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We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.

Increased mobilization of circulating endothelial progenitor cells may represent a new biological hallmark of myeloproliferative neoplasms. We measured circulating endothelial colony forming cells (ECFCs) in 106 patients with primary myelofibrosis, fibrotic stage, 49 with prefibrotic myelofibrosis, 59 with essential thrombocythemia or polycythemia vera, and 43 normal controls. Levels of ECFC frequency for patient's characteristics were estimated by using logistic regression in univariate and multivariate setting. The sensitivity, specificity, likelihood ratios, and positive predictive value of increased ECFC frequency were calculated for the significantly associated characteristics. Increased frequency of ECFCs resulted independently associated with history of splanchnic vein thrombosis (adjusted odds ratio = 6.61, 95% CI = 2.54-17.16), and a summary measure of non-active disease, i.e. hemoglobin of 13.8 g/dL or lower, white blood cells count of 7.8×10(9)/L or lower, and platelet count of 400×10(9)/L or lower (adjusted odds ratio = 4.43, 95% CI = 1.45-13.49) Thirteen patients with splanchnic vein thrombosis non associated with myeloproliferative neoplasms were recruited as controls. We excluded a causal role of splanchnic vein thrombosis in ECFCs increase, since no control had elevated ECFCs. We concluded that increased frequency of ECFCs represents the biological hallmark of a non-active myeloproliferative neoplasm with high risk of splanchnic vein thrombosis. The recognition of this disease category copes with the phenotypic mimicry of myeloproliferative neoplasms. Due to inherent performance limitations of ECFCs assay, there is an urgent need to arrive to an acceptable standardization of ECFC assessment.

Reelin is a large secreted extracellular matrix glycoprotein which contributes to positioning, migration and laminar organization of several central nervous system structures during neurodevelopment. Recent studies reported the expression of Reelin and its intracellular adapter protein DAB1 in neuroblastoma, where it appears to be involved in cell motility and invasiveness. Interestingly, our data obtained by immunolocalization analysis show the expression of Reelin in both tumor and peritumoral area of glioblastoma (GBM). It is known that many solid tumors may originate from cancer stem cells (CSC) which are usually resistant to common therapies and might be involved in tumor progression. Therefore, we evaluated the expression of Reelin in CSC neurospheres isolated from both tumor (GCSC) and peritumoral area (PCSC) of GBM. Immunocytochemistry analysis showed the expression of Reelin in both cell types, suggesting that this protein may contribute to neurosphere tridimensional organization and possibly to cell migration during tumor progression. This is the first evidence of Reelin expression in human GBM which might indicate a pivotal role of this protein in the regulation of tumor development. Our data may open potential avenues for GBM treatment by targeting Reelin signaling activity., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Since SARS-CoV-2 outbreak in December 2019, world health-system has been severely impacted with increased hospitalization, Intensive-Care-Unit (ICU) access and high mortality rates, mostly due to severe acute respiratory failure and multi-organ failure. Excessive and uncontrolled release of proinflammatory cytokines (cytokine release/storm syndrome, CRS) have been linked to the development of these events. The recent advancements of immunotherapy for the treatment of hematologic and solid tumors shed light on many of the molecular mechanisms underlying this phenomenon, thus rendering desirable a multidisciplinary approach to improve COVID-19 patients’ outcome. Indeed, currently available therapeutic-strategies to overcome CRS, should be urgently evaluated for their capability of reducing COVID-19 mortality. Notably, COVID-19 shares different pathogenic aspects with acute graft-versus-host-disease (aGVHD), hemophagocytic-lymphohistiocytosis (HLH), myelofibrosis, and CAR-T-associated CRS. Specifically, similarly to aGVHD, an induced tissue damage (caused by the virus) leads to increased cytokine release (TNFα and IL-6) which in turn leads to exaggerated dendritic cells, macrophages (like in HLH) and lymphocytes (as in CAR-T) activation, immune-cells migration, and tissue-damage (including late-stage fibrosis, similar to myelofibrosis). Janus Kinase (JAK) signaling represents a molecular hub linking all these events, rendering JAK-inhibitors suitable to limit deleterious effects of an overwhelming inflammatory-response. Accordingly, ruxolitinib is the only selective JAK1 and JAK2-inhibitor approved for the treatment of myelofibrosis and aGVHD. Here, we discuss, from a molecular and hematological point of view, the rationale for targeting JAK signaling in the management of COVID-19 patients and report the clinical results of a patient admitted to ICU among the firsts to be treated with ruxolitinib in Italy.

Background The Sars-CoV-2 can cause severe pneumonia with multiorgan disease; thus, the identification of clinical and laboratory predictors of the progression towards severe and fatal forms of this illness is needed. Here, we retrospectively evaluated and integrated laboratory parameters of 45 elderly subjects from a long-term care facility with Sars-CoV-2 outbreak and spread, to identify potential common patterns of systemic response able to better stratify patients’ clinical course and outcome. Methods Baseline white blood cells, granulocytes’, lymphocytes’, and platelets’ counts, hemoglobin, total iron, ferritin, D-dimer, and interleukin-6 concentration were used to generate a principal component analysis. Statistical analysis was performed by using R statistical package version 4.0. Results We identified 3 laboratory patterns of response, renamed as low-risk, intermediate-risk, and high-risk, strongly associated with patients’ survival (p Conclusions Our data suggest that a combination of few laboratory parameters, i.e. iron status, D-dimer and lymphocyte/monocyte count at admission and during the hospital stay, can predict clinical progression in COVID-19.

New insights into the field of iron metabolism within the tumor microenvironment have been uncovered in recent years. Iron promotes the production of reactive oxygen species, which may either trigger ferroptosis cell death or contribute to malignant transformation. Once transformed, cancer cells divert tumor-infiltrating immune cells to satisfy their iron demand, thus affecting the tumor immunosurveillance. In this review, we highlight how the bioavailability of this metal shapes complex metabolic pathways within the tumor microenvironment and how this affects both tumor-associated macrophages and tumor-infiltrating lymphocytes functions. Furthermore, we discuss the potentials as well as the current clinical controversies surrounding the use of iron metabolism as a target for new anticancer treatments in two opposed conditions: (i) the “hot” tumors, which are usually enriched in immune cells infiltration and are extremely rich in iron availability within the microenvironment, and (ii) the “cold” tumors, which are often very poor in immune cells, mainly due to immune exclusion.

Adult stem/progenitor are a small population of cells that reside in tissue-specific niches and possess the potential to differentiate in all cell types of the organ in which they operate. Adult stem cells are implicated with the homeostasis, regeneration, and aging of all tissues. Tissue-specific adult stem cell senescence has emerged as an attractive theory for the decline in mammalian tissue and organ function during aging. Cardiac aging, in particular, manifests as functional tissue degeneration that leads to heart failure. Adult cardiac stem/progenitor cell (CSC) senescence has been accordingly associated with physiological and pathological processes encompassing both non-age and age-related decline in cardiac tissue repair and organ dysfunction and disease. Senescence is a highly active and dynamic cell process with a first classical hallmark represented by its replicative limit, which is the establishment of a stable growth arrest over time that is mainly secondary to DNA damage and reactive oxygen species (ROS) accumulation elicited by different intrinsic stimuli (like metabolism), as well as external stimuli and age. Replicative senescence is mainly executed by telomere shortening, the activation of the p53/p16INK4/Rb molecular pathways, and chromatin remodeling. In addition, senescent cells produce and secrete a complex mixture of molecules, commonly known as the senescence-associated secretory phenotype (SASP), that regulate most of their non-cell-autonomous effects. In this review, we discuss the molecular and cellular mechanisms regulating different characteristics of the senescence phenotype and their consequences for adult CSCs in particular. Because senescent cells contribute to the outcome of a variety of cardiac diseases, including age-related and unrelated cardiac diseases like diabetic cardiomyopathy and anthracycline cardiotoxicity, therapies that target senescent cell clearance are actively being explored. Moreover, the further understanding of the reversibility of the senescence phenotype will help to develop novel rational therapeutic strategies.

// Alessio D’Alessio 1 , Gabriella Proietti 1 , Gina Lama 1 , Filippo Biamonte 1 , Libero Lauriola 2 , Umberto Moscato 3 , Angelo Vescovi 4 , Annunziato Mangiola 5 , Cristiana Angelucci 1 , Gigliola Sica 1 1 Institute of Histology and Embryology, “A. Gemelli” Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy 2 Institute of Pathology, “A. Gemelli” Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy 3 Institute of Public Health, Hygiene Division,“A. Gemelli” Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy 4 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy 5 Institute of Neurosurgery, "A. Gemelli" Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy Correspondence to: Alessio D’Alessio, email: alessio.dalessio@unicatt.it Keywords: angiogenesis, glioblastoma, peritumoral tissue, cancer stem cells, hypoxia Received: April 22, 2016 Accepted: September 25, 2016 Published: October 01, 2016 ABSTRACT The formation of new blood vessels represents a crucial event under both physiological and pathological circumstances. In this study, we evaluated by immunohistochemistry, and/or Western blotting and/or quantitative real time-PCR the expression of HIF1α, HIF2α, VEGF, VEGFR1 and VEGFR2 in surgical glioblastoma multiforme (GBM) and peritumoral tissue samples obtained from 50 patients as well as in cancer stem cells (CSCs) isolated from GBM (GCSCs) and peritumoral tissue (PCSCs) of 5 patients. We also investigated the contribution of both GCSCs and PCSCs on the behavior of endothelial cells (ECs) in vitro . Immunohistochemistry demonstrated the expression of angiogenesis markers in both GBM and peritumoral tissue. In addition, in vitro tube formation assay indicated that both GCSCs and PCSCs stimulate EC proliferation as well as tube-like vessel formation. An increased migration aptitude was mainly observed when ECs were cultured in the presence of GCSCs rather than in the presence of PCSCs. These findings suggest that relevant neoangiogenetic events may occur in GBM. In particular, VEGF/VEGFR co-expression in PCSCs leads to hypothesize the involvement of an autocrine signaling. Moreover, our results suggest that both GCSCs and PCSCs own the skill of activating the “angiogenic switch” and the capability of modulating EC behavior, indicating that both cell types are either responsive to angiogenic stimuli or able to trigger angiogenic response. Together with our previous findings, this study adds a further piece to the challenging puzzle of the characterization of peritumoral tissue and of the definition of its real role in GBM pathophysiology.

The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

Purpose To investigate the effect of 18 months' parathyroid hormone 1-84 (PTH 1-84) treatment on serum levels of bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF), in postmenopausal women with established osteoporosis.Methods Thirty-seven postmenopausal women with osteoporosis (mean age 72.9 +/- 8.1 years old) and 23 healthy controls (mean age 68.9 +/- 9.9 years old) were enrolled. Patients were treated with daily subcutaneous injections of PTH (1-84) 100 mcg for 18 months, plus calcium 1 gr and vitamin D 800 IU per os daily. Blood samples were taken every 6 months during the study.Results At baseline, there were no differences considering anthropometric parameters, co-morbidities, current medications used between patients and controls. Mean serum VEGF levels were significantly higher in osteoporotic patients compared to controls (436.7 +/- 259.7 vs. 260.3 +/- 184.3 pg/ml, p = 0.006), while there were no differences in mean serum values of BMP4 (5.3 +/- 1.7 vs. 5.7 +/- 1.6 pg/ml, p = 0.40). Serum VEGF levels increased by approximately 20% after 12 months of PTH (1-84) treatment compared to baseline (p = 0.03) and by 22% after 18 months (p = 0.01). A significant increase of 10% in mean serum BMP4 levels was observed after 18 months of PTH (1-84) treatment compared to baseline (p = 0.02). In the control group we found no differences after 18 months compared to baseline in BMP4 (5.7 +/- 1.6 vs. 6.0 +/- 1.5 pg/ml, p = 0.53) and VEGF (260.3 +/- 184.3 vs. 257.4 +/- 107.1 pg/ ml, p = 0.94).Conclusions PTH (1-84) treatment increased serum levels of VEGF and BMP4 in postmenopausal women with severe osteoporosis.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/ stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (α-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.

Background Metastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions. Methods 3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2D to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines. Results We found that the transition from 2D to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production and cell viability. Furthermore, both the number and size of spheroids produced by H460, HCC827, MCF7, and T47D cell lines were significantly reduced upon ALDOC and ENO2 knockdown. Conclusions Our results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid regardless of the tumor type and nutrient environmental availability, thus suggesting the possible general involvement of this mechanism in cancer metastasis. The pan-cancer validation of this vulnerability could potentially help to slow or prevent cancer progression.