Circulating Mesenchymal Stromal Cells in Patients with Infantile Hemangioma: Evaluation of Their Functional Capacity and Gene Expression Profile.

We previously published that in patients with infantile hemangioma (IH) at the onset (T0) colony forming unit-fibroblasts (CFU-Fs) are present in in vitro cultures from PB. Herein, we characterize these CFU-Fs and investigate their potential role in IH pathogenesis, before and after propranolol therapy. The CFU-F phenotype (by flow cytometry), their differentiation capacity and ability to support angiogenesis (by in vitro cultures) and their gene expression (by RT-PCR) were evaluated. We found that CFU-Fs are actual circulating MSCs (cMSCs). In patients at T0, cMSCs had reduced adipogenic potential, supported the formation of tube-like structures in vitro and showed either inflammatory (IL1ß and ESM1) or angiogenic (F3) gene expression higher than that of cMSCs from CTRLs. In patients receiving one-year propranolol therapy, the cMSC differentiation in adipocytes improved, while their support in in vitro tube-like formation was lost; no difference was found between patient and CTRL cMSC gene expressions. In conclusion, in patients with IH at T0 the cMSC reduced adipogenic potential, their support in angiogenic activity and the inflammatory/angiogenic gene expression may fuel the tumor growth. One-year propranolol therapy modifies this picture, suggesting cMSCs as one of the drug targets.

Preclinical Validation of an Advanced Therapy Medicinal Product Based on Cytotoxic T Lymphocytes Specific for Mutated Nucleophosmin (NPM1mut) for the Treatment of NPM1mut-Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with nucleophosmin (NPM1) genetic mutations is the most common subtype in adult patients. Refractory or relapsed disease in unfit patients or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has a poor prognosis. NPM1-mutated protein, stably expressed on tumor cells but not on normal tissues, may serve as an ideal target for NPM1-mutated AML immunotherapy. The study aim was to investigate the feasibility of producing mutated-NPM1-specific cytotoxic T cells (CTLs) suitable for somatic cell therapy to prevent or treat hematologic relapse in patients with NPM1-mutated AML. T cells were expanded or primed from patient or donor peripheral blood mononuclear cells by NPM1-mutated protein-derived peptides, and tested for leukemia antigen-targeted cytotoxic activity, cytokine production and hematopoietic precursor inhibitory effect. We found that mutated-NPM1-specific CTLs, displaying specific cytokine production and high-level cytotoxicity against patients' leukemia blasts, and limited inhibitory activity in clonogenic assays, could be obtained from both patients and donors. The polyfunctional mutated-NPM1-specific CTLs included both CD8+ and CD4+ T cells endowed with strong lytic capacity. Our results suggest that mutated-NPM1-targeted CTLs may be a useful therapeutic option to control low-tumor burden relapse following conventional chemotherapy in older NPM1-mutated AML patients or eradicate persistent MRD after HSCT.

Myelofibrosis-type megakaryocyte dysplasia (MTMD) as a distinct category of BCR::ABL-negative myeloproliferative neoplasms. Challenges and perspectives.

In this Perspective, we discuss criteria for defining a new disease entity or variant of a recognized disease or disorder. We do so in the context of the current topography of the BCR::ABL-negative myeloproliferative neoplasms (MPNs) where two new variants are reported: clonal megakaryocyte dysplasia with normal blood values (CMD-NBV) and clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). The cardinal feature of these variants is bone marrow megakaryocyte hyperplasia and atypia corresponding the WHO histological criteria for primary myelofibrosis (myelofibrosis-type megakaryocyte dysplasia-MTMD). Persons with these new variants have a different disease course and features from others in the MPN domain. In a broader context we suggest myelofibrosis-type megakaryocyte dysplasia defines a spectrum of related MPN variants including CMD-NBV, CMD-IT, pre-fibrotic myelofibrosis and overt myelofibrosis, which differ from polycythemia vera and essential thrombocythemia. Our proposal needs external validation and we stress the need for a consensus definition of the megakaryocyte dysplasia which is the hallmark of these disorders.

Clonal Megakaryocyte Dysplasia with Isolated Thrombocytosis Is a Distinct Myeloproliferative Neoplasm Phenotype.

INTRODUCTION: About 15% of people with a myeloproliferative neoplasm (MPN) are identified as MPN, unclassifiable using the 2016 WHO classification. METHODS: We tested whether persons with platelet concentration ≥450 × 10E+9/L, bone marrow megakaryocyte morphology typical of prefibrotic/early myelofibrosis (pre-MF), and no minor criteria of pre-MF should be classified as a distinct MPN subtype, clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). RESULTS: 139 subjects meet these criteria who we compared with primary myelofibrosis (PMF) including 402 with pre-MF and 521 with overt myelofibrosis. CMD-IT subjects were more likely female and younger. They had lower frequencies of JAK2V617F compared with persons with PMF (55% vs. 70%; p < 0.001) and higher frequencies of CALR mutations (37% vs. 17%; p < 0.001). They also had lower frequency of variations associated with JAK2V617F susceptibility, JAK2 46/1 (35% vs. 47%; p = 0.021), and VEGFA rs3025039 (12% vs. 17%; p = 0.030). Subjects with CMD-IT had lower incidences of thrombotic events compared with those with pre-MF (9.7% vs. 26%; p < 0.001) and longer survival (median, not reached vs. 23 years; HR = 0.34 (0.10, 0.30); p < 0.001). CONCLUSION: Our data indicate CMD-IT is a distinct MPN subtype and should be included in the classification of myeloid neoplasms.

Cells coexpressing both myeloid and endothelial markers are detectable in the spleen and bone marrow of patients with primary myelofibrosis.

Different bodies of evidence support the existence of a common origin of hematopoietic and endothelial lineages; moreover, recent studies have indicated the presence of a hemogenic endothelium and a common hemato-endothelial precursor both in the embryo and in the cord blood. Conversely, to our knowledge, there is no evidence of such bipotential cells in human postnatal tissues or blood. In this study, we investigated the presence and phenotype of "transitional" cells in different tissues of patients with primary myelofibrosis (PMF). Using confocal microscopy and flow cytometry, we identified a rare cell population in the bone marrow and spleen of patients with PMF, which coexpresses the endothelial marker CD144 (vascular endothelial (VE)-cadherin), the pan-hematopoietic marker CD45, the early myeloid marker CD33, and CD34, a common endothelial and hematopoietic antigen.

Elevated plasma EDA fibronectin in primary myelofibrosis is determined by high allele burden of JAK2V617F mutation and strongly predicts splenomegaly progression.

In primary myelofibrosis, extra-domain A fibronectin (EDA-FN), the result of alternative splicing of FN gene, sustains megakaryocyte proliferation and confers a pro-inflammatory phenotype to bone marrow cell niches. In this work we assessed the levels of circulating EDA-FN in plasma samples of 122 patients with primary myelofibrosis. Patients with a homozygous JAK2V617F genotype displayed the higher level of plasma EDA-FN. Increased EDA-FN levels were associated with anemia, elevated high-sensitivity C-reactive protein, bone marrow fibrosis and splanchnic vein thrombosis at diagnosis. While no correlation was observed with CD34+ hematopoietic stem cell mobilization, elevated blood level of EDA-FN at diagnosis was a predictor of large splenomegaly (over 10 cm from the left costal margin) outcome. Thus, EDA-FN expression in primary myelofibrosis may represent the first marker of disease progression, and a novel target to treat splenomegaly.

Clonal Megakaryocyte Dysplasia with Normal Blood Values Is a Distinct Myeloproliferative Neoplasm.

INTRODUCTION: In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity. METHODS: This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis. RESULTS: Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41-54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. JAK2V617F was present in 10 subjects and CALR mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40-160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5-4.0) and 5.0 events (4.6-5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis. CONCLUSION: Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.

VEGFA rs3025020 Polymorphism Contributes to CALR -Mutation Susceptibility and Is Associated with Low Risk of Deep Vein Thrombosis in Primary Myelofibrosis.

Background Single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor A ( VEGFA ) are associated with susceptibility to several diseases including cancer. Correlations between VEGFA rs3025020 genotypes with clinical and laboratory features of primary myelofibrosis (PMF) are unstudied. Methods DNA was analyzed by real-time polymerase chain reaction for VEGFA rs3025020 genotypes in a cohort of 844 subjects with PMF and in two cohorts of normal subjects ( N = 247 and N = 107). Results Frequency of rs3025020 minor allele (T) was not significantly different in subjects with PMF compared with normals; however, the T-allele was more frequent in PMF subjects with a calreticulin ( CALR )-mutated genotype compared with normals (35 vs. 27%; OR = 1.47 [95% CI, 1.09, 1.98] p = 0.011), especially in subjects with a CALR- type 2/type 2-like mutation (43 vs. 27%; OR = 2.01 [1.25, 3.24] p = 0.004). CALR mutants with the rs3025020 TT genotype had higher CXCR4 expression on CD34-positive blood cells, and those who carried CT/TT genotypes had lower platelet concentrations compared with other genotypes at diagnosis. Overall, subjects with the rs3025020 CT/TT genotype had a lower cumulative incidence of deep vein thrombosis in typical sites (1.6 vs. 4.2%; OR = 0.37 [0.15, 0.90] p = 0.029) and longer interval from diagnosis to first thrombosis (HR = 0.37 [0.14, 0.95] p = 0.039). Conclusion Persons with PMF and the VEGFA rs3025020 minor T-allele are more likely to have a CALR mutation compared with other somatic driver mutations and lower cumulative incidence and hazard for deep vein thrombosis in typical sites.

New Markers of Disease Progression in Myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm due to the clonal proliferation of a hematopoietic stem cell. The vast majority of patients harbor a somatic gain of function mutation either of JAK2 or MPL or CALR genes in their hematopoietic cells, resulting in the activation of the JAK/STAT pathway. Patients display variable clinical and laboratoristic features, including anemia, thrombocytopenia, splenomegaly, thrombotic complications, systemic symptoms, and curtailed survival due to infections, thrombo-hemorrhagic events, or progression to leukemic transformation. New drugs have been developed in the last decade for the treatment of PMF-associated symptoms; however, the only curative option is currently represented by allogeneic hematopoietic cell transplantation, which can only be offered to a small percentage of patients. Disease prognosis is based at diagnosis on the classical International Prognostic Scoring System (IPSS) and Dynamic-IPSS (during disease course), which comprehend clinical parameters; recently, new prognostic scoring systems, including genetic and molecular parameters, have been proposed as meaningful tools for a better patient stratification. Moreover, new biological markers predicting clinical evolution and patient survival have been associated with the disease. This review summarizes basic concepts of PMF pathogenesis, clinics, and therapy, focusing on classical prognostic scoring systems and new biological markers of the disease.

Increased Plasma Levels of lncRNAs LINC01268, GAS5 and MALAT1 Correlate with Negative Prognostic Factors in Myelofibrosis.

Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients' cohort, it could be used for further studies to design an updated classification model for MF patients.

Clinical Relevance of VEGFA (rs3025039) +936 C>T Polymorphism in Primary Myelofibrosis: Susceptibility, Clinical Co-Variates, and Outcomes.

We evaluated the association of VEGFA rs3025039 polymorphism with clinical co-variates and outcomes in 849 subjects with primary myelofibrosis (PMF) and 250 healthy controls. Minor T-allele frequency was higher in subjects with JAK2V617F compared with those without JAK2V617F (18% vs. 13%; p = 0.014). In subjects with JAK2V617F, the TT genotype was associated at diagnosis with lower platelet concentrations (p = 0.033), higher plasma LDH concentration (p = 0.005), higher blood CD34-positive cells (p = 0.027), lower plasma cholesterol concentration (p = 0.046), and higher concentration of high-sensitivity C-reactive protein (p = 0.018). These associations were not found in subjects with PMF without JAK2V617F. In subjects with the TT genotype, risk of death was higher compared with subjects with CC/CT genotypes (HR = 2.12 [1.03, 4.35], p = 0.041). Finally, the TT genotype was associated with higher frequency of deep vein thrombosis in typical sites (12.5% vs. 2.5%; OR = 5.46 [1.51, 19.7], p = 0.009). In conclusion, in subjects with PMF, the VEGFA rs3025039 CT or TT genotypes are more common in those with JAK2V617F than in those without JAK2V67F mutation and are associated with disease severity, poor prognosis, and risk of deep vein thrombosis.

The human amniotic fluid stem cell secretome triggers intracellular Ca2+ oscillations, NF-κB nuclear translocation and tube formation in human endothelial colony-forming cells.

Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CMHypo ) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+ -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca2+ release and store-operated Ca2+ entry (SOCE). hAFS-CMHypo -induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+ -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF-κB activity prevented hAFS-CMHypo -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury.

Impact of the rs1024611 Polymorphism of CCL2 on the Pathophysiology and Outcome of Primary Myelofibrosis.

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2-one of the most potent immunomodulatory chemokines-to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.

Gene expression profile correlates with molecular and clinical features in patients with myelofibrosis.

Myelofibrosis (MF) belongs to the family of classic Philadelphia-negative myeloproliferative neoplasms (MPNs). It can be primary myelofibrosis (PMF) or secondary myelofibrosis (SMF) evolving from polycythemia vera (PV) or essential thrombocythemia (ET). Despite the differences, PMF and SMF patients are currently managed in the same way, and prediction of survival is based on the same clinical and genetic features. In the last few years, interest has grown concerning the ability of gene expression profiles (GEPs) to provide valuable prognostic information. Here, we studied the GEPs of granulocytes from 114 patients with MF, using a microarray platform to identify correlations with patient characteristics and outcomes. Cox regression analysis led to the identification of 201 survival-related transcripts characterizing patients who are at high risk for death. High-risk patients identified by this gene signature displayed an inferior overall survival and leukemia-free survival, together with clinical and molecular detrimental features included in contemporary prognostic models, such as the presence of high molecular risk mutations. The high-risk group was enriched in post-PV and post-ET MF and JAK2V617F homozygous patients, whereas pre-PMF was more frequent in the low-risk group. These results demonstrate that GEPs in MF patients correlate with their molecular and clinical features, particularly their survival, and represent the proof of concept that GEPs might provide complementary prognostic information to be applied in clinical decision making.

Long-term safety and efficacy of givinostat in polycythemia vera: 4-year mean follow up of three phase 1/2 studies and a compassionate use program.

Polycythemia vera (PV) is a BCR-ABL1-negative myeloproliferative neoplasm (MPN) characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow, mainly due to a Janus kinase 2 gene mutation (JAK2V617F). Givinostat, a histone-deacetylase inhibitor that selectively targets JAK2V617F cell growth, has demonstrated good efficacy and safety in three phase 1/2 studies in patients with PV. This manuscript focuses on the 4-year mean (2.8 year median) follow-up of an open-label, long-term study that enrolled 51 patients with PV (out of a total of 54 with MPN) who received clinical benefit from givinostat in these previous studies or on compassionate use, and who continued to receive givinostat at the last effective and tolerated dose. The primary objectives are to determine givinostat's long-term safety and tolerability, and efficacy evaluated by the investigators according to internationally recognized response criteria. During follow-up, only 10% of PV patients reported Grade 3 treatment-related adverse events (AEs), while none had Grade 4 or 5 treatment-related AEs. The overall response rate for the duration of follow-up was always greater than 80% in patients with PV. In conclusion, givinostat demonstrated a good safety and efficacy profile in patients with PV, data supporting long-term use in this population.

Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe ß-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

Reduced CXCR4-expression on CD34-positive blood cells predicts outcomes of persons with primary myelofibrosis.

The expression of the CXCR4 chemokine receptor on CD34-positive blood cells is reduced in persons with primary myelofibrosis (PMF). We analyzed the relevance of cytofluorimetric assessment of the percentage of CD34-positive blood cells that had a positive CXCR4 surface expression (CD34/CXCR4-se) in a large cohort of subjects with myeloproliferative neoplasms. Mean CD34/CXCR4-se was lower in subjects with PMF compared with those with essential thrombocythemia (ET) or polycythemia vera (PV). A cutoff value of 39% was associated with a diagnosis of pre-fibrotic PMF vs. ET with a positive predictive value of 97%. In PMF male sex, older age, and MPL mutation were independent correlates of reduced CD34/CXCR4-se and associated with a briefer interval to development of severe anemia, large splenomegaly, thrombocytopenia, leukopenia, elevated CD34-positive blood cells, blast transformation and death. We constructed a prognostic model including age >65 years, hemoglobin < 100 g/L, CD34-positive blood cells > 50 × 106/L, and CD34/CXCR4-se <39% at diagnosis. The model identified three risk cohorts with greater accuracy compared with the International Prognostic Scoring System. In conclusion, CD34/CXCR4-se is a highly sensitive marker of disease activity and a new potential diagnostic and prognostic biomarker in PMF.