-date largest, contemporary cohort of MPN-SVT, VKA were confirmed effective in preventing recurrence, unlike cytoreduction, and safe; the major risk factor for bleeding was esophageal varices that therefore represent a major therapeutic target.

To describe an algorithm-based approach, whenever available, to the diagnosis, the risk stratification criteria informing therapy and the current management of polycythemia vera and essential thrombocythemia.Description of recurrent genetic abnormalities in driver genes, including Janus Kinase 2 (JAK2), myeloproliferative leukemia and calreticulin, a better appreciation of the key diagnostic role of bone marrow features, results of large epidemiologic studies and a few but landmark controlled clinical trials produced in the last decade, all resulted in a reappraisal of the approach to polycythemia vera and essential thrombocythemia.The revised 2017 WHO classification of polycythemia vera and essential thrombocythemia allows early diagnosis and accurate distinction from other chronic myeloproliferative neoplasms, particulary prefibrotic myelofibrosis. The prognostic value of selected mutations is being appreciated and JAK2V617F mutation is currently incorporated as risk variable in prognostic system for essential thrombocythemia. Risk-adjusted stratification is used to select therapeutic approaches that include target agents. However, there is not yet a curative approach to these hematologic neoplasms, and although their management has much improved in the last decades, the associated morbidity and mortality remains significant and may be worsened by toxicities of therapeutic agents. Therefore, several clinically relevant endpoints remain unmet.

Splenic hematopoiesis is a major feature in the course of myelofibrosis (MF). In fact, the spleen of patients with MF contains malignant hematopoietic stem cells retaining a complete differentiation program, suggesting both a pivotal role of the spleen in maintaining the disease and a tight regulation of hematopoiesis by the splenic microenvironment, in particular by mesenchymal stromal cells (MSCs). Little is known about splenic MSCs (Sp-MSCs), both in normal and in pathological context. In this work, we have in vitro expanded and characterized Sp-MSCs from 25 patients with MF and 13 healthy subjects (HS). They shared similar phenotype, growth kinetics, and differentiation capacity. However, MF Sp-MSCs expressed significant lower levels of nestin, and favored megakaryocyte (Mk) differentiation in vitro at a larger extent than their normal counterpart. Moreover, they showed a significant upregulation of matrix metalloprotease 2 (MMP2) and fibronectin 1 (FN1) genes both at mRNA expression and at protein level, and, finally, developed genetic abnormalities which were never detected in HS-derived Sp-MSCs. Our data point toward the existence of a defective splenic niche in patients with MF that could be responsible of some pathological features of the disease, including the increased trafficking of CD34+ cells and the expansion of the megakaryocytic lineage.

WOS: 000426644800007, PubMed ID: 29396713, Ruxolitinib was well tolerated and superior to best available therapy (including interferon [IFN]) in controlling hematocrit without phlebotomy eligibility, normalizing blood counts, and improving polycythemia vera-related symptoms in the Study of Efficacy and Safety in Polycythemia Vera Subjects Who Are Resistant to or Intolerant of Hydroxyurea: JAK Inhibitor INC424 (INCB018424) Tablets Versus Best Available Care (RESPONSE) studies. This ad hoc analysis focuses on ruxolitinib in relation to IFN in the RESPONSE studies, with attention on the following: (1) safety and efficacy of ruxolitinib and best available therapy in patients who received IFN before study randomization, (2) safety and efficacy of IFN during randomized treatment in best available therapy arm, and (3) use of ruxolitinib after crossover from best available therapy in IFN-treated patients. IFN exposure before randomization had little effect on the efficacy or safety of ruxolitinib. In the randomized treatment arms, ruxolitinib was superior to IFN in efficacy [hematocrit control (RESPONSE = 60% of ruxolitinib vs 23% of IFN patients; RESPONSE-2 = 62% of ruxolitinib vs 15% of IFN patients)] and was tolerated better in hydroxyurea-resistant or hydroxyurea-intolerant patients. After crossing over to receive ruxolitinib, patients who had initially received IFN and did not respond had improved hematologic and spleen responses (62% of patients at any time after crossover) and an overall reduction in phlebotomy procedures. Rates and incidences of the most common adverse events decreased after crossover to ruxolitinib, except for infections (primarily grade 1 or 2). These data suggest that ruxolitinib is efficacious and well tolerated in patients who were previously treated with IFN. The RESPONSE (NCT01243944) and RESPONSE-2 (NCT02038036) studies were registered at clinicaltrials.gov., Novartis (CH); Novartis Pharmaceuticals Corporation (US) [Not applicable]

Purpose To develop a prognostic system for transplantation-age patients with primary myelofibrosis (PMF) that integrates clinical, cytogenetic, and mutation data. Patients and Methods The study included 805 patients with PMF age ≤ 70 years recruited from multiple Italian centers and the Mayo Clinic (Rochester, MN), forming two independent learning and validation cohorts. A Cox multivariable model was used to select from among a list of 22 variables those that were predictive of overall survival (OS). Integrated clinical and genetic prognostic models with (MIPSS70-plus) or without (MIPSS70) cytogenetic information were developed. Results Multivariable analysis identified the following as significant risk factors for OS: hemoglobin < 100 g/L, leukocytes > 25 × 109/L, platelets < 100 × 109/L, circulating blasts ≥ 2%, bone marrow fibrosis grade ≥ 2, constitutional symptoms, absence of CALR type-1 mutation, presence of high–molecular risk mutation (ie, ASXL1, EZH2, SRSF2, IDH1/ 2), and presence of two or more high–molecular risk mutations. By assigning hazard ratio (HR)–weighted points to these variables, three risk categories were delineated for the MIPSS70 model; 5-year OS was 95% in low-risk, 70% in intermediate-risk, and 29% in high-risk categories, corresponding to median OS of 27.7 years (95% CI, 22 to 34 years), 7.1 years (95% CI, 6.2 to 8.1 years), and 2.3 years (95% CI, 1.9 to 2.7 years), respectively. In the MIPSS70-plus model, which included cytogenetic information, four risk categories were delineated, with 5-year OS of 91% in low-risk, 66% in intermediate-risk (HR, 3.2; 95% CI, 1.9 to 5.2), 42% in high-risk (HR, 6.4; 95% CI, 4.1 to 10.0), and 7% very high–risk categories (HR, 17.0; 95% CI, 9.8 to 29.2). Both models remained effective after inclusion of older patients in the analysis. Conclusion MIPSS70 and MIPSS70-plus provide complementary systems of risk stratification for transplantation-age patients with PMF and integrate prognostically relevant clinical, cytogenetic, and mutation data.

The new edition of the 2016 World Health Organization (WHO) classification system for tumors of the hematopoietic and lymphoid tissues was published in September 2017. Under the category of myeloproliferative neoplasms (MPNs), the revised document includes seven subcategories: chronic myeloid leukemia, chronic neutrophilic leukemia, polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia-not otherwise specified and MPN, unclassifiable (MPN-U); of note, mastocytosis is no longer classified under the MPN category. In the current review, we focus on the diagnostic criteria for JAK2/CALR/MPL mutation-related MPNs: PV, ET, and PMF. In this regard, the 2016 changes were aimed at facilitating the distinction between masked PV and JAK2-mutated ET and between prefibrotic/early and overtly fibrotic PMF. In the current communication, we (i) provide practically useful resource tables and graphs on the new diagnostic criteria including outcome, (ii) elaborate on the rationale for the 2016 changes, (iii) discuss the complementary role of mutation screening, (iv) address ongoing controversies and propose solutions, (v) attend to the challenges of applying WHO criteria in routine clinical practice, and (vi) outline future directions from the perspectives of the clinical pathologist.

A total of 410 patients with blast phase myeloproliferative neoplasm (MPN-BP) were retrospectively reviewed: 248 from the Mayo Clinic and 162 from Italy. Median survival was 3.6 months, with no improvement over the last 15 years. Multivariable analysis performed on the Mayo cohort identified high risk karyotype, platelet count 65 years and transfusion need as independent risk factors for survival. Also in the Mayo cohort, intensive chemotherapy resulted in complete remission (CR) or CR with incomplete count recovery (CRi) rates of 35 and 24%, respectively; treatment-specified 3-year/5-year survival rates were 32/10% for patients receiving allogeneic stem cell transplant (AlloSCT) (n = 24), 19/13% for patients achieving CR/CRi but were not transplanted (n = 24), and 1/1% in the absence of both AlloSCT and CR/CRi (n = 200) (p

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms that are characterized by thrombohemorrhagic complications, symptom burden, and impaired survival mainly due to thrombosis, progression to myelofibrosis, and transformation to acute leukemia. In this manuscript, we will review the most recent changes in diagnostic criteria, the improvements in risk stratification, and the “state of the art” in the daily management of these disorders. The role of conventional therapies and novel agents, interferon α and the JAK2 inhibitor ruxolitinib, is critically discussed based on the results of a few basic randomized clinical studies. Several unmet needs remain, above all, the lack of a curative approach that might overcome the still burdensome morbidity and mortality of these hematologic neoplasms, as well as the toxicities associated with therapeutic agents.

Background. The tendency towards thrombosis in myeloproliferative neoplasms (MPNs) is linked to the JAK2-mutant clone which leads to hypercellularity and functional interplay between abnormal erythrocytes, platelets, leukocytes and dysfunctional endothelium. The resulting cell activation that also involves stromal cells in their microenvironment, has been shown associated with chronic, systemic, subclinical pro-inflammatory state, and has been implicated in the pathogenesis of thrombosis in MPN. Neutrophil to lymphocyte ratio (NLR) is a novel inflammatory marker found to be associated with the severity and prognosis of cardiovascular diseases but there is little evidence for its prognostic significance in MPN. We investigated whether NLR could predict the onset of arterial and venous thrombosis in polycythemia vera (PV). Methods. Subjects of this study were 1508 patients included in the ECLAP trail with NLR evaluation available. In addition to standard statistical methods, we used proportional hazards additive models (GAM) as they can provide an excellent fit in the presence of nonlinear relationships, for the prediction of total, arterial and venous thrombosis as smooth functions with cubic splines of different blood parameters. Results. After a median follow-up of 2.76 years, 169 thrombotic events (10.3%) were objectively diagnosed and analyzed: 87 arterial (MI, Stroke, TIA, PAT) and 88 venous thrombosis (DVT±PE, superficial thrombophlebitis). In univariate analysis, arterial thrombosis was associated with age (p=0.003) and previous thrombosis, especially if arterial (p Conclusions. The NLR is an inexpensive and easily accessible test compared to other inflammatory markers. We found that NLR-alone is an independent predictor of venous thrombosis and could be included in a new scoring system. In addition to guiding the stratification of thrombotic risk, these data confirm that inflammation is a relevant target of antithrombotic therapy in PV. Figure 1 Figure 1. Disclosures Vannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background The detrimental effect of leukocytosis on survival in myeloproliferative neoplasms (MPN) has been well established for primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET ) (JCO. 2018;36:310; BJH. 2020;189:291) Previous studies have also implicated leukocytosis as a risk factor for leukemic transformation (Mayo Clin Proc. 2017;92:1118) and thrombosis in MPN (Blood Adv. 2019;3:1729). However, it is currently not clear as to which component(s) of white blood cells is responsible for these observations. In the current study, we sought to examine the individual prognostic contribution of absolute neutrophil (ANC), lymphocyte (ALC) and monocyte (AMC) counts, on overall (OS), leukemia-free (LFS), and myelofibrosis-free (MFFS) survival and in ET. Methods Study patients (n=349) were retrospectively recruited from the Mayo Clinic MPN database of 1,249 WHO-defined ET patients, evaluated over five decades (1967-2021), based on availability of information on ANC, ALC and AMC. Conventional criteria were used for diagnosis and definitions of major complications, including leukemic or fibrotic transformation (Blood 2016;127:2391). Conventional statistical methods were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC. Multivariable analyses included previously established risk factors for survival. Results 349 patients (median age 57 years, range 18-89; females 61%) with ET were included in the study: 46% JAK2, 34% CALR, 16% triple-negative and 4% MPL mutated; IPSET risk category high 24%, intermediate 41%, and low 35%; presenting median (range) values were 13.8 g/dL (11.1-16.4) for hemoglobin, 8.2 x 10(9)/L (3.2-52) for leukocyte count, and 859 x 10(9)/L (451-3460) for platelet count; palpable splenomegaly was present in 48 (14%); median followup was 10 years (range 0-47), during which time 118 deaths, 52 fibrotic progressions, and 14 leukemic transformations were documented. Multivariable analysis identified older age (p60 years, 3.1 (2.1-4.6; p60 years), 2 for increased ANC (≥8 x 10(9)/L) and 1 for ALC ( An external validation cohort from the University of Florence (n=485) confirmed the independent survival risk contribution from age >60 years (p Conclusions: The current study identifies increased ANC and decreased ALC as age-independent risk factors for survival in ET, thus allowing the development of a globally applicable simple to use "Triple A" risk model that is based on Age, ANC and ALC. Decreased ALC also predicted fibrotic and leukemic progression. Our observations suggest potential value for immune profiling as an additional prognostic tool in MPN. Figure 1 Figure 1. Disclosures Szuber: Novartis: Honoraria. Vannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Introduction Hereditary alpha-Tryptasemia (HαT) is a group of genetically defined traits that share increased copy number of TPSAB1 gene encoding for both the α- and β-alleles (Lyons et al 2018). Increased copy number (CN) of the α-tryptase coding sequence in TPSAB1 on one or both alleles represents the genetic base of HαT (Lyons et al 2016). HαT is characterized by mild elevation in serum tryptase levels and a variety of mast cell (MC) activation symptoms, including recurrent anaphylaxis. Prevalence in the general population is up to 5%, that increases up to 20% in systemic mastocytosis (SM); it has been suggested that HαT might be a germline variant predisposing to SM development. In SM, HαT correlated with higher incidence of mediator-related symptoms (Greiner G et al, Blood 2021). Due to its complexity, the assay for TPSAB1 CN is performed in few centers and the actual prevalence of HαT in selected subsets is still to be elucidated. To this end, we screened for HαT 2 groups of subjects, the first with MC activation symptoms and no evidence of SM, the second with diagnosis of SM according to WHO-2016. Methods Droplet digital PCR (ddPCR) was used to measure CN variation (CNV) in TPSAB1 by adapting standard CNV ddPCR protocol to genotype for both TPSAB1 and TPSB2 (Fig.1A). The high homology between α and β encoding isoforms and the presence of paralogous genes in a single locus makes TPSAB1 CNV detection very challenging. ddPCR was performed on genomic DNA with/without BamHI, using the PrimePCR ddPCR Copy Number reference AP3B1 (BioRad). Accuracy and precision of the ddPCR protocol was assessed by analyzing 10 samples in triplicate in 3 separate experiments. Data robustness and repeatability can be appreciated in Fig 1B. Results We studied 41 subjects with mediator-related symptoms and augmented basal serum tryptase (BST) (cohort 1) and 150 patients with ascertained diagnosis of mastocytosis (cohort 2). The BST threshold established for cohort 1 was equal or higher than 11 mcg/L. Median age was 64.7 yr, males 54%; median BST levels was 15.3 (range 12.3-21 mcg/L); 29% of the pts had history of anaphylaxis. In cohort 2, 134/150 (89.3%) pts had a diagnosis of SM, whereas 13/150 (8,6%) were Cutaneous Mastocytosis (CM). Among SM patients, 113(84.3%) presented with non-advanced SM variants. Advanced forms including aggressive SM (ASM) and SM with an associated hematological neoplasm (SM-AHN) were diagnosed in 6 (4.5%) and 8 (6%) respectively. In 3 pts, SM subtype was not available. Median age was 49 yr, males 55%; 41.7% of the pts had history of anaphylaxis. HαT was documented in 27 (65.9%) subjects in cohort 1, and 14 (9.3%) in cohort 2. In cohort 1, 3 α-tryptase (3α) copy number was observed more frequently (59.2% of HαT+ pts); conversely 3α and 2α-tryptase copy number were observed at a similar rate (42,8%) in cohort 2. HαT+ pts in cohort 1 presented significantly higher BST (17.1 vs 12.05 mgc/L, P In cohort 2, BST levels were similar in HαT+ and HαT wt pts (24.6 and 24.3 mcg/L), as were anaphylaxis episodes (50% and 41%, respectively). A trend for lower MC burden in HαT+ as assessed by flow cytometry was demonstrated (% of bone marrow MC: 0.01% in HαT+ vs 0.07%) whereas no meaningful differences emerged regarding the symptom burden. In addition, a lower prevalence of KIT 816V mutation was observed in HαT+ (71.4% vs 89.5%; p=0.073). Conclusions In our study HαT+ was observed in around 10% of patients with SM, a prevalence lower than previously reported (Greiner et al, Blood 2021) and remarkably lower than in a selected cohort of subjects with raised BTL and history of mediator-released symptoms (66%). ddPCR represents a suitable method to investigate the presence of CNV in the α-tryptase coding sequence. Genetic testing for HαT+ should be considered in the diagnostic workout of patients presenting with anaphylaxis or MC mediator-related symptoms and no suspicion/evidence of SM. The clinical correlates of HαT in SM remain to be fully ascertained. Supported by IMH no.GR-2016-02362631 and AIRC, Mynerva project no21267 Figure 1 Figure 1. Disclosures Elena: CELGENE: Other: funding for meeting participation; PFIZER: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees. Vannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Introduction: Myelofibrosis -either primary (PMF) or post-polycythemia vera/essential thrombocythemia (collectively, secondary MF [SMF])- is characterized by biologic and clinical heterogeneity, with some patients (pts) presenting with features of myeloproliferation (MyP) and others exhibiting a myelodepletive (MyD) phenotype. Although not well defined, the latter is marked by cytopenias involving one or more hematopoietic lineages, in some respects mimicking a myelodysplastic or bone marrow (BM) failure syndrome. MyD features have been associated with poor prognosis in patients with MF, but cytopenias have been usually considered individually. In the current study, we aimed at investigating the phenotypic and prognostic correlates of a MyD phenotype in a large cohort of MF patients. Methods: After IRB approval, 704 consecutive patients with WHO-defined MF referring to CRIMM (Florence), were included in the study. There were 442 (63%) PMF and 262 (37%) SMF. Cytopenias at diagnosis were defined as follows: white blood cell (WBC) 15×10 9/L, Hb >16.5 g/dL for male and >16 g/dL for female, Plt >450×10 9/L). Pts not included in the MyD group were considered as having a MyP phenotype. Mutational analysis by targeted NGS was available for 594/704 patients (PMF 368/442; SMF 226/262). Results: Overall, 166 pts were diagnosed as having a MyD MF, including 108 (24%) PMF and 58 (22%) SMF. Among PMF patients, leukopenia, sex-adjusted anemia and thrombocytopenia were present in 38%, 84% and 35%, respectively; in SMF, corresponding figures were 24%, 91% and 24%. Two or more cytopenias were found in 11% and 6% of PMF and SMF patients, respectively. In PMF, a MyD phenotype was associated with male gender (P=.0468), older age (P=.0002), lower peripheral blast count (P=.0006), higher prevalence of splenomegaly (P=0.0142), constitutional symptoms (P Phenotypic differences between MyD and MyP patients were less evident in SMF, a part for older age of the former (P=.0207). The molecular background was likewise different: a MyD feature was enriched in mutations in TP53 (P=.0024), U2AF1 (P Conclusion: The current study provides a comprehensive analysis of the MyD phenotype in pts with PMF and SMF, partly recapitulating findings of previous studies (Marcellino BK et al. Clin Lymphoma Myeloma Leuk 2020). We showed that the MyD phenotype is associated with high-risk clinical and molecular features, particularly in PMF, and correlates with inferior OS. Of interest, U2AF1 mutations emerged as a distinct molecular marker of MyD in both PMF and SMF, while mutations in TP53 appear prevalent in MyD SMF. Figure 1 Figure 1. Disclosures Vannucchi: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND The rate of major arterial and venous thrombosis in primary myelofibrosis (PMF) and post-ET (PET) and post-PV (PPV) secondary myelofibrosis has been evaluated in a limited number of studies. In the present paper we describe the clinical epidemiology of thrombosis in a large series of patients with overt PMF and PPV/PET MF looking at the rate and risk factors. Moreover, we report findings on thrombosis rate in two cohorts of patients treated with Hydroxyurea (HU) or Ruxolitinib (Ruxo). METHODS Patients were registered in the European Registry for Myeloproliferative Neoplasms (ERNEST). This project, promoted by the European LeukemiaNet, is coordinated by FROM - Foundation for Research, Papa Giovanni XXIII Hospital, Bergamo (Italy) and supported by Novartis through a research collaboration . Patients were diagnosed in 6 Centers from Italy, Spain and Sweden, between Jan, 2001 and Dec, 2012, with the required follow-up information. Patients (n= 1010) with PMF (n=584, 59%), PET-MF (n=207, 20%) and PPV-MF (n=219, 21%) were evaluated for incident thrombosis as primary endpoint. Considering death as a competitive event, uni-and multivariate analyses were performed by applying Fine & Gray competing-risk regression models. RESULTS After a median follow-up of 3.8 years (IQR: 1.8-7.1) from diagnosis, 108 thromboses (10.7%) occurred, for an overall incidence rate of 2.0% pts-yr (95% CI: 1.7-2.5). Arterial thromboses were found in 50 patients (46.3%) including cerebral (n=21, 19.4%), myocardial infarction (n=13, 12.0%) and peripheral events (n=9, 8.3%). Venous thromboses were 58 (53.7%), of which 25 (23.0%) were DVT ± PE and 11 (10.2%) were splanchnic. Thrombosis rate was 1.91, 1.60 and 2.79% pts-yr in PMF, PET-MF and PPV-MF, respectively. In univariate analysis, factors significantly associated with an increased thrombotic risk in PMF were age (p=0.013) and the presence of the JAK2 mutation (p=0.003); in addition, a significant higher proportion of PMF patients at low and intermediate-1 vs intermediate-2 or high risk IPSS score, had thrombosis during the follow-up (p=0.008). In multivariate analysis, only JAK2 mutation retained statistical significance (SHR=3.12, 95% CI: 1.40-6.94, p=0.005). Conversely, neither in univariate nor in multivariable analysis, significant risk factors were not found.To investigate the possible interaction of IPSS score and JAK2 mutation we created a model whose results are presented in Fig. 1A: the cumulative incidence function (CIF) of thrombosis was significantly lower in patients with JAK2 wild-type and intermediate-2 or high IPSS score (CIF: 4% projected at 10 years; SHR=1 [reference category]), while patients at the highest risk for thrombosis harbored JAK2 mutation and were categorized at low or intermediate-1 by IPSS score (CIF: 20% projected at 10 years, SHR=7.13, p=0.008). Of note, thrombosis had a significant impact on mortality. After adjusting for sex, age, year of diagnosis, type of MF and IPPS, HR was 1.51, (95% CI. 1.15-1.98, p=0.003).The influence of drug exposure to incident thrombosis was investigated in two cohorts of 559 consecutive patients exposed to HU (n=470) or to Ruxo (n=89), median treatment 2.6 and 3.0 years, respectively. HU- compared to Ruxo-treated patients were older (median age 67 vs. 63 years, p=0.001), more frequently triple negatives (12% vs. 2%, p=0.036), less splenomegalic (spleen length >10 cm: 30% vs. 88%, p CONCLUSION IPSS score, in addition to the survival risk assessment, may be useful, if associated with the JAK2 mutation, to recognize patients at vascular risk and to suggest appropriate anti-thrombotic prophylaxis. The trend towards a benefit of Ruxo, compared to HU, warrants a study in larger case series. Figure 1 Figure 1. Disclosures Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Passamonti: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Speakers Bureau. Vannucchi: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Lectures; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Lectures.

Background: Thrombosis is the main cause of morbidity and mortality in pts with Polycythemia Vera (PV). Current risk stratification is based on 2 variables: age >60y and history of thrombosis. Additional thrombotic risk factors in PV are generic cardiovascular risk factors and leukocytosis. JAK2V617F (JAK2VF) variant allele frequency (VAF) at diagnosis is highly heterogeneous. A VAF>75% was associated with higher rate of all thrombosis after diagnosis (Vannucchi AM et al, Leukemia 2007), and a VAF ≥ 60% correlated with increased rate of venous thrombosis (VT) in high-risk pts (Guglielmelli P et al, ASH 2018); however, predictive role of JAK2VF VAF is still debated. Aim: To evaluate the impact of JAK2VF VAF on rate of arterial and venous thrombosis in PV pts. Patients and methods: A cohort of 576 strictly 2016 WHO-defined PV pts followed at Univ. of Florence (1981-2020) were included. All pts were annotated for JAK2VF VAF, determined Results: Median age was 61.4 y (range, 16.2-91.8), 58.2% were male; 62% were high-risk based on current classification. Median JAK2VF VAF was 41.5% (range, 0.3-100). A total of 76 (13.2%) pts had an AT event before/at PV diagnosis and 49 (8.5%) pts had an AT during FU. As regards VT, 64 (11.1%) and 39 (6.8%) pts had a VT before/at or after PV diagnosis, respectively. We found that JAK2 VAF as a continue variable was correlated with the risk of VT in FU (p=0.003) but not with AT (p=0.8). ROC analysis to determine the best cut-off level for JAK2 VAF predicting VT had an AUC of 0.72 and a best cut-off value of VAF=50%. VT at FU were significantly enriched in pts with VAF >50%: 14.5% versus 2.4%, p=50% (HR 4, CI 1.9-8.6, p50%, univariate analysis for VT-FS identified history of VT (HR 2.9; CI 1.4-6.1, p=0.006), leukocytosis ≥11x10 9/L (HR 1.9; CI 1.1-3.4, p=0.02) and palpable splenomegaly (HR 1.9, CI 1-3.6; p=0.04) as risk factors. Multivariable analysis confirmed VAF>50% (HR 3.8, CI 1.8-8.1, p=0.0006) and previous VT (HR 2.4, CI 1.1-5.1; p=0.02) as independent risk factors for future VT. In contrast, univariate analysis for AT-free survival (AT-FS) identified history of AT (HR 2.5; CI 1.3-4.9, p=0.007), diabetes (HR 3.3; CI 1.6-6.5, p=0.0007), hyperlipidemia (HR 3.1; CI 1.7-5.6, p=0.0003) and hypertension (HR 2, CI 1.1-3.8; p=0.03) as predictors of future AT; age >60y showed only a trend (p=0.08). Multivariable analysis for AT-FS identified diabetes (HR 2.4, CI 1.2-5; p=0.02), hyperlipidemia (HR 2.3; CI 1.2-4.3, p=0.01) and previous AT (HR 2.1, CI 1-4.2; p=0.04) as independent predictors of future AT. Validation: Our findings were validated in an independent cohort of 315 2016-WHO defined PV pts from Policlinico Gemelli, Catholic Univ., Rome. After exclusion of 26 pts with SVT, analysis was conducted on 289 pts, 38 of them with thrombosis as heralding event (21 AT and 17 VT). Multivariable analysis confirmed JAK2VF VAF >50% (HR 2.3, CI 1.03-5.0, p=0.04) and previous VT (HR 4.5, CI 2.0-10.1; p=0.0003) as independent risk factors for future VT. In pts with VAF >50%, the rate of VT at FU was 19.9% vs 7.7%, P=0.005. KM curve showed that VT-FS was significantly shorter in pts with a JAK2VF VAF >50% (HR 2.2, CI 1.2-4.2; p=0.01) (Figure 1B). Of note, impact of JAK2 VAF>50% on VT at FU was statistically significant particularly in conventionally low-risk pts, accounting for an HR of 9.4 (CI 1.2-72) and HR 3.6 (CI 1.3-10) in Florence and Rome cohorts, respectively. Conclusions: These data support JAK2VF VAF as a strong independent predictor for future venous thrombosis in PV, in association with history of prior venous events, reinforcing that AT and VT are associated with unique risk factors in pts with PV. Supported by AIRC, Project Mynerva n.21267 Figure 1 Figure 1. Disclosures Vannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell, characterized by an abnormal proliferation of largely mature cells driven by mutations in JAK2, CALR, and MPL. All these mutations lead to a constitutive activation of the JAK-STAT signaling, which represents a target for therapy. Beyond driver ones, most patients, especially with myelofibrosis, harbor mutations in an array of “myeloid neoplasm-associated” genes that encode for proteins involved in chromatin modification and DNA methylation, RNA splicing, transcription regulation, and oncogenes. These additional mutations often arise in the context of clonal hematopoiesis of indeterminate potential (CHIP). The extensive characterization of the pathologic genome associated with MPN highlighted selected driver and non-driver mutations for their clinical informativeness. First, driver mutations are enlisted in the WHO classification as major diagnostic criteria and may be used for monitoring of residual disease after transplantation and response to treatment. Second, mutation profile can be used, eventually in combination with cytogenetic, histopathologic, hematologic, and clinical variables, to risk stratify patients regarding thrombosis, overall survival, and rate of transformation to secondary leukemia. This review outlines the molecular landscape of MPN and critically interprets current information for their potential impact on patient management.

In patients with polycythemia vera (PV), an elevated JAK2 p.V617F allele burden is associated with indicators of more severe disease (e.g., leukocytosis, splenomegaly, and increased thrombosis risk); however, correlations between allele burden reductions and clinical benefit in patients with PV have not been extensively evaluated in a randomized trial. This exploratory analysis from the multicenter, open-label, phase 3 Randomized Study of Efficacy and Safety in Polycythemia Vera With JAK Inhibitor INCB018424 Versus Best Supportive Care trial evaluated the long-term effect of ruxolitinib treatment on JAK2 p.V617F allele burden in patients with PV. Evaluable JAK2 p.V617F-positive patients randomized to ruxolitinib (n = 107) or best available therapy (BAT) who crossed over to ruxolitinib at week 32 (n = 97) had consistent JAK2 p.V617F allele burden reductions throughout the study. At all time points measured (up to weeks 208 [ruxolitinib-randomized] and 176 [ruxolitinib crossover]), mean changes from baseline over time in JAK2 p.V617F allele burden ranged from −12.2 to −40.0% (ruxolitinib-randomized) and −6.3 to −17.8% (ruxolitinib crossover). Complete or partial molecular response was observed in 3 patients (ruxolitinib-randomized, n = 2; ruxolitinib crossover, n = 1) and 54 patients (ruxolitinib-randomized, n = 33; ruxolitinib crossover, n = 20; BAT, n = 1), respectively. Among patients treated with interferon as BAT (n = 13), the mean maximal reduction in allele burden from baseline was 25.6% after crossover to ruxolitinib versus 6.6% before crossover. Collectively, the data from this exploratory analysis suggest that ruxolitinib treatment for up to 4 years provides progressive reductions in JAK2 p.V617F allele burden in patients with PV who are resistant to or intolerant of hydroxyurea. The relationship between allele burden changes and clinical outcomes in patients with PV remains unclear. Electronic supplementary material The online version of this article (doi:10.1007/s00277-017-2994-x) contains supplementary material, which is available to authorized users.

Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. Results PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. Conclusions These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Splanchnic vein thrombosis (SVT) is one of the vascular complications of myeloproliferative neoplasms (MPN). We designed a phase 2 clinical trial to evaluate safety and efficacy of ruxolitinib in reducing splenomegaly and improving disease-related symptoms in patients with MPN-associated SVT. Patients diagnosed with myelofibrosis (12 cases), polycythemia vera (5 cases) and essential thrombocythemia (4 cases) received ruxolitinib for 24 weeks in the core study period. Spleen volume was assessed by magnetic resonance imaging (MRI) and splanchnic vein circulation by echo-Doppler analysis. Nineteen patients carried JAK2V617F, one had MPLW515L, and one CALRL367fs*46 mutation. Eighteen patients had spleno-portal-mesenteric thrombosis, two had Budd-Chiari syndrome, and one had both sites involved; 16 patients had esophageal varices. Ruxolitinib was well tolerated with hematological toxicities consistent with those of patients without SVT and no hemorrhagic adverse events were recorded. After 24 weeks of treatment, spleen volume reduction ≥35% by MRI was achieved by 6/21 (29%) patients, and a ≥50% spleen length reduction by palpation at any time up to week 24 was obtained by 13/21 (62%) patients. At week 72, 8 of the 13 (62%) patients maintained the spleen response by palpation. No significant effect of treatment on esophageal varices or in splanchnic circulation was observed. MPN-related symptoms, evaluated by MPN-symptom assessment form (SAF) TSS questionnaire, improved significantly during the first 4 weeks and remained stable up to week 24. In conclusion, this trial shows that ruxolitinib is safe in patients with MPN-associated SVT, and effective in reducing spleen size and disease-related symptoms.

BACKGROUND RUX, a JAK 1/2 inhibitor, is a second-line treatment for symptom management and control of trilineage expansion in patients (pts) with PV who are resistant or intolerant to hydroxyurea (HU). In the RESPONSE study, RUX showed a superior and durable response in controlling hematocrit (HCT) and improving splenomegaly in PV pts resistant or intolerant to HU. RESPONSE-2 (NCT02038036) is a multicenter, open-label, Phase 3 trial comparing RUX with best available therapy (BAT) in HU-resistant/intolerant PV pts without splenomegaly. The primary analysis at Week (Wk) 28 and follow-ups at Wks 80 and 156 proved superiority of RUX over BAT. Here we present the final study results of the 5-year follow-up (Wk 260). METHODS Pts were randomized (1:1) to RUX 10 mg twice daily or BAT; crossover from BAT to RUX was allowed from Wk 28. The final analysis at Wk 260 evaluated durability of HCT control, complete hematologic response (CHR), and safety. Durability of HCT control was defined as absence of phlebotomy eligibility from Wk 8 to Wk 260 with ≤1 phlebotomy eligibility post-randomization to Wk 8. CHR was defined as HCT control with a white blood cell count RESULTS Pts were randomized to RUX (n=74) or BAT (n=75). A total of 59/74 RUX pts and 61/75 BAT pts completed the treatment duration. Among the 75 BAT pts, 58 pts crossed over to RUX, with 38 of them completing the study, allowing for long-term follow-up of 97 pts treated with RUX. Median (range) exposure was 260.0 (0.1 - 273.1) wks in RUX, 28.4 (6.7 - 83.0) wks in BAT, and 224.7 (2.7 - 236.1) wks in crossover arms. At Wk 260, 21.6% of RUX pts (16/74) achieved durable HCT control. The Kaplan-Meier (KM) -estimated median duration of HCT control was not reached (Fig. A). Durable CHR was achieved in 24.3% of RUX pts (18/74; estimated median duration, 34.0 wks). Crossover pts derived RUX benefit by achieving HCT control with HCT levels decreasing over time (median HCT was 46.2% at the crossover baseline and 38.9% at the end of treatment). JAK2 V617F allele burden reduced over time for pts treated with RUX from baseline with a median decrease of 14.8% in RUX (59 evaluable pts) and 13.5% in crossover arms (42 evaluable pts). RUX showed durable improvement in PV-associated symptoms; about 45% of RUX pts continued to achieve ≥50% reduction in MPN-SAF TSS at Wk 260. RUX pts also continued to experience improvements in all 5 dimensions of the EQ-5D-5L assessment. Total number of phlebotomies was 60 in the RUX arm (at Wk 260) vs 106 for BAT (Wk 80). At Wk 260, RUX- (vs BAT-) treated pts required >0 − ≤2 phlebotomies in 16.2% (vs 38.7%), >2 − ≤4 in 9.5% (vs 21.3%), >4 − ≤6 in 5.4% vs (2.7%), and >6 − ≤8 in none (vs 1.3%). The most common AEs (>5%, exposure-adjusted rate) were anemia, arthralgia, and increased weight in the RUX arm and anemia, and hypertension in pts after crossover. Of note, exposure-adjusted rates of herpes zoster were 3.9 in both RUX (Grade 3/4, 0.9) and crossover pts (no Grade 3/4) and none in the BAT arm. The exposure-adjusted rate of thromboembolic events was higher with BAT than RUX (3.7 vs 1.5). As expected given prior HU exposure, non-melanoma skin cancer was the most common secondary malignancy in RUX (exposure-adjusted rates: randomized, 2.7; crossover, 2.9) and BAT-treated pts (1.9). No RUX pts developed AML, whereas myelofibrosis was reported in 2 pts (exposure-adjusted rate, 0.6%). On-treatment death was reported in 5 pts: 1 each in the RUX arm (metastatic melanoma) and BAT arm (septic shock), and 3 in crossover (1 pt each - cardiac disorder, general health deterioration, and PV progression). The KM-estimated median overall survival was not reached in both RUX (95.8%; 95% CI: 87.4, 98.6) and BAT arms (90.7%; 95% CI: 80.3, 95.7; Fig. B) at ≥260 wks. Transformation-free survival (KM-estimated, 95% CI) in the RUX arm was 94.18% (85.23, 97.78) at ≥260 wks. CONCLUSIONS In line with the RESPONSE study outcomes, final analysis from the 5-yr follow-up of RESPONSE 2 proved superiority of RUX over BAT with a safety profile that remained consistent with previous reports. RUX provided durable HCT control with a decrease in the need for phlebotomies, CHR, improved PV-associated symptoms and PRO scores, with fewer thromboembolic events in PV pts without splenomegaly, who are HU resistant or intolerant. Disclosures Palandri: Novartis: Consultancy, Honoraria. Callum:Canadian Blood Services: Research Funding; Octapharma Canada: Research Funding. Devos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Vannucchi:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Zor:Novartis: Current Employment. Gilotti:Novartis: Current Employment. Zhang:Novartis: Current Employment. Griesshammer:Celgene: Honoraria, Speakers Bureau; AOP Orphan: Honoraria, Speakers Bureau; CTI: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.