Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition.

BACKGROUND: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. METHODS: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. RESULTS: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. CONCLUSIONS: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results.

Transient Daratumumab-Induced Refractive Shift in Multidrug-Resistant Multiple Myeloma: Case Report and Literature Review.

Daratumumab, an anti-CD38 monoclonal antibody, is worldwide approved for treatment of newly diagnosed and relapsed/refractory multiple myeloma (MM) patients and is available as intravenous or subcutaneous formulations. Intravenous daratumumab is associated with frequent infusion-related reactions, while eye complications, especially refractive shifts, are very rare, with only previously reported cases. Here, we described a rare case of multi-refractory MM who developed transient myopic shift during intravenous daratumumab infusion successfully treated only with cycloplegic collyrium not requiring infusion rate lowering or drug discontinuation. This conservative therapeutic approach allowed termination of induction therapy and autologous hematopoietic stem-cell transplantation resulting in durable complete remission.

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Plasminogen System in the Pathophysiology of Sepsis: Upcoming Biomarkers.

Severe hemostatic disturbances and impaired fibrinolysis occur in sepsis. In the most serious cases, the dysregulation of fibrinolysis contributes to septic shock, disseminated intravascular coagulation (DIC), and death. Therefore, an analysis of circulating concentrations of pro- and anti-fibrinolytic mediators could be a winning strategy in both the diagnosis and the treatment of sepsis. However, the optimal cutoff value, the timing of the measurements, and their combination with coagulation indicators should be further investigated. The purpose of this review is to summarize all relevant publications regarding the role of the main components of the plasminogen activation system (PAS) in the pathophysiology of sepsis. In addition, the clinical value of PAS-associated biomarkers in the diagnosis and the outcomes of patients with septic syndrome will be explored. In particular, experimental and clinical trials performed in emergency departments highlight the validity of soluble urokinase plasminogen activator receptor (suPAR) as a predictive and prognostic biomarker in patients with sepsis. The measurements of PAI-I may also be useful, as its increase is an early manifestation of sepsis and may precede the development of thrombocytopenia. The upcoming years will undoubtedly see progress in the use of PAS-associated laboratory parameters.

Secretory and Membrane-Associated Biomarkers of Mast Cell Activation and Proliferation.

Mast cells (MCs) are immune cells distributed in many organs and tissues and involved in the pathogenesis of allergic and inflammatory diseases as a major source of pro-inflammatory and vasoactive mediators. MC-related disorders are heterogeneous conditions characterized by the proliferation of MC within tissues and/or MC hyper-reactivity that leads to the uncontrolled release of mediators. MC disorders include mastocytosis, a clonal disease characterized by tissue MC proliferation, and MC activation syndromes that can be primary (clonal), secondary (related to allergic disorders), or idiopathic. Diagnosis of MC disorders is difficult because symptoms are transient, unpredictable, and unspecific, and because these conditions mimic many other diseases. Validation of markers of MC activation in vivo will be useful to allow faster diagnosis and better management of MC disorders. Tryptase, being the most specific MC product, is a widely used biomarker of proliferation and activation. Other mediators, such as histamine, cysteinyl leukotrienes, and prostaglandin D2, are unstable molecules and have limitations in their assays. Surface MC markers, detected by flow cytometry, are useful for the identification of neoplastic MC in mastocytosis but, so far, none of them has been validated as a biomarker of MC activation. Further studies are needed to identify useful biomarkers of MC activation in vivo.

Macrophages and Urokinase Plasminogen Activator Receptor System in Multiple Myeloma: Case Series and Literature Review.

The microenvironment plays an essential role in multiple myeloma (MM) development, progression, cell proliferation, survival, immunological escape, and drug resistance. Mesenchymal stromal cells and macrophages release tolerogenic cytokines and favor anti-apoptotic signaling pathway activation, while the urokinase plasminogen activator receptor (uPAR) system contributes to migration through an extracellular matrix. Here, we first summarized the role of macrophages and the uPAR system in MM pathogenesis, and then we reported the potential therapeutic effects of uPAR inhibitors in a case series of primary MM-derived adherent cells. Our preliminary results showed that after uPAR inhibitor treatments, interleukein-6 (mean ± SD, 8734.95 ± 4169.2 pg/mL vs. 359.26 ± 393.8 pg/mL, pre- vs. post-treatment; p = 0.0012) and DKK-1 levels (mean ± SD, 7005.41 ± 6393.4 pg/mL vs. 61.74 ± 55.2 pg/mL, pre- vs. post-treatment; p = 0.0043) in culture medium were almost completely abolished, supporting further investigation of uPAR blockade as a therapeutic strategy for MM treatment. Therefore, uPAR inhibitors could exert both anti-inflammatory and pro-immunosurveillance activity. However, our preliminary results need further validation in additional in vitro and in vivo studies.

All Roads Lead to Interferon-γ: From Known to Untraveled Pathways in Acquired Aplastic Anemia.

Bone marrow failure (BMF) syndromes are a heterogeneous group of benign hematological conditions with common clinical features including reduced bone marrow cellularity and peripheral blood cytopenias. Acquired aplastic anemia (AA) is caused by T helper(Th)1-mediated immune responses and cytotoxic CD8+ T cell-mediated autologous immune attacks against hematopoietic stem and progenitor cells (HSPCs). Interferon-γ (IFNγ), tumor necrosis factor-α, and Fas-ligand are historically linked to AA pathogenesis because they drive Th1 and cytotoxic T cell-mediated responses and can directly induce HSPC apoptosis and differentiation block. The use of omics technologies has amplified the amount of data at the single-cell level, and knowledge on AA, and new scenarios, have been opened on "old" point of view. In this review, we summarize the current state-of-art of the pathogenic role of IFNγ in AA from initial findings to novel evidence, such as the involvement of the HIF-1α pathway, and how this knowledge can be translated in clinical practice.

Implementation of International Prognostic Index with flow cytometry immunophenotyping for better risk stratification of chronic lymphocytic leukemia.

BACKGROUND: Current chronic lymphocytic leukemia (CLL) International Prognostic Index (IPI) stratifies patients based on clinical, molecular, and biochemical features; however, B-cell markers also influence CLL outcomes. Here, prognostic roles of CD11c, CD38, and CD49d were first evaluated, and then an immunophenotypic score was combined with CLL-IPI for risk stratification of CLL patients. METHODS: A total of 171 CLL subjects were included, and surface marker expression was assessed by flow cytometry. Levels ≥30% were chosen as cut-off of positivity to a marker; then values of 1 (for CD11c and CD38) or 3 (for CD49d) were assigned and scores determined for each patient's clone immunophenotype. RESULTS: CD49d positivity was significantly associated with simultaneous expression of CD11c and/or CD38, unmutated IGHV status, and higher ß2-microglobulin levels compared to those with CD49d negativity. Moreover, CD49d+ patients experienced a shorter progression-free survival and time to treatment. When the immunophenotypic score was combined with CLL-IPI, patients with high-risk immunophenotype had a significantly lower time-to-treatment regardless CLL-IPI. CONCLUSIONS: Our results suggested clinical utility of an integrated prognostic score for better risk stratification of CLL patients. These results require further validation in prospective larger studies.

Giant plasma cells with multiple immature nuclei in a young woman with newly diagnosed multiple myeloma.

BACKGROUND: Multiple myeloma with giant multinucleated plasma cells is a very rare entity and mostly reported cases are dated. This plasma cell morphology has been observed after monoclonal antibody treatments, such as daratumumab, and patients have experienced a worse prognosis with partial responses and a high rate of relapse. RESULTS: Here, we showed a newly diagnosed multiple myeloma with giant plasma cells with multiple (up to 13) immature nuclei who achieved a complete remission after a first line therapy and underwent to autologous hematopoietic stem cell transplantation, as per international guidelines.

Real-world experience with the Sydney System on 1458 cases of lymph node fine needle aspiration cytology.

INTRODUCTION: Lymph node (LN) fine needle aspiration cytology (FNAC) is a safe, quick, inexpensive, reliable, and minimally invasive technique for the diagnosis of lymphadenopathies. Recently, an international committee of experts proposed guidelines for the performance, classification, and reporting of LN-FNAC: the Sydney System. We set out to analyse the diagnostic performance of the Sydney System in a retrospective study. METHODS: We retrieved 1458 LN-FNACs, reformulated the diagnoses according to the Sydney System, and compared them to the histological control where available (n = 551, 37.8%). RESULTS: The risk of malignancy for each of the five categories was 66.7% for inadequate/insufficient, 9.38% for benign (overall: 0.84%), 28.6% for atypical, 100% for suspicious and 99.8% for malignant. LN-FNAC showed a sensitivity of 97.94%, a specificity of 96.92%, a positive predictive value of 99.58%, and a negative predictive value of 86.30%. CONCLUSIONS: These data support the usage of LN-FNAC as an agile first-level technique in the diagnosis of lymphadenopathies. The Sydney System supports and enhances this role of LN-FNAC, and its adoption is encouraged. In negative cases, coupled with ancillary techniques, LN-FNAC can reassure the clinician regarding the benignity of a lymphadenopathy and indicate the need for clinical follow-up, which will catch possible false negatives. In positive cases, LN-FNAC can provide sufficient information, including predictive biomarkers, to initiate management and obviate the need for subsequent, more invasive procedures. Given its speed, minimal invasiveness, and low cost, LN-FNAC can be performed in most cases, even when more invasive techniques are not feasible.

Innovative Anti-CD38 and Anti-BCMA Targeted Therapies in Multiple Myeloma: Mechanisms of Action and Resistance.

CD38 and B-cell maturation antigens (BCMAs) are prevalently expressed on neoplastic plasma cells in multiple myeloma (MM), making them ideal therapeutic targets. Anti-CD38 monoclonal antibodies, such as approved daratumumab and isatuximab, are currently the milestone in MM treatment because they induce plasma cell apoptosis and kill through several mechanisms, including antibody-dependent cellular cytotoxicity or phagocytosis. BCMA is considered an excellent target in MM, and three different therapeutic strategies are either already available in clinical practice or under investigation: antibody-drug conjugates, such as belantamab-mafodotin; bispecific T cell engagers; and chimeric antigen receptor-modified T cell therapies. Despite the impressive clinical efficacy of these new strategies in the treatment of newly diagnosed or multi-refractory MM patients, several mechanisms of resistance have already been described, including antigen downregulation, the impairment of antibody-dependent cell cytotoxicity and phagocytosis, T- and natural killer cell senescence, and exhaustion. In this review, we summarize the current knowledge on the mechanisms of action and resistance of anti-CD38 and anti-BCMA agents and their clinical efficacy and safety.

Sex-tailored pharmacology and COVID-19: Next steps towards appropriateness and health equity.

Making gender bias visible allows to fill the gaps in knowledge and understand health records and risks of women and men. The coronavirus disease 2019 (COVID-19) pandemic has shown a clear gender difference in health outcomes. The more severe symptoms and higher mortality in men as compared to women are likely due to sex and age differences in immune responses. Age-associated decline in sex steroid hormone levels may mediate proinflammatory reactions in older adults, thereby increasing their risk of adverse outcomes, whereas sex hormones and/or sex hormone receptor modulators may attenuate the inflammatory response and provide benefit to COVID-19 patients. While multiple pharmacological options including anticoagulants, glucocorticoids, antivirals, anti-inflammatory agents and traditional Chinese medicine preparations have been tested to treat COVID-19 patients with varied levels of evidence in terms of efficacy and safety, information on sex-targeted treatment strategies is currently limited. Women may have more benefit from COVID-19 vaccines than men, despite the occurrence of more frequent adverse effects, and long-term safety data with newly developed vectors are eagerly awaited. The prevalent inclusion of men in randomized clinical trials (RCTs) with subsequent extrapolation of results to women needs to be addressed, as reinforcing sex-neutral claims into COVID-19 research may insidiously lead to increased inequities in health care. The huge worldwide effort with over 3000 ongoing RCTs of pharmacological agents should focus on improving knowledge on sex, gender and age as pillars of individual variation in drug responses and enforce appropriateness.

Bone Marrow Failure Syndromes, Overlapping Diseases with a Common Cytokine Signature.

Bone marrow failure (BMF) syndromes are a heterogenous group of non-malignant hematologic diseases characterized by single- or multi-lineage cytopenia(s) with either inherited or acquired pathogenesis. Aberrant T or B cells or innate immune responses are variously involved in the pathophysiology of BMF, and hematological improvement after standard immunosuppressive or anti-complement therapies is the main indirect evidence of the central role of the immune system in BMF development. As part of this immune derangement, pro-inflammatory cytokines play an important role in shaping the immune responses and in sustaining inflammation during marrow failure. In this review, we summarize current knowledge of cytokine signatures in BMF syndromes.

Stem Cell and Macrophage Roles in Skeletal Muscle Regenerative Medicine.

In severe muscle injury, skeletal muscle tissue structure and functionality can be repaired through the involvement of several cell types, such as muscle stem cells, and innate immune responses. However, the exact mechanisms behind muscle tissue regeneration, homeostasis, and plasticity are still under investigation, and the discovery of pathways and cell types involved in muscle repair can open the way for novel therapeutic approaches, such as cell-based therapies involving stem cells and peripheral blood mononucleate cells. Indeed, peripheral cell infusions are a new therapy for muscle healing, likely because autologous peripheral blood infusion at the site of injury might enhance innate immune responses, especially those driven by macrophages. In this review, we summarize current knowledge on functions of stem cells and macrophages in skeletal muscle repairs and their roles as components of a promising cell-based therapies for muscle repair and regeneration.

Olive Trees By-Products as Sources of Bioactive and Other Industrially Useful Compounds: A Systematic Review.

The need to produce an ever-increasing quantity of material products and food resulting from the planet globalization process has contributed to the spread of modern agriculture based on a linear production resulting in the generation of tons of waste. This huge amount of waste is generally accumulated in landfills, causing different environmental problems. Hence, researchers moved on to study the processes used to recover agro-industrial by-products within a circular and sustainable bio-economy concept. A systematic quest on Scopus and PubMed databases was performed to identify the data available to date on recycling agro-industrial by-products of Olea europaea L. This systematic review summarizes the knowledge regarding the use of olive trees by-products for producing animal feed, biocomposites, bioethanol, cellulose pulp, activated carbon, and as a fuel source for energy production. Furthermore, the data regarding the potential biological activity of extracts from olive roots, wood, bark, and pruning were analyzed. Olive trees by-products are, indeed, rich in molecules with antioxidant, antimicrobial, cardioprotective, and anticancer activity, representing a promising candidate for treat several human diseases.

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.

Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases.

Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases.

Axitinib in Ponatinib-Resistant B-Cell Acute Lymphoblastic Leukemia Harboring a T315L Mutation.

Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph) is a hematological aggressive disease with a fatal outcome in more than 50% of cases. Tyrosine kinase inhibitors (TKIs) targeting the activity of BCR-ABL1 protein have improved the prognosis; however, relapses are frequent because of acquired somatic mutations in the BCR-ABL1 kinase domain causing resistance to first, second and third generation TKIs. Axitinib has shown in vitro and ex vivo activity in blocking ABL1; however, clinical trials exploring its efficacy in ALL are missing. Here, we presented a 77-year-old male with a diagnosis of Ph positive ALL resistant to ponatinib and carrying a rare threonine to leucine (T315L) mutation on BCR-ABL1 gene. The patient was treated with axitinib at 5 mg/twice daily as salvage therapy showing an immediate although transient benefit with an overall survival of 9.3 months. Further dose-finding and randomized clinical trials are required to assess the real efficacy of axitinib for adult Ph positive ALL resistant to third generation TKIs.

Circulating S100A8 and S100A9 protein levels in plasma of patients with acquired aplastic anemia and myelodysplastic syndromes.

The alarmin family members S100A8 and S100A9 are acute phase inflammation proteins, but they also have been proposed as biomarkers in many malignant and non-malignant diseases. In this study, circulating S100A8 and S100A9 homodimers and S100A8/A9 heterodimers in plasma were systematically investigated by ELISA in aplastic anemia (AA) and myelodysplastic syndromes (MDS). Plasma was obtained from 58 severe AA (SAA) and 30 MDS patients, and from 47 age- and sex-matched healthy donors. In 40 out of the 58 AA subjects, S100A protein levels were measured before and 6 months after immunosuppressive therapy (IST). No differences were observed in AA patients at diagnosis compared to healthy controls for circulating S100A homodimers and heterodimers. After therapy, SAA-responders showed significantly increased circulating S100A8. Non-responding patients had significantly higher levels of circulating S100A8/A9 compared to responders and healthy controls, but without variations of S100A8 and S100A9 homodimers. In MDS patients, circulating S100A8 was significantly elevated compared to those of AA and/or healthy controls. By Pearson correlation analysis of protein levels and blood counts, multiple correlations were found. However, as S100A8 and S100A9 are abundantly present in white blood cells and platelets, correlations with blood counts likely mirror the higher number of cells in the blood of some patients. In conclusion, our findings indicate that circulating S100A8 is increased in MDS but not in AA, and that may be useful to distinguish these diseases in the differential diagnosis of bone marrow failure syndromes. Clinicaltrials.gov identifiers: NCT00260689, NCT00604201, NCT01328587, NCT01623167, NCT00001620, NCT00001397.