Long-term pharmacodynamic and clinical effects of twice- versus once-daily low-dose aspirin in essential thrombocythemia: The ARES trial.

Patients with essential thrombocythemia (ET) are treated with once-daily low-dose aspirin to prevent thrombosis, but their accelerated platelet turnover shortens the antiplatelet effect. The short-term Aspirin Regimens in EsSential Thrombocythemia trial showed that twice-daily aspirin dosing restores persistent platelet thromboxane (TX) inhibition. However, the long-term pharmacodynamic efficacy, safety and tolerability of twice-daily aspirin remain untested. We performed a multicenter, randomized, open-label, blinded-endpoint, phase-2 trial in which 242 patients with ET were randomized to 100 mg aspirin twice- or once-daily and followed for 20 months. The primary endpoint was the persistence of low serum TXB2, a surrogate biomarker of antithrombotic efficacy. Secondary endpoints were major and clinically relevant non-major bleedings, serious vascular events, symptom burden assessed by validated questionnaires, and in vivo platelet activation. Serum TXB2 was consistently lower in the twice-daily versus once-daily regimen on 10 study visits over 20 months: median 3.9 ng/mL versus 19.2 ng/mL, respectively; p < .001; 80% median reduction; 95% CI, 74%-85%. No major bleeding occurred. Clinically relevant non-major bleedings were non-significantly higher (6.6% vs. 1.7%), and major thromboses lower (0.8% vs. 2.5%) in the twice-daily versus once-daily group. Patients on the twice-daily regimen had significantly lower frequencies of disease-specific symptoms and severe hand and foot microvascular pain. Upper gastrointestinal pain was comparable in the two arms. In vivo platelet activation was significantly reduced by the twice-daily regimen. In patients with ET, twice-daily was persistently superior to once-daily low-dose aspirin in suppressing thromboxane biosynthesis and reducing symptom burden, with no detectable excess of bleeding and gastrointestinal discomfort.

Acute myeloid leukemia: from NGS, through scRNA-seq, to CAR-T. dissect cancer heterogeneity and tailor the treatment.

Acute myeloid leukemia (AML) is a malignant blood cancer with marked cellular heterogeneity due to altered maturation and differentiation of myeloid blasts, the possible causes of which are transcriptional or epigenetic alterations, impaired apoptosis, and excessive cell proliferation. This neoplasm has a high rate of resistance to anticancer therapies and thus a high risk of relapse and mortality because of both the biological diversity of the patient and intratumoral heterogeneity due to the acquisition of new somatic changes. For more than 40 years, the old gold standard "one size fits all" treatment approach included intensive chemotherapy treatment with anthracyclines and cytarabine.The manuscript first traces the evolution of the understanding of the pathology from the 1970s to the present. The enormous strides made in its categorization prove to be crucial for risk stratification, enabling an increasingly personalized diagnosis and treatment approach.Subsequently, we highlight how, over the past 15 years, technological advances enabling single cell RNA sequencing and T-cell modification based on the genomic tools are affecting the classification and treatment of AML. At the dawn of the new millennium, the advent of high-throughput next-generation sequencing technologies has enabled the profiling of patients evidencing different facets of the same disease, stratifying risk, and identifying new possible therapeutic targets that have subsequently been validated. Currently, the possibility of investigating tumor heterogeneity at the single cell level, profiling the tumor at the time of diagnosis or after treatments exist. This would allow the identification of underrepresented cellular subclones or clones resistant to therapeutic approaches and thus responsible for post-treatment relapse that would otherwise be difficult to detect with bulk investigations on the tumor biopsy. Single-cell investigation will then allow even greater personalization of therapy to the genetic and transcriptional profile of the tumor, saving valuable time and dangerous side effects. The era of personalized medicine will take a huge step forward through the disclosure of each individual piece of the complex puzzle that is cancer pathology, to implement a "tailored" therapeutic approach based also on engineered CAR-T cells.

Hematologic Neoplasms Associated with Down Syndrome: Cellular and Molecular Heterogeneity of the Diseases.

The molecular basis of Down syndrome (DS) predisposition to leukemia is not fully understood but involves various factors such as chromosomal abnormalities, oncogenic mutations, epigenetic alterations, and changes in selection dynamics. Myeloid leukemia associated with DS (ML-DS) is preceded by a preleukemic phase called transient abnormal myelopoiesis driven by GATA1 gene mutations and progresses to ML-DS via additional mutations in cohesin genes, CTCF, RAS, or JAK/STAT pathway genes. DS-related ALL (ALL-DS) differs from non-DS ALL in terms of cytogenetic subgroups and genetic driver events, and the aberrant expression of CRLF2, JAK2 mutations, and RAS pathway-activating mutations are frequent in ALL-DS. Recent advancements in single-cell multi-omics technologies have provided unprecedented insights into the cellular and molecular heterogeneity of DS-associated hematologic neoplasms. Single-cell RNA sequencing and digital spatial profiling enable the identification of rare cell subpopulations, characterization of clonal evolution dynamics, and exploration of the tumor microenvironment's role. These approaches may help identify new druggable targets and tailor therapeutic interventions based on distinct molecular profiles, ultimately improving patient outcomes with the potential to guide personalized medicine approaches and the development of targeted therapies.

Association of Platelet Thromboxane Inhibition by Low-Dose Aspirin With Platelet Count and Cytoreductive Therapy in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once-daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once- vs. twice- or three-times daily low-dose aspirin in inhibiting platelet thromboxane (TX) A2 production, as reflected by serum (s) TXB2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once-daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P < 0.01, n = 84), but not in patients on cytoreduction. Patients in the lowest sTXB2 quartile were older, more often on cytoreductive drugs, had lower platelet count and Janus-Associated Kinase2 (JAK2)-V617F allele frequency as compared with patients in the upper sTXB2 quartiles. After 2 weeks of a twice- or 3-times daily aspirin regimen, the association between the platelet count and sTXB2 became similar in cytoreduced and non-cytoreduced patients. In conclusion, the platelet count appears the strongest determinant of TXA2 inhibition by once-daily low-dose aspirin in ET, with different patterns depending of cytoreductive treatment. More frequent aspirin dosing restores adequate platelet inhibition and reduces interindividual variability, independently of cytoreduction.

Essential Thrombocythemia in Children and Adolescents.

This paper reviews the features of pediatric essential thrombocythemia (ET). ET is a rare disease in children, challenging pediatric and adult hematologists alike. The current WHO classification acknowledges classical Philadelphia-negative MPNs and defines diagnostic criteria, mainly encompassing adult cases. The presence of one of three driver mutations (JAK2V617F, CALR, and MPL mutations) represent the proof of clonality typical of ET. Pediatric ET cases are thus usually confronted by adult approaches. These can fit only some patients, because only 25-40% of cases present one of the driver mutations. The diagnosis of hereditary, familial thrombocytosis and the exclusion of reactive/secondary thrombocytosis must be part of the diagnostic process in children and can clarify most of the negative cases. Still, many children present a clinical, histological picture of ET, with a molecular triple wild-type status. Moreover, prognosis seems more benign, at least within the first few decades of follow-up. Thrombotic events are rare, and only minor hemorrhages are ordinarily observed. As per the management, the need to control symptoms must be balanced with the collateral effects of lifelong drug therapy. We conclude that these differences concert a compelling case for a very careful therapeutic approach and advocate for the importance of further cooperative studies.

Extremely Old Patients Hospitalized in Internal Medicine: What about Their Anemia?

In western countries, about half of the hospitalized patients are anemic. Generally, these patients are old, often with multiple diseases, and anemia worsens the prognosis, finally increasing the risk of death. We describe a monocentric observational study that evaluates 249 consecutive adult patients (160 women and 89 men) with anemia admitted in the internal medicine department over five months. They represent 71.5% of all patients admitted in the study period. Demographic, historical, and clinical data, laboratory tests, duration of hospitalization, readmission at 30 days, and death were recorded. Patients were stratified by age (75-84=old, >85 years=oldest-old), anemia severity, and etiology of anemia. Anemia was found in 67.5% of old and in 77.2% of oldest-old patients. In 37% of old and 32% of oldest-old patients, anemia was mild, in 43% old and 59% of oldest-old moderate and in 20% old and 9% of oldest-old severe in agreement with WHO criteria. Moderate anemia was significantly more common in the oldest-old (p=0.01). The causes of anemia were iron deficiency in 10.6% of patients, other deficiencies in 2.8%, chronic diseases in 38.2%, hematologic neoplasms in 6.1%, multifactorial in 24.1%, and undetermined in 19.9%. The oldest-old have a higher frequency of multifactorial anemia (p=0.04), while hematologic neoplasms were more common in old patients (p=0.03). Most patients with undetermined anemia had mild/moderate forms. An anti-anemic treatment, mainly blood transfusion, was adopted in 100% of oldest-old patients and in 60% of old (p= 0.04). Anemia (and/or its treatment) was reported in the discharge letter in 19% of old and in 28.2% of oldestold patients. From a general point of view, physicians seem to disregard anemia in the context of more important pathologic conditions. In oldest-old patients, multifactorial anemia seems to be considered only "one more cause of disability." When borderline anemia occurs, even if it can represent a relevant adverse condition in frailty, it is poorly considered.

A randomized double-blind trial of 3 aspirin regimens to optimize antiplatelet therapy in essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the twice-daily and thrice-daily regimens showed substantially reduced interindividual variability and lower median (interquartile range) values for sTXB2 (ng/mL) compared with the once-daily arm: 4 (2.1-6.7; n = 79), 2.5 (1.4-5.65, n = 79), and 19.3 (9.7-40; n = 85), respectively. Urinary PGIM was comparable in the 3 arms. Urinary TXM was reduced by 35% in both experimental arms. Patients in the thrice-daily arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75 to 100 once daily for cardiovascular prophylaxis appears to be largely inadequate in reducing platelet activation in the vast majority of patients with ET. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement with further reductions (EudraCT 2016-002885-30).

Splanchnic vein thromboses associated with myeloproliferative neoplasms: An international, retrospective study on 518 cases.

Myeloproliferative Neoplasms (MPN) course can be complicated by thrombosis involving unusual sites as the splanchnic veins (SVT). Their management is challenging, given their composite vascular risk. We performed a retrospective, cohort study in the framework of the International Working Group for MPN Research and Treatment (IWG-MRT), and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). A total of 518 MPN-SVT cases were collected and compared with 1628 unselected, control MPN population, matched for disease subtype. Those with MPN-SVT were younger (median 44 years) and enriched in females compared to controls; PV (37.1%) and ET (34.4%) were the most frequent diagnoses. JAK2V617F mutation was highly prevalent (90.2%), and 38.6% of cases had an additional hypercoagulable disorder. SVT recurrence rate was 1.6 per 100 patient-years. Vitamin K-antagonists (VKA) halved the incidence of recurrence (OR 0.48), unlike cytoreduction (OR 0.96), and were not associated with overall or gastrointestinal bleeding in multivariable analysis. Esophageal varices were the only independent predictor for major bleeding (OR 17.4). Among MPN-SVT, risk of subsequent vascular events was skewed towards venous thromboses compared to controls. However, MPN-SVT clinical course was overall benign: SVT were enriched in PMF with lower IPSS, resulting in significantly longer survival than controls; survival was not affected in PV and slightly reduced in ET. MPN-U with SVT (n = 55) showed a particularly indolent phenotype, with no signs of disease evolution. In the to-date largest, contemporary cohort of MPN-SVT, VKA were confirmed effective in preventing recurrence, unlike cytoreduction, and safe; the major risk factor for bleeding was esophageal varices that therefore represent a major therapeutic target.

Systemic Mastocytosis and Essential Thrombocythemia: Case Report and Literature Overview.

Mastocytosis is a rare disease in which heightened amounts of mast cells accumulate in the skin, bone marrow, and other visceral organs. Upon activation, mast cells release a wide variety of preformed or newly synthesized mediators which can induce allergic symptoms and inflammatory reactions. Mastocytosis is diagnosed by biopsy and can be divided into cutaneous and systemic mastocytosis (SM). The first one affects the skin and is relatively benign, whilst SM, which involves bone marrow and other organs, may be aggressive and associate with both myelodisplastic and myeloproliferative diseases. Here we present a case of SM associated with essential thrombocythemia and complicated by severe osteoporosis, successfully treated with hydroxyurea, low-dose aspirin and zolendronic acid.

Contemporary management of essential thrombocythemia in children.

INTRODUCTION: Essential thrombocythemia (ET) is a disease which is extremely rare in children. Only recently, data on pediatric ET have become available. Areas covered: In children with sustained platelet count over 450 x 109/L, secondary thrombocytosis must be ruled out. ET workup comprehends research of JAK2V617F, CALR and MPL mutations and bone marrow biopsy (BM). In asymptomatic children wait and watch is the best option. Aspirin controls headache and other microvascular disturbances. Patients with venous thrombosis need anticoagulation. Cytoreductive drugs in children with ET should be prescribed as a last choice. Hydroxyurea and IFN-a are first-line therapy at any age including children; Anagrelide is not licensed as first-line therapy for ET in Europe. New JAK2-inhibitors are not clearly useful in ET and hence not approved for ET. Expert opinion: The most challenging problem is to understand if a child with prolonged not secondary thrombocytosis really has ET. Diagnostic workup requires molecular and histological studies. The rare children with clonal ET have features like those of adults. Patients with ET have long expected survival and the treatment in children must be long-term efficacious and well tolerated.

Thrombotic events in severe FXII deficiency in comparison with unaffected family members during a long observation period.

To investigate the occurrence of thrombotic events (myocardial infarction, deep vein thrombosis or ischemic stroke) in a group of 39 cases of severe FXII deficiency during a mean 22.5 years follow-up. All patients seen in Padua during the years 1968-2006 will the object of this investigation. FXII was less than or 1% of normal in all cases. Factor FXII activity in unaffected family members was 98% (range 90-140%). No patient or control had a thrombotic event in the past and none were on anticoagulant therapy. FV Leiden was present in one patient and in two controls whereas the G to A20210 prothrombin polymorphism was absent in both groups. There was one death among the patients (breast cancer) and one among the control (car accident). There were two thrombotic events (myocardial infarction and deep vein thrombosis) in the patient group and three (myocardial infarction and two deep vein thrombosis) in the control group. Heterozygous FV Leiden was present in the patient who had venous thrombosis, One of the two control subjects who developed venous thrombosis had heterozygous FV Leiden and was on oral contraception. The second control subject who developed venous thrombosis was on oral contraception and had varicose veins. No ischemic stroke was observed in the patients or controls. Periods of immobilization were 42 days and 38 days, respectively for FXII deficient patients and for the controls. Patients with severe FXII deficiency may present thrombotic events but these are similar to these presented by unaffected family members. As a consequence it may be stated that severe FXII deficiency does not appear to effect thrombotic events.

The Aspirin Regimens in Essential Thrombocythemia (ARES) phase II randomized trial design: Implementation of the serum thromboxane B2 assay as an evaluation tool of different aspirin dosing regimens in the clinical setting.

Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of patients with essential thrombocytemia (ET). This practice is based on data extrapolated from other high-risk patients and an aspirin trial in polycythemia vera, with the assumption of similar aspirin pharmacodynamics in the two settings. However, the pharmacodynamics of low-dose aspirin is impaired in ET, reflecting accelerated renewal of platelet cyclooxygenase (COX)-1. ARES is a parallel-arm, placebo-controlled, randomized, dose-finding, phase II trial enrolling 300 ET patients to address two main questions. First, whether twice or three times 100 mg aspirin daily dosing is superior to the standard od regimen in inhibiting platelet thromboxane (TX)A2 production, without inhibiting vascular prostacyclin biosynthesis. Second, whether long-term persistence of superior biochemical efficacy can be safely maintained with multiple vs. single dosing aspirin regimen. Considering that the primary study end point is serum TXB2, a surrogate biomarker of clinical efficacy, a preliminary exercise of reproducibility and validation of this biomarker across all the 11 participating centers was implemented. The results of this preliminary phase demonstrate the importance of controlling reproducibility of biomarkers in multicenter trials and the feasibility of using serum TXB2 as a reliable end point for dose-finding studies of novel aspirin regimens.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Benefit-risk profile of cytoreductive drugs along with antiplatelet and antithrombotic therapy after transient ischemic attack or ischemic stroke in myeloproliferative neoplasms.

We analyzed 597 patients with myeloproliferative neoplasms (MPN) who presented transient ischemic attacks (TIA, n = 270) or ischemic stroke (IS, n = 327). Treatment included aspirin, oral anticoagulants, and cytoreductive drugs. The composite incidence of recurrent TIA and IS, acute myocardial infarction (AMI), and cardiovascular (CV) death was 4.21 and 19.2%, respectively at one and five years after the index event, an estimate unexpectedly lower than reported in the general population. Patients tended to replicate the first clinical manifestation (hazard ratio, HR: 2.41 and 4.41 for recurrent TIA and IS, respectively); additional factors for recurrent TIA were previous TIA (HR: 3.40) and microvascular disturbances (HR: 2.30); for recurrent IS arterial hypertension (HR: 4.24) and IS occurrence after MPN diagnosis (HR: 4.47). CV mortality was predicted by age over 60 years (HR: 3.98), an index IS (HR: 3.61), and the occurrence of index events after MPN diagnosis (HR: 2.62). Cytoreductive therapy was a strong protective factor (HR: 0.24). The rate of major bleeding was similar to the general population (0.90 per 100 patient-years). In conclusion, the long-term clinical outcome after TIA and IS in MPN appears even more favorable than in the general population, suggesting an advantageous benefit-risk profile of antithrombotic and cytoreductive treatment.

Thromboses and hemorrhages are common in MPN patients with high JAK2V617F allele burden.

The most common causes of morbidity and mortality in myeloproliferative neoplasms (MPN) are thrombotic and hemorrhagic complications. The JAK2V617F mutation, commonly found in MPN, correlates with several clinical and laboratory characteristics even if the relevance of JAK2V617F allele burden in the natural history of these diseases is unclear. In this study we searched, a relation between thrombotic and hemorrhagic complications and JAK2V617F allele burden level in MPN patients. We evaluated 253 consecutive MPN [121 essential thrombocythemia (ET), 124 polycythemia vera (PV), and 8 primary myelofibrosis (PMF)] patients in whom the JAK2V617F allele burden was available, all studied and followed (median 8.8 years) in our department. Patients were stratified accordingly to their JAK2V617F allele burden, into four quartiles (1st <25%, 2nd 26-50%, 3rd 51-75%, and 4th >75%). Significantly higher incidence of thromboses (p = 0.001) and hemorrhages (p < 0.001) during follow-up has been observed in higher quartiles when compared to lower ones. Thrombosis- and hemorrhage-free survivals were poorer in patients belonging to the highest quartile. Our data suggest that MPN patients with JAK2V617F allele burden higher than 75% have to be considered as high risk patients, being prone to develop thrombo-hemorrhagic complications during the disease course.