Circulating CD34+ cells of primary myelofibrosis patients contribute to myeloid-dominant hematopoiesis and bone marrow fibrosis in immunodeficient mice.

INTRODUCTION: Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how these aberrant myeloid cells and fibroblasts are produced remains unclear. AIM AND METHODS: In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination. RESULTS: Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms. CONCLUSION: A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.

Data-driven analysis of the kinetics of the JAK2V617F allele burden and blood cell counts during hydroxyurea treatment of patients with polycythemia vera, essential thrombocythemia, and primary myelofibrosis.

BACKGROUND: Hydroxyurea (HU) treatment of patients with essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) (MPNs) normalizes elevated blood cell counts within weeks in the large majority of patients. Studies on the impact of HU upon the kinetics of the JAK2V617F allele burden, leukocyte, and platelet counts over time are scarce. PURPOSE: Using data-driven analysis as a novel tool to model the kinetics of the JAK2V617F allele burden and blood cell counts over time during treatment with HU. MATERIAL AND METHODS: Using serial measurements of JAK2V617F and correlation analysis of routine hematological values (the Hb-concentration, leukocyte count, platelet count, and lactic dehydrogenase), we present a detailed description and analysis of the kinetics of the JAK2V617F, leukocyte, and platelet counts and lactic dehydrogenase in 27 patients (PV = 18; ET = 7; PMF = 2), who were followed in the Danish randomized trial (DALIAH). To further analyze the JAK2V617F kinetics, we use a machine learning clustering algorithm to group the response patterns. RESULTS: Response patterns were highly heterogeneous, with clustering resulting in 3 groups and 3 outliers. In the large majority of patients, HU treatment was initially associated with a modest decline in the JAK2V617F allele burden in concert with a decline in leukocyte and platelet counts. However, HU did not induce a sustained and continuous decrease in the JAK2V617F allele burden. CONCLUSION: Using data-driven analysis of the JAK2V617F allele burden, leukocyte, and platelet kinetics during treatment with HU, we have shown that HU does not induce a sustained decrease in the JAK2V617F allele burden and neither induces sustained normalization of elevated cell counts in MPN patients. Our results may explain why MPN patients during treatment with HU still have a substantially increased risk of thrombosis.

Automated diagnostic support system with deep learning algorithms for distinction of Philadelphia chromosome-negative myeloproliferative neoplasms using peripheral blood specimen.

Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-negative MPNs) such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis are characterized by abnormal proliferation of mature bone marrow cell lineages. Since various non-hematologic disorders can also cause leukocytosis, thrombocytosis and polycythemia, the detection of abnormal peripheral blood cells is essential for the diagnostic screening of Ph-negative MPNs. We sought to develop an automated diagnostic support system of Ph-negative MPNs. Our strategy was to combine the complete blood cell count and research parameters obtained by an automated hematology analyzer (Sysmex XN-9000) with morphological parameters that were extracted using a convolutional neural network deep learning system equipped with an Extreme Gradient Boosting (XGBoost)-based decision-making algorithm. The developed system showed promising performance in the differentiation of PV, ET, and MF with high accuracy when compared with those of the human diagnoses, namely: > 90% sensitivity and > 90% specificity. The calculated area under the curve of the ROC curves were 0.990, 0.967, and 0.974 for PV, ET, MF, respectively. This study is a step toward establishing a universal automated diagnostic system for all types of hematology disorders.

Abnormally Short Erythrocyte LifeSpan in Three Patients with Primary Myelofibrosis Despite Successful Control of Splenomegaly.

Splenectomy is an elective operation for refractory anemia in patients with primary myelofibrosis (PMF). We found that 3/3 patients with PMF in our department continued to have very shortened erythrocyte (RBC) lifespans (35 days, 66 days, and 37 days, respectively) after treatment-alleviated splenomegaly. These outcomes suggest that intravascular hemolysis predominantly independent of hypersplenism may underlie, at least to some extent, peripheral hemolysis in patients with PMF. More cases studies are needed to elucidate the role of splenomegaly in PMF-associated anemia.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

Prognostic value of blasts in peripheral blood in myelofibrosis in the ruxolitinib era.

BACKGROUND: Circulating blasts (peripheral blood [PB] blasts) ≥1% have long been considered an unfavorable feature for patients with primary myelofibrosis. Whether further quantification of PB blasts and their correlation with bone marrow (BM) blasts have incremental value with regard to patient prognostication is unclear. Similarly, the role of the JAK1/JAK2 inhibitor ruxolitinib (RUX) is not well defined in patients who have increased blasts. METHODS: The authors retrospectively studied 1316 patients with myelofibrosis who presented at their institution between 1984 and 2018 and had available PB and BM blasts. RESULTS: The PB blast percentage influenced overall survival (OS) only among patients who had BM blasts <5%, with a median OS of 64 months for patients with 0% PB blasts, 48 months for those with 1% to 3% PB blasts, and 22 months for those with 4% PB blasts (P < .01). Patients who had 4% PB blasts and 5% to 9% BM/PB blasts had clinical features similar to those of patients who had 10% to 19% blasts. Although the OS of the former patients was longer than in patients who had 10% to 19% blasts, it was not statistically different (median OS: 22, 26, and 13 months, respectively; P > .05). Forty-four percent of patients received RUX throughout their disease course. All patients who had <10% blasts (PB or BM) and received treatment with RUX had superior OS compared with those who did not receive RUX within the same group. PB blasts ≥4% and BM blasts ≥5% were significant for predicting inferior survival in multivariate analysis. CONCLUSIONS: The current results provide comprehensive insight into the role of peripheral blasts in patients with myelofibrosis and indicates that patients who have PB blasts ≥4% have an unfavorable prognosis. RUX provides a survival benefit to patients who have PB blasts <10%.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

Primary analysis of JUMP, a phase 3b, expanded-access study evaluating the safety and efficacy of ruxolitinib in patients with myelofibrosis, including those with low platelet counts.

Ruxolitinib is a potent Janus kinase (JAK) 1/JAK2 inhibitor approved for the treatment of myelofibrosis (MF). Ruxolitinib was assessed in JUMP, a large (N = 2233), phase 3b, expanded-access study in MF in countries without access to ruxolitinib outside a clinical trial, which included patients with low platelet counts (<100 × 109 /l) and patients without splenomegaly - populations that have not been extensively studied. The most common adverse events (AEs) were anaemia and thrombocytopenia, but they rarely led to discontinuation (overall, 5·4%; low-platelet cohort, 12·3%). As expected, rates of worsening thrombocytopenia were higher in the low-platelet cohort (all grades, 73·2% vs. 53·5% overall); rates of anaemia were similar (all grades, 52·9% vs. 59·5%). Non-haematologic AEs, including infections, were mainly grade 1/2. Overall, ruxolitinib led to meaningful reductions in spleen length and symptoms, including in patients with low platelet counts, and symptom improvements in patients without splenomegaly. In this trial, the largest study of ruxolitinib in patients with MF to date, the safety profile was consistent with previous reports, with no new safety concerns identified. This study confirms findings from the COMFORT studies and supports the use of ruxolitinib in patients with platelet counts of 50-100 × 109 /l. (ClinicalTrials.gov identifier NCT01493414).

Data-driven analysis of JAK2V617F kinetics during interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms.

Treatment with PEGylated interferon-alpha2 (IFN) of patients with essential thrombocythemia and polycythemia vera induces major molecular remissions with a reduction in the JAK2V617F allele burden to undetectable levels in a subset of patients. A favorable response to IFN has been argued to depend upon the tumor burden, implying that institution of treatment with IFN should be as early as possible after the diagnosis. However, evidence for this statement is not available. We present a thorough analysis of unique serial JAK2V617F measurements in 66 IFN-treated patients and in 6 untreated patients. Without IFN treatment, the JAK2V617F allele burden increased exponentially with a period of doubling of 1.4 year. During monotherapy with IFN, the JAK2V617F allele burden decreased mono- or bi-exponentially for 33 responders of which 28 patients satisfied both descriptions. Bi-exponential description improved the fits in 19 cases being associated with late JAK2V617F responses. The decay of the JAK2V617F allele burden during IFN treatment was estimated to have half-lives of 1.6 year for the monoexponential response and 1.0 year in the long term for the bi-exponential response. In conclusion, through data-driven analysis of the JAK2V617F allele burden, we provide novel information regarding the JAK2V617F kinetics during IFN-treatment, arguing for early intervention.

Life after ruxolitinib: Reasons for discontinuation, impact of disease phase, and outcomes in 218 patients with myelofibrosis.

BACKGROUND: After discontinuing ruxolitinib, the outcome of patients with myelofibrosis reportedly has been poor. The authors investigated whether disease characteristics before the receipt of ruxolitinib may predict drug discontinuation in patients with myelofibrosis and whether reasons for drug discontinuation, disease phase at discontinuation, and salvage therapies may influence the outcome. METHODS: A centralized electronic clinical database was created in 20 European hematology centers, including clinical and laboratory data for 524 patients who received ruxolitinib for myelofibrosis. RESULTS: At 3 years, 40.8% of patients had stopped ruxolitinib. Baseline predictors of drug discontinuation were: intermediate-2-risk/high-risk category (Dynamic International Prognostic Score System), a platelet count <100 ×109 per liter, transfusion dependency, and unfavorable karyotype. At last contact, 268 patients (51.1%) had discontinued therapy, and the median drug exposure was 17.5 months. Fifty patients (18.7%) died while taking ruxolitinib. The reasons for discontinuation in the remaining 218 patients were the lack (22.9%) or loss (11.9%) of a spleen response, ruxolitinib-related adverse events (27.5%), progression to blast phase (23.4%), ruxolitinib-unrelated adverse events (9.2%), and allogeneic transplantation during response (5.1%). The median survival after ruxolitinib was 13.2 months and was significantly better in the 167 patients who discontinued ruxolitinib in chronic phase (27.5 vs 3.9 months for those who discontinued in blast phase; P < .001). No survival differences were observed among patients who discontinued ruxolitinib in chronic phase because of lack of response, loss of response, or ruxolitinib-related adverse events. The use of investigational agents and/or ruxolitinib rechallenge were associated with improved outcome. CONCLUSIONS: The survival of patients with myelofibrosis after discontinuation of ruxolitinib is poor, particularly for those who discontinue in blast phase. Salvage therapies can improve outcome, emphasizing the need for novel therapies.

Decreased Activity of Blood Acid Sphingomyelinase in the Course of Multiple Myeloma.

Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway. It catalyzes the breakdown of sphingomyelin into ceramide, a bioactive lipid that, among other roles, is involved in regulation of apoptosis. Dry drop blood test (DBS) and colorimetric 2-step enzymatic assay were used to assess the activity of human blood aSMase, beta-galactosidase, and beta-glucosidase, these enzymes are lysosomal hydrolases that catalyze the degradation of related sphingolipids, of sphingolipid signaling molecules. Blood was collected from a group of healthy volunteers and patients that were diagnosed with multiple myeloma (MM) in various stages of the disease. Additionally, activity of those enzymes in patients diagnosed with other hematological cancers was also assessed. We found that aSMase activity in the blood of patients with MM (at the time of diagnosis) was 305.43 pmol/spot*20 h, and this value was significantly lower (p < 0.030) compared to the healthy group 441.88 pmol/spot*20 h. Our collected data suggest a possible role of aSMase in pathogenesis of MM development.

Vitamin B6 deficiency is prevalent in primary and secondary myelofibrosis patients.

Vitamin B6 (VB6) deficiency contributes to oncogenesis and tumor progression in certain cancers, and is prevalent in cancer patients in general. VB6 is also an essential element of heme synthesis, and deficiency can lead to anemia. Primary myelofibrosis (PMF) and secondary myelofibrosis (sMF) are myeloproliferative neoplasms often presenting with anemia along with other cytopenias. We performed a prospective study to determine whether PMF and sMF patients suffer from VB6 deficiency, and whether VB6-deficient patients show improvement of anemias with VB6 supplementation. Twelve PMF patients and 11 sMF patients were analyzed. A total of 16 of 23 patients (69.6%) were found to have VB6 deficiency, but VB6 supplementation with pyridoxal phosphate hydrate did not elevate hemoglobin levels in deficient patients. None of the patients presented with vitamin B12, iron, or copper deficiencies. Four patients showed serum folate levels below the lower limit of normal and eight patients showed serum zinc levels below the lower limit of normal; however, these deficiencies were marginal and unlikely to contribute to anemia. Compared to VB6-sufficient patients, VB6-deficient patients showed significantly lower serum folate levels and higher serum copper levels. Studies elucidating the relationship of VB6 deficiency and etiology of PMF/sMF are warranted.