ALBERT

Description: Sorafenib is an antitumor drug for treatment of advanced hepatocellular carcinoma. It acts as a multikinase inhibitor suppressing cell proliferation and angiogenesis. Human microRNA-125a-5p (miR-125a) is endowed with similar activities and is frequently downregulated in hepatocellular carcinoma. Looking for a potential microRNA-based mechanism of action of the drug, we found that sorafenib increases cellular expression of miR-125a in cultured HuH-7 and HepG2 hepatocellular carcinoma cells. Upregulation of the microRNA inhibited cell proliferation by suppression of sirtuin-7, a NAD(+)-dependent deacetylase, and p21/p27-dependent cell cycle arrest in G1. Later, recruitment of miR-125a in the antiproliferative activity of sorafenib was inquired by modulating its expression in combination with the drug treatment. This analysis showed that intracellular delivery of miR-125a had no additive effect on the antiproliferative activity of sorafenib, whereas a miR-125a inhibitor could counteract it. Finally, evaluation of other oncogenic targets of miR-125a revealed its ability to interfere with the expression of matrix metalloproteinase-11, Zbtb7a proto-oncogene, and c-Raf, possibly contributing to the antiproliferative activity of the drug. This article is protected by copyright. All rights reserved

Description: Venous thromboembolism (VTE) in children is a rare occurrence, although in recent decades we have seen an increase due to several factors, such as the rise in survival of subjects with chronic conditions, the use of catheters, and the increased sensitivity of diagnostic tools. Besides inherited thrombophilia, acquired conditions such as cardiovascular diseases, infections, chronic disorders, obesity and malignancy are also common risk factors for paediatric VTE. The treatment of paediatric VTE consists of the use of heparins and/or vitamin K antagonists to prevent dissemination, embolization, and secondary VTE. Randomized clinical trials of direct oral anticoagulants in paediatric VTE are ongoing, with the aim to improve the compliance and the care of patients. We reviewed the physiological and pathological mechanisms underlying paediatric thrombosis and updated the current diagnosis and treatment options.

Description: Objectives—Failure to thrive (FTT) in infants is characterized by growth failure. Although, cow’s milk allergy (CMA) may have an impact on growth and leads to FTT, data are still limited. We focused on FTT as a possible clinical marker for an early diagnosis of CMA. The aim of the present study was to evaluate the implications of cow’s milk hypersensitivity in infants with FTT and the growth catch-up after a cow’s milk-free diet (CMFD). Methods—A cross-sectional study of all consecutive infants evaluated at the Pediatric Nutrition and Allergy Unit of the University Hospital of Bari (Italy) from January 2016 to April 2018 with a medical-driven diagnosis of FTT. Eligible infants were investigated for possible IgE mediated or non-IgE mediated CMA. Results—43 infants were included, mean age 5.7 months. 33/43 (77%) FTT presented a CMA related disease: 3/43 (7%) were diagnosed as presenting an IgE mediated CMA, 30 (93%) had a non IgE-mediated CMA, confirmed by the elimination diet for diagnostic purposes, that led to a significant improvement of symptoms and recrudescence after milk reintroduction. A total of 29 out of 30 patients (one patient was lost at follow-up) moved up to their original growth percentile after dietary changes. Growth z-scores were computed based on WHO anthropometric data. In 10 out of 43 patients (23%) were diagnosed with gastro-esophageal reflux disease (GERD). Conclusions—when evaluating an infant with FTT, physicians should include in their evaluation an extensive search for IgE mediated and non IgE mediated CMA. When in vivo and in vitro analysis are not conclusive, a 4- to 8-weeks trial of CMFD and a consecutive re-introduction of milk proteins may be helpful in less common diagnoses.

Description: Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

Description: Introduction. Despite the increasing of number of patients with Sickle Cell Disease (SCD) in Italy, due to multi-ethnic migratory phenomena, a large percentage of Caucasian sickle population is already present in Italy mainly with b-thal/HbS genotype. Red cell transfusion is one effective treatment for both acute and chronic complications of SCD, while hydroxycarbamide (HC) is used to reduce the frequency of painful vaso-occlusive crises (VOCs) and decrease the need for blood transfusion. Through the National Comprehensive Reference Centers for SCD, the Italian Society of Thalassemia and Hemoglobinopathies (SITE), in collaboration with the Society Italian Transfusion Medicine and Immunohematology (SIMTI) and the Italian Association of Hematology and Pediatric Oncology (AIEOP) conducted a national survey to collect information on different therapeutic approaches used for SCD patients. Aim. To assess therapeutic approaches used a large Italian cohort of patients with SCD, accounting for age, genotype and ethnicity. Patients and Methods. Observational Longitudinal Systemic Multicentre Study (https://clinicaltrials.gov/ct2/show/NCT03397017). Data were collected from 2015 to 2018 through a standard web-based application (www.SITE-italia.org) encrypted by the Central Server. All the SCD patients, treated or not treated, were included in order to identify the overall number and all gave written informed consent. The study was approved by Ethics Committee of Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico of Milan, Italy. Results. Thirty-four centers were involved from 14 Italian regions and 1,579 patients were enrolled (802 male and 777 female; median age 23 years - IQR, 25th-75th 10-41 yrs). Genotype, age and ethnicity distribution are shown in Table 1A. As expected, the median age of non-Caucasian patients, mainly HbSS, is significantly lower than Caucasian ones (p

Description: While Essential Thrombocytemia (ET) is a typical chronic myeloproliferative disease (MPD) in adults of median/advanced age, it is rarely seen in children. Literature is scarce on this issue and mostly focused on case reports. Current diagnostic criteria in adults are those of WHO2008; new biological markers show a growing clinical relevance. In contrast, we suggested from individual Institutions’ experience that these criteria are questionable for children. Therapeutic options in pediatric age are far from established, as demonstrated by the heterogeneous treatments reported in the literature. We thus analyzed a large series of thrombocytemic children, diagnosed and followed in the Units of Italian Association of Pediatric Hematology and Oncology (AIEOP), to evaluate their clinical and biological features in comparison with adult counterparts. 90 children with sporadic ET were reported. Children with familiar history of MPD were excluded. Diagnosis was performed initially according to the PVSG criteria and since 2001 to the WHO criteria. All patients underwent bone marrow aspirate; biopsy was performed only in one third of children. Main clinical data, age at diagnosis, length of follow up (f-up), possible treatment and complications were recorded for all cases. Since 2002, most patients were analyzed for significant biological features, either retrospectively or at diagnosis. V617FJAK2mutation was analyzed by allele specific PCR on DNA extracted from granulocytes. Erythroid colonies (EEC) were obtained with and without addition of erythropoietin. Clonality was studied on female patients according to HUMARA method. The mutations of thrombopoietin (TPO) and its receptor (c-MPL) genes were performed by sequencing methods. Prevalence of females (62 girls and 28 boys) was seen as in adult ET. Median age at diagnosis was 6.75 years (range 1 mo-17 yrs); median f-up was 5 years (range 2–11 yrs). Platelets counts at diagnosis were 611–4020 × 109/l (median 1260); haemoglobin was normal, leukocytosis was frequent. Splenomegaly was present in 21% of cases. In 57 patients thrombocytemia was a casual finding. Headache was the most frequent complaint(25%). 10 patients presented minor symptoms. Major problems were seen at diagnosis in 2 infants (deep venous thrombosis with Budd-Chiari syndrome) and during f-up in 2 more children (deep venous thrombosis and splenic infarction with venous thrombosis); no major hemorrhagic events were recorded. One girl had autoimmune peripheral neuropathy. Treatment was heterogeneous and often modified during f-up: 28 children did not receive any treatment; 29 cases used low dose ASA alone or in association. Cytoreduction was obtained by hydroxyurea (29 cases), interferon alfa (17cases) and anagrelide (26 cases), aiming at platelets level 400–600 × 109/l. Treatment was stopped for toxicity in 1 case of IFN and 3 cases of Anagrelide. The V617F JAK2 mutation, present in about 50% of adult ET, was found in 16 out of 65 children (24%). Of 28 informative female patients, 17 (60%) had clonal myelopoiesis. Spontaneous EEC grew in 15 out of 32 cases (47%). We could not find any mutation of TPO or cMPL genes in sporadic patients. In conclusion, this represents the largest reported series of pediatric ET. Differences from adult cases are shown: most cases have little or no clinical symptoms; the incidence of thrombotic events is low. The primitive proliferative nature of these cases is not always confirmed: V617FJAK2 mutation is less frequent than in adults and its correlation with the clinical severity is not firm. This heterogeneity requires the definition of specific criteria for diagnosis and treatment of ET in children. Main features of pediatric ET Features Symptoms Treatment patients 90 None 52 (55%) None 28 M/F 28/62 Budd Chiari 2 ASA 29 Age at Dx 6.75 y (1m–17y) Deep Venous Thrombosis 2 Anagrelide 26 Follow-up 5 y (2–11) Haedache 22 (25%) a-IFN 17 Plts × 109/L 1260 (611–4020) Paresthesia 3 OHU 29 WBC × 109/L 11.75 Nosebleed 2 JAK2 V617F 16/65 (24%) Abdominal pain 4 EEC pos 17/28 (60%) Splenomegaly 19 (21%) Monoclonality 15/32 (47%) Peripheral neuropathy 1 MPL/TPO mutation none

Description: Abstract 4660 One of the serious complication in hemophilia therapy is the development of high titre inhibitors to FVIII and less often to others coagulation factors. It makes treatment of bleeds very challenging. We report a case of hemarthrosis in hemophilia A pediatric patient with inhibitors, treated with sequential infuson of rFVIIa (rFVIIa, NOVOSEVEN; Novo Nordisk A/S, Bagsvaerd, Denmark) and plasma activated prothrombin complex concentrate (pd- aPCC, FEIBA; Baxter AG Vienna Austria). rFVIIa and plasma activated prothrombin complex concentrate are, indeed, used as haemostatic bypassing agents to prevent eaemorrages, with the goal of limiting sequelae as arthropathy, or to control quickly heamostasis as intensive on–demand treatment. A 3 years old male patient affected by haemophilia A with inhibitors came to our observation for a traumatic hemarthrosis of the left knee. Clinic examination showed swelling and pain. His inhibitor titre was 29 Bethesda Units. First we infused rFVIIa for seven consecutive days at the dose of 90 ug/kg every 3 hours. This therapy didn't determinate any clinical improvement. Then we infused plasma activated prothrombin complex concentrate for the next consecutive seven days at the dose of 60 UI/kg every 12 hours. At the end of treatment we noticed pain disappearance and reducing swelling. Medical literature recently describes similar paediatric cases treated with sequential infusion of rFVIIa and plasma activated prothrombin complex concentrate. Our positive experience could stimulate to use haemostatic bypassing agents because apparently safe. We encourage to use this therapeutic scheme because it seem to reduce healing times of acute events. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4662 Replacement therapy is a very hard challenge in haemophilia B with inhibitor. We describe the case of a child with severe haemophilia B and with a family history positive for development of inhibitor to factor IX (FIX) and for occurrence of allergic reaction after replacement therapy. Genetic analysis demonstrated an almost complete deletion of FIX gene. The child received replacement therapy first in his life when he was 5 years old because of the occurrence of a large haematoma of the left thigh. He was treated with recombinant FIX concentrate at the dosage of 30 IU/kg daily for three days and no inhibitor to FIX was evidenced after this therapy. Two months later the patient was treated with a single dose of recombinant FIX concentrate at the same dosage for the occurrence of a post-traumatic joint bleeding of the right knee. An inhibitor towards FIX (1.7 B.U.) was detected two weeks after this treatment and confirmed in a subsequent analysis performed after ten days (1.4 B.U.). One month later the patient was hospitalized for a post traumatic hemarthrosis of the right shoulder. In this occasion it was planned to treat the patient with recombinant FIX under careful monitoring in intensive care unit considering of the inhibitor. After the slow endovenous infusion of 200 IU of recombinant FIX concentrate we stopped immediately the administration because the child presented cough, mild respiratory failure, tachycardia, tongue and lips oedema, lips cyanosis, diffuse vasodilatation, psychomotor agitation. He received also hydrocortisone, antihistaminic by intramuscular injection and oxygen by facial mask. The presence of inhibitor towards FIX and anaphylactic reaction occurrence preclude forever any replacement therapy with FIX both recombinant and plasmatic (PCC and/or aPCC). After this episode the patient needed another hospitalization for a tonsil bleeding with severe anaemization. We treat him with recombinant activated FVII (rFVIIa) first at the dosage of 270 mcg/kg in bolus, and after at the dosage of 90 mcg/kg every three hours for one day with complete bleeding remission. We encourage the careful monitoring of inhibitor towards FIX in haemophilic B patients especially with large FIX gene deletion. It could prevent severe anaphylactic reaction during replacement therapy. Considering the previous anaphylactic reaction, in this child rFVIIa represents the only therapeutic option for bleeding management. Disclosures: No relevant conflicts of interest to declare.

Description: Background The development of an immunogenic response to FVIII and the appearance of neutralizing antibodies - "inhibitors" - against FVIII is the most important adverse event in hemophilia treatment. Immune Tolerance Induction (ITI) via the long-term, intravenous administrations of high-dose FVIII is the only proven strategy to eradicate inhibitors. The success rate is about 60-70% and no formal demonstration of its mechanism of action has been provided yet. Indeed, the mechanisms underlying successful ITI have been unclear, and not much is known about the major factors determinant of success vs. failure. Our aim is to understand the process of activation and immune regulation in response to FVIII in patients undergoing ITI, and comparing the immunological events in patients who successfully eradicate anti-FVIII antibody to those in patients failing to achieve FVIII tolerance. Methods This is a multicenter, observational, prospective cohort study enrolling severe HA patients who developed high-titer inhibitors, candidate to ITI. A blood sample has been collected before starting and during the course of ITI, until ITI ends (5 blood samples during the first year, 3 during the second year if ITI treatment is not already ended, plus a final sample 30 days after ITI conclusion). Total PBMCs have been used to establish cell cultures where cells are re-stimulated with the same FVIII used during the ITI or medium alone. To identify candidate genes, key proteins and cell subsets associated to differential outcomes to ITI, immune cells are used for gene expression profiling (Human Inflammation & Immunity Transcriptome Targeted RNA Panel, Qiagen) together with extensive phenotypic characterization of immune cells via the CyTOF (Cytometry by Time Of Flight mass spectrometry) technology. To identify potential biomarkers predictors of ITI outcomes metabolomics analysis and multiplex cytokine arrays are performed in plasma collected at each time point during ITI and in supernatants from cells cultures. Plasma samples, are collected at each time point and stored at -80°C for cytokine determination and metabolomic analysis. Total anti-FVIII antibody (and their isotypes) and inhibitor titer are also determined in plasma samples by Nijmegen methodology in the central laboratory as described previously (1). Results Currently, 18 subjects have been enrolled among 8 Hemophilia Centers. Among these 5 subjects, two patients reached the study endpoint of ITI success by study criteria (negative inhibitor titer, a normal FVIII recovery, a normal FVIII half-life and the absence of anamnesis upon further FVIII exposure) while three were assessed as ITI failure. The analysis of anti-FVIII antibody isotypes revealed the presence of IgG4 as the most relevant component of the total anti-FVIII antibodies. The inhibitor clearance was also accompanied by the disappearance of IgG4 anti-FVIII antibodies in both tolerized patients, while those who failed to eradicate the inhibitor showed a sustained IgG4 anti-FVIII response (Fig. 1). The evaluation of the cytokines in the supernatants from in vitro cultured cells showed a consistent increase in production of pro-inflammatory cytokines in response to FVIII (IL-1β, IL-6, IL-12, IL-17A, IL-15) at all time points only in patients who continued to produce high-titer inhibitors. Conclusions Here we report an interim analysis of a prospective study of the immunological mechanisms of immune tolerance induction. The preliminary results obtained so far suggest that anti-FVIII IgG4 are a major component of the total anti-FVIII antibodies and their persistence is associated with unfavourable ITI outcome. We expect that the ongoing immune gene expression profiling, immune-phenotypic characterization and determination of soluble marker of activation/regulation of the immune system in supernatants and plasma in this cohort of patients will provide substantial knowledge to increase our current understanding of the complex immunological pathways involved in the development of tolerance to FVIII. Ultimately, this could lead to the discovery of biomarkers of ITI success/failure and to the generation of focused and strategic intervention to modulate the immune system during this treatment. References 1. Matino D, Gargaro M, et al. "IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII". J Clin Invest. 2015 Oct 1;125(10):3766-81. doi: 10.1172/JCI81859. Figure 1. Figure 1. Disclosures Matino: Sobi: Speakers Bureau. Peyvandi:Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Roche: Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Sobi: Speakers Bureau; Sobi: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Shire: Speakers Bureau; Octapharma US: Honoraria; Grifols: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy. Santagostino:Pfizer: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Iorio:NovoNordisk: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Novo Nordisk; Shire: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Shire; CSL: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with CSL; Grifols: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Grifols; Octapharma: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Octapharma; Bayer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Bayer; Pfizer: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Pfizer; Roche: Other: Alfonso Iorio's Institution has received project based funding via research or service agreements with Roche.