Does Double Mean Trouble? Coexistence of Myeloproliferative and Lymphoproliferative Neoplasms.

Background: The occurrence of myeloproliferative neoplasms (MPNs) that evolve into each other is well-described, as is this occurrence of lymphoproliferative neoplasms (LPNs). However, less is known about rare MPN/LPN coexistence, and the aim of our study was to analyze charachteristics of these patients after long term follow-up. Methods: Fourteen patients with MPN/LPN coexistence were diagnosed and treated according to guidelines at a single university center across two decades. Results: The overall median age was 53 years (22-69). MPNs patients with subsequent LPNs had a shorter period of second malignancy development and a more aggressive course of LPN, which can cause fatal outcomes. Polycythemia vera and chronic lymphocytic leukemia were most commonly associated (36%). The JAK2V617F mutation had 2/3 and cytogenetic abnormalities occurred in 1/3 of patients. MPN/LPN coexistence cases had significantly higher thrombotic potential (42.8%) and a higher third malignancy accruement frequency (21.4%) versus those without such malignancies. Conclusions: Considering the younger ages at MPN diagnosis, it is recommended to check regularly for blood lymphocytosis or lymphadenopathy occurrences and organomegaly progression faster than expected for MPN, with the aim of timely LPN diagnoses. The presence of molecular-cytogenetic abnormalities in a majority of patients indicate possible genetic instability and increased risk of development of multiple neoplasms, thus elevating thrombotic risk.

Easily Applicable Predictive Score for Differential Diagnosis of Prefibrotic Primary Myelofibrosis from Essential Thrombocythemia.

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (prePMF) initially have a similar phenotypic presentation with thrombocytosis. The aim of our study was to determine significant clinical-laboratory parameters at presentation to differentiate prePMF from ET as well as to develop and validate a predictive diagnostic prePMF model. This retrospective study included 464 patients divided into ET (289 pts) and prePMF (175 pts) groups. The model was built using data from a development cohort (229 pts; 143 ET, 86 prePMF), which was then tested in an internal validation cohort (235 pts; 146 ET, 89 prePMF). The most important prePMF predictors in the multivariate logistic model were age ≥ 60 years (RR = 2.2), splenomegaly (RR = 13.2), and increased lactat-dehidrogenase (RR = 2.8). Risk scores were assigned according to derived relative risk (RR) for age ≥ 60 years (1 point), splenomegaly (2 points), and increased lactat-dehidrogenase (1 point). Positive predictive value (PPV) for pre-PMF diagnosis with a score of ≥points was 69.8%, while for a score of ≥3 it was 88.2%. Diagnostic performance had similar values in the validation cohort. In MPN patients with thrombocytosis at presentation, the application of the new model enables differentiation of pre-PMF from ET, which is clinically relevant considering that these diseases have different prognoses and treatments.

Comprehensive Next-Generation Sequencing Testing in a Patient with TEMPI Syndrome.

TEMPI syndrome is a new and poorly understood disease that is currently considered a type of plasma cell neoplasm with paraneoplastic manifestations. The TEMPI acronym defines the hallmarks of the syndrome: T for telangiectasia; E for erythrocytosis with elevated erythropoietin; M, monoclonal gammopathy; P, perinephric collections; and I, intrapulmonary shunting. Due to the marked erythrocytosis as the most common presenting feature, TEMPI is often misdiagnosed as polycythemia vera. However, unlike polycythemia vera, TEMPI is not associated with a JAK2 mutation. The pathogenesis of TEMPI syndrome is unknown, although a few hypothetical disease mechanisms have been previously discussed. Here we present a new case of TEMPI syndrome, discuss results of a next-generation sequencing (NGS) panel covering 1,425 known cancer-related genes, and review the current literature with focus on an update of the genetics of TEMPI syndrome. This is the first report of TEMPI that includes results of comprehensive NGS testing.

Correlation between leukocyte-platelet aggregates and thrombosis in myeloproliferative neoplasms.

INTRODUCTION: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. METHODS: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. RESULTS: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P < .05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89 ng/mL vs 286.39 ng/mL, P = .034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR = 1.561, 95% CI: 1.007-2.420, P = .046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR = 1.975, 95%CI: 1.215-3.212, P = .006) for thrombosis occurrence. CONCLUSION: Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

Prediction of optimal cytogenetic responses at 6 and 12 months in patients with chronic myeloid leukemia in chronic phase treated with imatinib.

PURPOSE: Imatinib mesylate transformed the treatment and paradigms of chronic myeloid leukemia. European LeukemiaNet (ELN) group has defined specific treatment milestones with an optimal outcome to be achieved in patients. METHODS: In a retrospective cohort study, we evaluated the impact of clinical and biological variables on achieving an optimal response at 6 and 12 months according to ELN recommendations. We included 106 patients with chronic phase chronic myeloid leukemia (CML) with appropriate bone marrow aspirate and biopsy for immunohistochemistry. RESULTS: The number of white blood cells (WBC), the percentage of peripheral blast, the values of Sokal and ELTS scores and the percentage of Ki-67+ cells in the bone marrow predicted a complete cytogenetic response (CCyR) at 6 months, but only WBC and EUTOS score predicts CCyR at 12 months. We found that Sokal score below 0.775 was very sensitive to achieve of CCyR at 6 months (m) and that all patients with a value <0.550 achieved CCyR-6m. Patients with a low percentage of blast in the peripheral blood (≤1.5%) or in the bone marrow (≤5%) together with lower WBC (≤100×109/L) were likely to have significantly higher CCyR rates at 6 and 12 months respectively. Also, patients with a higher number of Ki67+ cells in the leukemic areas of the bone marrow had a significantly better outcome. Unfortunately, our investigation did not reveal that bone marrow fibrosis (MF grade), microvascular density, percentage of CD34+, CD61+ or PTCH1+ cells could have any effect on achievement of CCyR at 6 or 12 months. CONCLUSION: Our investigation has shown that only a few biological characteristics in patients with CML can predict the optimal treatment outcome after imatinib.

Investigation of a healthcare-associated Candida tropicalis candidiasis cluster in a haematology unit and a systematic review of nosocomial outbreaks.

BACKGROUND: Non-albicans Candida spp. are an emerging cause of hospital-acquired bloodstream infections, associated with high mortality due to the challenges in diagnosis and delayed treatment. OBJECTIVES: We aimed to investigate a cluster of healthcare-associated invasive candidiasis caused by C tropicalis and review the literature of healthcare-associated outbreaks or clusters caused by C tropicalis. METHODS: An investigation was performed to determine clinical presentation, treatment outcomes and the factors contributing to C tropicalis candidemia occurrence. We searched the Medline database via PubMed and Ovid using the keywords of "Candida tropicalis" combined with "outbreak" or "clustering" or "clusters," and we limited the search to studies conducted from January 1989 to January 2019. RESULTS: We report two related cases of C tropicalis candidemia among patients with AML following a period of neutropenia, who had erythematous skin rash as a first manifesting sign of candidiasis. C tropicalis was isolated from blood and skin cultures of both patients, which were identical by pulsed-field gel electrophoresis typing. Our systematic review of outbreaks caused by C tropicalis suggests that (a) most reported outbreaks have occurred in neonatal and adult ICUs; (b) patients who receive total parenteral therapy, antibiotics and those who have indwelling catheters and recent surgery are at high risk of infection; and (c) environmental and healthcare personnel surveillance suggest that cross-contamination is a major risk factor. CONCLUSION: Control of nosocomial outbreaks caused by C tropicalis should include better infection control measures, education of healthcare professionals especially working in adult and neonatal intensive care and haematology units.

IL6 inhibition of inflammatory S100A8/9 proteins is NF-κB mediated in essential thrombocythemia.

This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34+ cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-κB, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-κB and inflammation signalling in MPN-derived CD34+ cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-κB and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. SIGNIFICANCE OF THE STUDY: We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-κB and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.

JAK2-mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.

Increased energy requirement and metabolic reprogramming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2-driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells, resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy, and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high-fat diet improved survival, whereas high-glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2-mutant hematopoietic stem and progenitor cells that were altered in comparison with wild-type controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPNs. These effects were additive with the JAK1/2 inhibitor ruxolitinib in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPNs and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPNs.

Genotype phenotype correlation in a pediatric population with antithrombin deficiency.

Inherited antithrombin (AT) deficiency is a rare autosomal dominant disorder, caused by mutations in the AT gene (SERPINC1). Considering that the genotype phenotype relationship in AT deficiency patients remains unclear, especially in pediatric patients, the aim of our study was to evaluate genotype phenotype correlation in a Serbian pediatric population. A retrospective cohort study included 19 children younger than 18 years, from 15 Serbian families, with newly diagnosed AT deficiency. In 21% of the recruited families, mutations affecting exon 4, 5, and 6 of the SERPINC1 gene that causes type I AT deficiency were detected. In the remaining families, the mutation in exon 2 causing type II HBS (AT Budapest 3) was found. Thrombosis events were observed in 1 (33%) of those with type I, 11 (85%) of those with AT Budapest 3 in the homozygous respectively, and 1(33%) in the heterozygous form. Recurrent thrombosis was observed only in AT Budapest 3 in the homozygous form, in 27% during initial treatment of the first thrombotic event. Abdominal venous thrombosis and arterial ischemic stroke, observed in almost half of the children from the group with AT Budapest 3 in the homozygous form, were unprovoked in all cases.Conclusion: Type II HBS (AT Budapest 3) in the homozygous form is a strong risk factor for arterial and venous thrombosis in pediatric patients. What is Known: • Inherited AT deficiency is a rare autosomal dominant disorder, caused by mutations in the SERPINC1gene. • The genotype phenotype correlation in AT deficiency patients remains unclear, especially in pediatric patients. What is New: • The genetic results for our paediatric population predominantly showed the presence of a single specific mutation in exon 2, that causes type II HBS deficiency (AT Budapest 3). • In this group thrombosis mostly occurred as unprovoked, in almost half of them as abdominal thrombosis or stroke with high incidence of recurrent thrombosis, in 27% during initial treatment.

IL-6 stimulation of DNA replication is JAK1/2 mediated in cross-talk with hyperactivated ERK1/2 signaling.

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.

ß-catenin and PPAR-γ levels in bone marrow of myeloproliferative neoplasm: an immunohistochemical and ultrastructural study.

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: ß-catenin, PPAR-γ and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of ß-catenin immunoreactive cells was significantly higher in PV then in ET (p < 0.01) or PMF group of patients (p < 0.01) and also in ET versus PMF group of patients (p < 0.01). Erythroid lineage showed absent ß-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-γ was localized mostly in megakaryocytes and the highest number of PPAR-γ immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed ß-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-γ immunopositive megakaryocytes characterized by abnormal maturation.

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

PURPOSE: Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. METHODS: We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. RESULTS: We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34+ cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV. CONCLUSIONS: From our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

The WHO diagnostic criteria for polycythemia vera-role of red cell mass versus hemoglobin/hematocrit level and morphology.

Regarding diagnosis of polycythemia vera (PV), discussion persists about hemoglobin (Hb) and/or hematocrit (Hct) threshold values as surrogate markers for red cell mass (RCM) and the diagnostic impact of bone marrow (BM) morphology. We performed a retrospective study on 290 patients with PV (151 males, 139 females; median age 65 years) presenting with characteristic BM features (initial biopsies, centralized evaluation) and endogenous erythroid colony (EEC) formations. This cohort included (1) a group of 229 patients when following the 2008 versus 256 patients diagnosed according to the 2016 World Health Organization (WHO) guidelines, all presented with increased RCM; (2) masked PV patients with low Hb (n = 143)/Hct (n = 45) recruited from the 2008 WHO cohort; (3) a cohort of 17 PV patients with elevated diagnostic Hb/Hct levels but low RCM; and (4) nine PV patients with increased RCM, opposing low Hb/Hct values. All patients were treated according to current PV guidelines (phlebotomies 87%, hydroxyurea 79%, and acetylsalicylic acid 87%). Applying the 2016 WHO criteria significantly increased concordance between RCM and Hb values compared with the 2008 WHO criteria (90 vs. 43% in males and 83 vs. 64% in females). Further analysis of the WHO 2016 PV cohort revealed that increased RCM is associated with increased Hb/Hct (93.8/94.6%). Our study supports and extends the diagnostic impact of the 2016 revised WHO classification for PV by highlighting the importance of characteristic BM findings and implies that Hb/Hct threshold values may be used as surrogate markers for RCM measurements.