Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patientyear. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; P 5 0.02) and spleen length 10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; P 5 0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, P < 0.001). In conclusion, IPSS-category and splenomegaly, emerged as the main risk factors for infections in MF. RUX-treated patients experienced significantly more infection episodes; however, future prospective studies are needed to isolate the confounding contribution of other risk factors such as disease stage.

We investigated the influence of molecular status on disease characteristics and clinical outcome in young patients (⩽ 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) or early/prefibrotic primary myelofibrosis (early-PMF). Overall, 217 patients with ET (number 197) and early-PMF (number 20) were included in the analysis. Median follow-up time was 10.2 years. The cumulative incidence of thrombosis, hemorrhages and disease evolution into myelofibrosis/acute leukemia were 16.6%, 8.6% and 3% at 15 years, respectively. No differences were detectable between ET and early-PMF patients, although the latter cohort showed a trend for worse combined-event free survival (EFS). Mutation frequency were 61% for JAK2V617F, 25% for CALR and 1% for MPLW515K, and were comparable across WHO diagnosis; however, JAK2V617F allele burden was higher in the early-PMF group. Compared with JAK2V617F-positive patients, CALR-mutated patients displayed higher platelet count and lower hemoglobin level. CALR mutations significantly correlated with lower thrombotic risk (9.1% versus 21.7%, P = 0.04), longer survival (100% versus 96%, P = 0.05) and better combined-EFS (86% versus 71%, P = 0.02). However, non-type 1/type 2 CALR mutations ('minor' mutations) and abnormal karyotype were found to correlate with increased risk of disease evolution. At last contact, six patients had died; in five cases, the causes of death were related to the hematological disease and occurred at a median age of 64 years (range: 53-68 years). Twenty-eight patients (13%) were unmutated for JAK2, CALR and MPL: no event was registered in these 'triple-negative' patients.

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2,13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2 mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1. Overall, we have demonstrated that in MF different mutations lead to phenotypic and functional alterations in different immune subsets that may have a potential role in disease progression and susceptibility to infections.

Along with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of Myelofibrosis (MF). Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived circulating CD34+ cells.We found that, regardless mutation status, IL-1β or TNF-α increases the survival of MF-derived CD34+ cells. In addition, along with stimulation of cell cycle progression to the S-phase, IL-1β or TNF-α ± TIMP-1 significantly stimulate(s) the in vitro clonogenic ability of CD34+ cells from JAK2V617 mutated patients. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α + TIMP-1 decreased the erythroid compartment of the CALR mutated patients. Megakaryocyte progenitors were stimulated by IL-1β (JAK2V617F mutated patients only) and inhibited by TNF-α. IL-1β + TNF-α + C-X-C motif chemokine 12 (CXCL12) ± TIMP-1 highly stimulates the in vitro migration of MF-derived CD34+ cells. Interestingly, after migration toward IL-1β + TNF-α + CXCL12 ± TIMP-1, CD34+ cells from JAK2V617F mutated patients show increased clonogenic ability.Here we demonstrate that the interplay of these inflammatory factors promotes and selects the circulating MF-derived CD34+ cells with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach.

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease.

Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%.Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (‘’Histology-based’’ diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (‘’Clinical-based’’ or ‘’Molecular-based’’ diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process.Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion.

Introduction Young adults with Essential Thrombocythemia (ET) or early Primary Myelofibrosis (early-PMF) are a category of patients projected to a prolonged survival but also to an extended utilization of medical resources. Mutations, including those in the calreticulin (CALR) gene, have been reported to affect main clinical features and outcome in large cohorts of patients with Ph-negative MPNs. However, no data are available on mutational status and long-term outcome in young MPN patients. Methods A clinic-pathologic database of ET patients followed in 5 Italian Hematology Centers was created. A total of 217 WHO-diagnosed ET or early-PMF patients ≤ 40 years at diagnosis was retrieved from the general database of 2635 patients. All bone marrow biopsies were reviewed at local institution. Baseline clinical/molecular characteristics and outcome measures (thrombosis, hemorrhages, secondary MF and AL, second neoplasia, death, overall and event-free survival) were evaluated. JAK2V617F allele-burden was assessed in granulocyte DNA by using ipsogen JAK2 MutaQuant Kit (qPCR). CALR mutations were identified by next generation sequencing (NGS) approach on GS Junior (Roche-454 platform); MPL mutations were evaluated by using ipsogen MPLW515L/K MutaScreen Kit. Results Overall, 197 WHO-defined ET and 20 early-PMF (age range: 16-40, median 34) were included in the study. Mutational frequencies were 61% for JAK2, 25% for CALR, 1% for MPL and 13% for triple negative. Baseline clinical characteristics and use of antiplatelet/cytoreductive therapies were comparable in ET and early-PMF, although frequency of triple negative was higher in the early-PMF cohort. Compared to the JAK2 positive population, both CALR and triple-negative patients showed higher platelet count and lower hemoglobin and hematocrit levels (Table 1). Median follow-up was 10.2 years (range: 0.5-37.5). During follow-up, 19 (9,6%) ET and 3 (15%) early-PMF patients experienced a total of 31 thrombotic (arterial: 38%) and 12 hemorrhagic events, with an incidence rate of 0.91% and 0.39% patients/yr, respectively. The cumulative incidence of thrombosis was 0,14% and 0,24% at 15 and at 20 years, respectively. Overall, 10 patients (4,6%) and 1 (0,4%) patients evolved to MF and AL, respectively; 10 developed a second neoplasia. The cumulative incidence of disease progression into MF/AL was 0,03% and 0,13% at 15 and at 20 years, respectively. At last contact, 6 (2,7%) patients had died, at a median age of 61 years (20-71), for an overall survival of 98% at 15 years. Causes of death were related to the myeloproliferative neoplasm (disease evolution or thrombotic complications) in all patients but one. Event-free survival was similar in the ET/early-PMF cohorts considering both every event separately and all together. In univariate analysis, male sex (p=0.003), previous thrombosis (p=0.001), splenomegaly (p=0.037), JAK2V617F (p=0.019) were associated with increased thrombotic risk; in multivariate Cox analysis, only previous thrombosis and male sex remained significant (p=0.012). Baseline splenomegaly was the only predictive factor for subsequent hemorrhages (p=0.017). Abnormal karyotype was associated with secondary MF (p=0.013) and also with second neoplasia (p<0.001). Together with JAK2V617F positivity and leukocytosis >11x109/L, abnormal karyotype was also associated with worse survival in univariate analysis. However, in multivariate analysis only JAK2V617F mutation remained as negative predictor of survival (p=0.019). Also, multivariable analysis confirmed JAK2 mutation and splenomegaly as independent risk factors for cumulative events. Conclusions With the limitations due to the low number of early-PMF, the outcome of young adults with early-PMF and true ET seemed to be comparable. The correlation of abnormal karyotype with MF transformation and second neoplasia suggests the need for an accurate cytogenetic analysis at diagnosis. Mutational status did influence disease phenotype, in terms of baseline characteristics and prognosis. Indeed, JAK2 mutational status confirmed a negative prognostic role for thrombosis and survival, while event-free survival was significantly better in triple-negative patients. Notably, causes of death were mostly related to the hematological malignancy, pointing out the substantial impact that this generally indolent disease may acquire in young adults. Disclosures Latagliata: Novartis: Consultancy; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy; Shire: Consultancy. Cavo:Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.