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Purpose: To evaluate the characteristics of cytopenias in pediatric kidney transplant recipients (KTRs), identify predictors and assess management and consequences. Methods: A retrospective, single-center, case-control study of pediatric KTRs between the years 2000-2019. Possible risk factor for cytopenias were compared in multivariate Cox regression, with the aim of finding predictors for post-transplant thrombocytopenia (PTT) and post-transplant neutropenia (PTN). PTNs were analyzed for the total study period, and for the period beyond 6-months post-transplant (late PTNs), to rule-out the confounding influence of induction and initial intensive therapy.Results: 89 children were included in the study. Prevalence of PTT was 22%, all cases were mild or moderate. Post-transplant infections and graft rejection were found to be significant risk factors for PTT (HR 6.06, 95% CI 1.6-22.9, and HR 5.82, 95% CI 1.27-26.6, respectively). Overall PTN prevalence was 60%; 30% were severe (ANC ≤ 500(. Pre-transplant dialysis and post-transplant infections were significant predictors for late PTN (HR 11.2, 95% CI 1.45-86.4, and HR 3.32, 95% CI 1.46-7.57, respectively). Graft rejection occurred in 10% of KTRs with cytopenia, all following neutropenia, within 3 months from cytopenia appearance. In all such cases, mycophenolate mofetil dosing had been held or reduced prior to the rejection. One case resulted in graft-loss. Conclusions: Post-transplant infections are substantial contributors for developing PTTs and PTNs. Pre-emptive transplantation appears to reduce risk for late PTN, the accompanying reduction in immunosuppressive therapy and the ensuing risk for graft rejection. An alternative response to PTN, possible with granulocyte colony stimulating factor (G-CSF), may diminish graft loss.

This is the first study examining real-life data of pediatric cancer patients treated with rivaroxaban. Children with thrombocytopenia and high bleeding risk were excluded from previous clinical trials. Data regarding the safety and efficacy of rivaroxaban in pediatric cancer-associated thrombosis are scarce. Our case series included 16 children aged 7.5-17 years. Thrombus resolution rate in our study was comparable to results of previous studies. However, higher rates of thrombotic and bleeding complications were seen in our study as compared to previous reports, especially among patients with relapsed or refractory disease.

We report the results of national retrospective study of 45 children with hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) in Israel between the years 2000–2018. Donors were either HLA-matched (n = 26), partially mismatched (n = 7), haploidentical (n = 8), or cord-blood (n = 4). Myeloablative conditioning (MAC) was used in 20 procedures, and reduced-intensity conditioning (RIC) in 25. Forty-two patients engrafted, two had primary graft failure (one successfully retransplanted), one died prior to engraftment, and two developed secondary graft failure. Of the eight patients who had mixed donor chimerism at day 30 (5–95%), five achieved stable mixed or full donor chimerism. The 5-year probabilities of overall survival and event-free survival (EFS) were 86% and 82%, respectively. Five-year EFS was lower for patients receiving RIC compared to MAC (72% vs. 100%, p = 0.018) and following alternative-donor transplant (68% vs. 92% for HLA-matched donors, p = 0.034), mostly due to increased transplant-related mortality (TRM). Thus, both HLA-matched and alternative donor transplant procedures may benefit form a myeloablative conditioning regimen.

Background In all, 15-30% of pediatric immune thrombocytopenia (ITP) patients will remain chronically thrombocytopenic at 1 year post diagnosis. All attempts to classify patients at diagnosis have proven unsuccessful. We hypothesized that a different pathophysiology is responsible for non-chronic versus chronic pediatric ITP. We aimed to examine differences in the apoptotic markers' presentation at diagnosis between non-chronic and chronic patients. Methods Blood samples were collected from 42 pediatric patients with newly diagnosed ITP prior to initiation of treatment. We incubated patients' sera with control platelets and compared the results among three research groups: healthy controls, chronic ITP, and non-chronic ITP patients. We measured apoptotic markers phosphatidylserine (PS) exposure and mitochondrial inner membrane potential (ΔΨm) by flow cytometry and the level of human apoptotic proteins by Human Apoptosis Array. Results We found increased platelet PS exposure and decreased ΔΨm in response to all ITP patients' sera compared to control subjects. Human Apoptotic Array revealed an increased expression of five apoptotic proteins: BIM, CD40, IGFBP2, P21, and SMAC, following sera incubation of non-chronic pediatric ITP patients, compared to chronic patients' sera, at diagnosis. Conclusions Our data contribute to knowledge on apoptosis markers that may aid in predicting the prognosis of children with ITP. Impact The key message of our article is that children with chronic ITP have a different apoptotic profile compared to non-chronic ITP. Addition to existing literature: This is the first study comparing apoptotic markers between children with chronic ITP to non-chronic ITP. Impact Our findings indicate that, in the future, apoptotic markers may help to classify ITP patients into non-chronic versus chronic ones, at diagnosis.

Background/Objective: Alpha-thalassemia is one of the most prevalent genetic diseases, with the –α3.7 deletion being the most common mutation. Molecular studies have suggested mechanisms to explain the mild phenotype of “silent carrier” heterozygotes. However, the correlation between the clinical laboratory picture and the –α3.7 heterozygous state remains unclear, thus we chose to investigate. Methods: We analyzed the medical files of 192 children evaluated for microcytosis at our tertiary center between 2007 and 2017 and diagnosed as heterozygotes for the –α3.7 deletion. Additional α-thalassemia mutations, iron deficiency anemia, and β-thalassemia were ruled out. Laboratory parameters were compared to age- and sex-matched reference values. Results: The –α3.7 carriers had significantly lower Hb and mean corpuscular volume (MCV) than the reference population, and significantly higher red blood cell counts across all age groups. The greatest reduction in Hb level appeared among male adolescents, while MCV was consistently 2 SDs lower than normal in most patients older than 2 years. Conclusion: Heterozygosity for the –α3.7 deletion was associated with clinically significant microcytosis and mild anemia in our pediatric population. In the absence of iron deficiency and β-thalassemia, this finding provides a diagnosis for mild microcytic anemia, making additional investigations of microcytosis unnecessary.

Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem cell transplantation prior to the development of leukemia. We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children. The study comprises children with persistent cytopenias, myelodysplastic syndrome, aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used Sanger sequencing of commonly mutated genes and a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias; the minority subsequently underwent whole exome sequencing. In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, nine (19.1%) had inherited thrombocytopenia predisposing to leukemia, and three each (6.4%) had predisposition to myelodysplastic syndrome or congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia. In summary, almost one third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome.

Fanconi anemia (FA), an inherited bone marrow failure (BMF) syndrome, caused by mutations in DNA repair genes, is characterized by congenital anomalies, aplastic anemia, high risk of malignancies and extreme sensitivity to alkylating agents. We aimed to study the clinical presentation, molecular diagnosis and genotype-phenotype correlation among patients with FA from the Israeli inherited BMF registry. Overall, 111 patients of Arab (57%) and Jewish (43%) descent were followed for a median of 15 years (range: 0.1-49); 63% were offspring of consanguineous parents. One-hundred patients (90%) had at least one congenital anomaly; over 80% of the patients developed bone marrow failure; 53% underwent hematopoietic stem-cell transplantation; 33% of the patients developed cancer; no significant association was found between hematopoietic stem-cell transplant and solid tumor development. Nearly 95% of the patients tested had confirmed mutations in the Fanconi genes FANCA (67%), FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%), including twenty novel mutations. Patients with FANCA mutations developed cancer at a significantly older age compared to patients with mutations in other Fanconi genes (mean 18.5 and 5.2 years, respectively, P=0.001); however, the overall survival did not depend on the causative gene. We hereby describe a large national cohort of patients with FA, the vast majority genetically diagnosed. Our results suggest an older age for cancer development in patients with FANCA mutations and no increased incidence of solid tumors following hematopoietic stem-cell transplant. Further studies are needed to guide individual treatment and follow-up programs.

Valganciclovir (2/d) therapy for 6 months in neonates with symptomatic congenital cytomegalovirus (cCMV) infection improves hearing and neurodevelopmental outcome. The only reported adverse event was neutropenia. Since 2009, our protocol for symptomatic cCMV infection was a 1-year treatment of 2/d for the first 3 months followed by 9 months of 1/d.A retrospective study. Infants with cCMV treated with valganciclovir for 1 year were recruited. Data of drug-related hematologic adverse events were collected.One hundred sixty infants were eligible; 46 (28.8%) had experienced at least 1 episode of neutropenia (58 episodes), the majority (39/46, 84.8%) during the first 3 months of treatment and 7 (15.2%) during the last 9 months of treatment. Grades 3 and 4 neutropenia occurred in 9 (5.6%) children, almost exclusively during the first 3 months of treatment. Anemia (hemoglobin9 g/dL) was recorded in 12 (7.5%) children during the first 3 months of 2/d treatment. Four children presented with hemoglobin levels7 g/dL and needed a blood transfusion. One child was diagnosed with transient pure red cell aplasia. No long-term adverse events were recorded.Although prolonged valganciclovir treatment for cCMV is safe, a close monitoring of the white blood cell count and hemoglobin levels is warranted. Much lower rates of grades 3 and 4 neutropenia were observed than previously reported, probably owing to our unique treatment protocol. Nevertheless, drug-induced anemia should be of primary concern. The optimal protocol assessing clinical outcome, concurrently with potential side effects, has not yet been determined.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome that may complicate hematologic malignancies (HMs). The appropriateness of current criteria for diagnosing HLH in the context of HMs is unknown because they were developed for children with familial HLH (HLH-2004) or derived from adult patient cohorts in which HMs were underrepresented (HScore). Moreover, many features of these criteria may directly reflect the underlying HM rather than an abnormal inflammatory state. To improve and potentially simplify HLH diagnosis in patients with HMs, we studied an international cohort of 225 adult patients with various HMs both with and without HLH and for whom HLH-2004 criteria were available. Classification and regression tree and receiver-operating curve analyses were used to identify the most useful diagnostic and prognostic parameters and to optimize laboratory cutoff values. Combined elevation of soluble CD25 (>3900 U/mL) and ferritin (>1000 ng/mL) best identified HLH-2004–defining features (sensitivity, 84%; specificity, 81%). Moreover, this combination, which we term the optimized HLH inflammatory (OHI) index, was highly predictive of mortality (hazard ratio, 4.3; 95% confidence interval, 3.0-6.2) across diverse HMs. Furthermore, the OHI index identified a large group of patients with high mortality risk who were not defined as having HLH according to HLH-2004/HScore. Finally, the OHI index shows diagnostic and prognostic value when used for routine surveillance of patients with newly diagnosed HMs as well as those with clinically suspected HLH. Thus, we conclude that the OHI index identifies patients with HM and an inflammatory state associated with a high mortality risk and warrants further prospective validation.

BACKGROUND Inflammatory manifestations (IM) are well described in adult patients with myelodysplastic syndrome (MDS), but the presentation is highly variable and no standardized treatment exists. This phenomenon is rarely reported in children. As more pediatric patients are hematopoietic stem cell transplantation (HSCT) candidates, the role of anti-inflammatory treatment in relation to HSCT should be defined. PROCEDURE Here, we report a series of five children from a tertiary center. We describe the clinical presentation, molecular findings, and treatment options. RESULTS All patients presented with advanced MDS with blast percentages ranging 10-30%, all had severe IM. One patient had MDS secondary to severe congenital neutropenia, the other four patients had presumably primary MDS. All four were found to harbor a PTPN11 gene driver mutation, which is found in 35% of cases of juvenile myelomonocytic leukemia (JMML). The mutation was present in the myeloid lineage but not in T lymphocytes. Three had symptoms of Behcet's-like disease with trisomy 8 in their bone marrow. All patients were treated with anti-inflammatory medications (mainly systemic steroids) in an attempt to bring them to allogeneic HSCT in a better clinical condition. All demonstrated clinical improvement as well as regression in their MDS status post anti-inflammatory treatment. All have recovered from both MDS and their inflammatory symptoms post HSCT. CONCLUSION Primary pediatric MDS with IM is driven in some cases by PTPN11 mutations, and might be on the clinical spectrum of JMML. Anti-inflammatory treatment may reverse MDS progression and improve the outcome of subsequent HSCT.

Background Osteonecrosis is a major cause of acute and long-lasting complications of acute lymphoblastic leukemia (ALL) therapy in children. Our study aimed to evaluate the prevalence, characteristics, risk factors, and outcome of osteonecrosis in children with ALL. Procedure The cohort included 559 children aged 1-20 years diagnosed with ALL between 2003 and 2018 at two tertiary medical centers in Israel and enrolled in two consecutive protocols: ALL-IC BFM 2002 and AIEOP-BFM ALL 2009. Symptomatic osteonecrosis was prospectively captured as an adverse event. Results Osteonecrosis occurred in 51 patients (9.1%). Ninety-four percent of the events were graded as moderate or severe (grades 3-4, Ponte di Legno Toxicity Working Group classification) and multiple bone involvement was common. Full resolution of osteonecrosis was documented in only 16% of the children (median follow-up 4.2 years). Stepwise logistic regression identified five risk factors for osteonecrosis, with a high predictive value (AUC = 0.88): older ageat ALL diagnosis, high-risk ALL group, T-cell immunophenotype, female gender, and a novel risk factor: bone pain at the time of leukemia diagnosis. In addition, osteonecrosis was less common among children of Arab ethnicity. Thrombophilia and an elevated age-adjusted body mass index were not confirmed as risk factors for osteonecrosis. Conclusion Due to the low rates of osteonecrosis resolution and its debilitating long-term impact, the identification of patients at high risk for osteonecrosis is important for their inclusion in further studies evaluating potential therapeutic adjustments.

Introduction Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. Aim We aimed to study the genetic cause of IPFD mimicking GT. Methods During 2017–2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. Results Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbβ3 and αvβ3, as well as normal integrin αIIbβ3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. Discussion Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. Conclusion Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.

Detection of somatic mutations may help verify the diagnosis of myelodysplastic syndrome (MDS) in patients with persistent cytopenias or with MDS-predisposition syndromes, prior to the development of overt leukemia. However, the spectrum and consequences of acquired changes in paediatric patients have not been fully evaluated, and especially not in the context of an underlying syndrome. We incorporated a targeted next-generation-sequencing panel of 54 genes for the detection of somatic mutations in paediatric and young adult patients with inherited or acquired cytopenias. Sixty-five patients were included in this study, of whom 17 (26%) had somatic mutations. We detected somatic mutations in 20% of individuals with inherited MDS-predisposition syndromes, including in patients with severe congenital neutropenia and Fanconi anaemia, and with germline mutations in SAMD9L. Thirty-eight per cent of children with acquired cytopenias and suspected MDS had somatic changes, most commonly in genes related to signal transduction and transcription. Molecularly abnormal clones often preceded cytogenetic changes. Thus, routine performance of somatic panels can establish the diagnosis of MDS and determine the optimal timing of haematopoietic stem cell transplantation, prior to the development of leukaemia. In addition, performing somatic panels in patients with inherited MDS-predisposition syndromes may reveal their unique spectrum of acquired mutations.

We report the results of national retrospective study of 45 children with hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) in Israel between the years 2000-2018. Donors were either HLA-matched (n = 26), partially mismatched (n = 7), haploidentical (n = 8), or cord-blood (n = 4). Myeloablative conditioning (MAC) was used in 20 procedures, and reduced-intensity conditioning (RIC) in 25. Forty-two patients engrafted, two had primary graft failure (one successfully retransplanted), one died prior to engraftment, and two developed secondary graft failure. Of the eight patients who had mixed donor chimerism at day 30 (5-95%), five achieved stable mixed or full donor chimerism. The 5-year probabilities of overall survival and event-free survival (EFS) were 86% and 82%, respectively. Five-year EFS was lower for patients receiving RIC compared to MAC (72% vs. 100%, p = 0.018) and following alternative-donor transplant (68% vs. 92% for HLA-matched donors, p = 0.034), mostly due to increased transplant-related mortality (TRM). Thus, both HLA-matched and alternative donor transplant procedures may benefit form a myeloablative conditioning regimen.

Splenectomy is considered therapeutic in various non-malignant haematologic diseases. Adverse events - specifically infections and thromboembolism - are not extensively documented in the paediatric population, maintaining the concern over risks-versus-benefits of the procedure. We studied a cohort of paediatric haematology patients undergoing splenectomy between 1977 and 2015 to determine short- and long-term complications. We summarised all the patients of the haematology clinic in our major Israeli tertiary centre undergoing splenectomy for therapeutic reasons, capturing infectious and thromboembolic events. The data of 103 patients, comprising 1657 follow-up years, were analysed. The cohort included 33 patients with transfusion-dependent thalassaemia, seven with non-transfusion-dependent thalassaemia, four with sickle-thalassaemia, 41 with hereditary spherocytosis, and 18 with immune thrombocytopenia. Standard presplenectomy vaccinations were noted in most. No typical cases of overwhelming postsplenectomy infection (OPSI) were identified, nor were typical OPSI bacteria isolated. Thalassaemics with central lines were most prone to infection and thrombosis. Beyond this subgroup, thrombotic events were anecdotal. This is the largest study to date to comprehensively analyse infectious and thrombotic complications of childhood splenectomy for the treatment of haematologic diseases. The use of splenectomy appears to be a relatively safe therapeutic option in paediatric patients with proper preoperative vaccination and follow-up care; use of central venous lines or catheters increase the risk in thalassaemic patients and should be avoided if possible.

Poor adherence of transfusion-dependent patients to chelation treatment is often the cause of persistent iron overload and ensuing morbidity. However, a tool to assess patient compliance with therapy is lacking in clinical practice. Labile plasma iron (LPI, the redox-active component of non-transferrin bound iron) has been studied as an indicator of systemic iron overload and of chelation efficacy, and may particularly reflect recent iron equilibrium. We considered the use of LPI as a potential indicator for recent chelation treatment in 18 transfusion-dependent pediatric patients. Samples were collected under chelation treatment or after a short interruption of the treatment, and LPI was measured by the FeROS assay (Aferrix, Tel Aviv, Israel). LPI was significantly higher after a short-term interruption of the chelation (median of 0.4 μM off-therapy [range:0-4] vs 0 μM on-therapy [range:0-2.8] (p .001)). Conversely, serum iron, serum ferritin and calculated transferrin saturation were not significantly higher in the "off-therapy" samples compared to "on-therapy". In addition, in multivariate logistic regression analysis LPI was the variable most significantly associated with recent chelation treatment (p = .001). We conclude that LPI could serve as a useful indicator of compliance to chelation therapy.

Background-Aim: Infection from SARS-CoV-2 has emerged as new pathological entity within the global medical community. One of the earliest questions was in relation to the ability of the immunocompromised patients to clear the infection. In COST EuNet-INNOCHRON we were interested in the impact of SARS-CoV-2 infection in patients with different types of chronic neutropenia (CNP). The aim of the current study is to understand the impact of SARS-CoV-2 infection and to identify any possible characteristic patterns of the clinical course in patients with CNP. Patients and Methods: The COST EuNet-INNOCHRON Action in collaboration with the European Haematology Association - Scientific Working Group (EHA-SWG) on Granulocytes and Constitutional Marrow Failure Syndromes has conducted an online survey on SARS-CoV-2 infection in patients with CNP. The EuNet-INNOCHRON participants from different countries got access to an on-line platform fulfilling the General Data Protection Regulation (GDPR) and could register adult and paediatric CNP patients who had been infected by SARS-CoV-2 from March 2020 to June 2021. Data on demographic characteristics, type of CNP, patients' background and SARS-CoV-2 infection history (symptoms, laboratory features, radiological appearance, therapeutic approach and outcome) were collected. Results: Twenty-six patients with diagnosis of CNP, 7 males and 19 females were registered. Patient age distribution as follows: 16 patients >18 years old (y.o.)5 patients 5-18 y.o, 4 patients < 5 y.o whereas age was not available for one of the patients. Nine of the patients were diagnosed with idiopathic CNP, 7 patients with congenital neutropenia (6 of them with severe congenital neutropenia), 3 with secondary CNP, 2 with suspected autoimmune neutropenia of infancy (although antineutrophil Ab were negative), one with autoimmune neutropenia, one with drug induced neutropenia and 3 with other types of CNP. Twelve patients were on treatment with G-CSF and 6 patients had a history of previous viral or bacterial infections. Clonal Cytopenia(s) of Undetermined Significance (CCUS) was excluded in the eight patients who were investigated. Twenty-four out of 26 patients had positive PCR and one was found incidentally with positive antibodies for SARS-CoV-2. One more patient was symptomatic with history of close contact with SARS-CoV-2 infected family members. The commonest observed symptoms were fever >38 oC (19 patients), cough (10 patients), rhinorrhoea (10 patients), sore throat (6 patients), musculoskeletal pains (7 patients), taste/smell loss (5 patients), headache (5 patients), dyspnoea (4 patients), chest pain (one patient) and none of them had gastrointestinal symptoms. No other associated respiratory viral or bacterial infections were reported. Four patients who had one or more underlying conditions (immune deficiency, heart/respiratory/kidney disease) were admitted in hospital and needed anti SARS-CoV-2 treatment. Two of them had non-invasive ventilation and one of them needed admission in intensive care unit (ICU); both recovered. Another patient with Fallot's tetralogy needed mechanical ventilation in ICU and sadly passed away. No other deaths were observed. Deterioration of the pre-existing neutropenia was seen in two patients, two patients developed thrombocytopenia, one patient developed worsening lymphopenia and one anaemia. Twelve patients had chest X-ray and consolidation was found in two of them. All three patients who had chest CT scans were found with ground-glass changes. During the observation period (up to two months), no re-infection from SARS-CoV-2 was found. The Stockholm, Sweden experience is similar to the above data. One hundred fifty-four patients with CNP were followed up, for 10 months (March 1 to December 31, 2020) for SARS-CoV-2. Seventeen of these (i.e. 11 %) were infected. None needed hospitalization and there were no fatalities. Conclusion: Although the relative susceptibility of neutropenic patients to contract SARS-CoV-2 needs to be assessed with further studies, the clinical course and severity of SARS-CoV-2 infection doesn't seem to be worse in CNP patients (regardless the type of neutropenia and the need for GCSF treatment) compared to the general population. Also, like what has been observed in non-neutropenic patients, underlying comorbidities is a significant risk factor for severe disease and adverse outcome. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Palmblad: Chiesi Ltd Sweden: Honoraria; Roche Sweden: Speakers Bureau; Chiesi Ltd Candada,: Honoraria.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyper-inflammatory syndrome which may occur in adults with hematologic malignancies (HM). The diagnosis of HLH in this context (HM-HLH) is hindered by a number of factors. First, the currently used HLH 2004 diagnostic criteria are derived from pediatric patients commonly with HLH-associated genetic lesions, a very different population than adults with cancer. Second, most parameters used for diagnosis of HLH are directly impacted by the underlying HM and may reflect the presence of the malignant clone itself rather than an inflammatory process. Finally, appropriate diagnostic cutoff values for laboratory abnormalities in HM-HLH have not been defined. In this study we determine the diagnostic value of the laboratory components of the HLH 2004 diagnostic criteria and establish optimal cutoffs for the diagnosis of HM-HLH in HM patients. Methods: This is a multicenter, retrospective study of adult patients with a hematologic malignancy in whom sCD25 was measured because of clinically suspected HM-HLH or as part of routine screening of patients with a newly diagnosed hematologic malignancy, between January 2012 and March 2020. We considered patients fulfilling the five of eight of the HLH 2004 diagnostic criteria to have HM-HLH. Patients fulfilling fewer than five criteria were assigned to the HM group. These cohorts were well balanced in terms of disease distribution. We established the optimal cutoffs for laboratory parameters used for the diagnosis of HM-HLH using receiver operating curves (ROC) in a discovery cohort and tested their performance in a validation cohort. In order to improve the results obtained using the individual ROC, we then created a combined ROC using parameters demonstrating the highest individual performance (highest area under the curve (AUC)), in order to develop a diagnostic index. Finally, we examined the performance of each parameter in each cohort by using a contingency table and Chi-square and Fisher's exact test to determine the positive predictive value (PPV), negative predictive value (NPV), sensitivity, specificity and likelihood ratio (LR) of disease for each parameter. Results: 212 adults with HM with or without HLH in whom testing for HLH was performed were included in the study. HMs were: B cell lymphoma (41%), T cell lymphoma (26%), Hodgkin lymphoma (9%), acute myeloid leukemia (8%), myelodysplastic syndrome (8%), myeloproliferative neoplasms (5%) and chronic lymphocytic leukemia (4%). 99 (47%) patients had HM-HLH. Despite considerable overlap in laboratory values between the patient groups, all parameters apart from fibrinogen were able to distinguish HM-HLH from HM alone, with ferritin and sCD25 having the greatest discriminatory power. ROC analysis revealed an optimal cutoff value of >5,600 U/mL for sCD25 (sensitivity/specificity 76%/78%, AUC=0.83) and >1,300 ng/ml for ferritin (sensitivity/specificity 76%/76%, AUC=0.83). Combining the two markers to create a novel inflammatory index (HM-INFL) yielded superior diagnostic ability (AUC =0.86). Using HLH 2004 cutoff levels the HM-INFL index had a sensitivity of 94% and NPV of 94% and when using the optimal cutoff levels, it had a specificity of 92% and PPV of 90% (Table 1). Conclusions: HM-INFL is an index comprising only ferritin and sCD25. Using the original HLH 2004 cutoffs the index is an effective screening tool. Using our newly defined cutoff levels obtained by ROC analysis it is highly specific and can be used as a confirmatory test for the diagnosis of HLH in HM patients. These findings also support the hypothesis that HLH in the context of HM is an inflammatory condition associated with immune dysregulation. Disclosures Miller: Foundation Medicines, Inc.: Consultancy. Daver:Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Jordan:Sobi: Consultancy.

Background Essential thrombocythemia (ET) is rare in children, and pediatric guidelines are lacking. Therefore, we aimed to evaluate ET diagnosis and treatment in a pediatric cohort. Procedure Data of patients with ET from three hospitals were reviewed. Molecular diagnosis included JAK2V617F, CALR, and MPL mutations. Patients were evaluated for acquired von Willebrand syndrome (AVWS). Follow-up included clinical symptoms, adverse events, and treatment. Results Twelve children (median age: 8 years, range 1-14.5) were included. Mean lag period between the first documentation of thrombocytosis until ET diagnosis was 36 months. Six patients were positive for JAK2V617F and two for CALR mutations. In six of nine patients, AVWS was diagnosed. At diagnosis, only 33% of patients started therapy with aspirin (n = 4) and hydroxyurea (n = 2). In three of eight untreated patients, therapy was added during follow-up. The cohort was followed for a median of 32.5 months (range: 4-108 months). Clinical follow-up disclosed vascular complications in 4 of 12 patients (deep vein thrombosis, n = 1; transient ischemic attack, n = 3). Two females experienced excessive bleeding; both were diagnosed with AVWS. Neither leukemia nor myelofibrosis evolved in our cohort. Conclusion Increased awareness to pediatric ET is warranted, as delayed diagnosis is common. Compared to adults, AVWS may be more prevalent among children with ET.

Alpha-thalassemia is one of the most prevalent genetic diseases, with the -α3.7 deletion being the most common mutation. Molecular studies have suggested mechanisms to explain the mild phenotype of "silent carrier" heterozygotes. However, the correlation between the clinical laboratory picture and the -α3.7 heterozygous state remains unclear, thus we chose to investigate.We analyzed the medical files of 192 children evaluated for microcytosis at our tertiary center between 2007 and 2017 and diagnosed as heterozygotes for the -α3.7 deletion. Additional α-thalassemia mutations, iron deficiency anemia, and β-thalassemia were ruled out. Laboratory parameters were compared to age- and sex-matched reference values.The -α3.7 carriers had significantly lower Hb and mean corpuscular volume (MCV) than the reference population, and significantly higher red blood cell counts across all age groups. The greatest reduction in Hb level appeared among male adolescents, while MCV was consistently 2 SDs lower than normal in most patients older than 2 years.Heterozygosity for the -α3.7 deletion was associated with clinically significant microcytosis and mild anemia in our pediatric population. In the absence of iron deficiency and β-thalassemia, this finding provides a diagnosis for mild microcytic anemia, making additional investigations of microcytosis unnecessary.

7563 Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome that may accompany hematologic malignancies (HM). The diagnosis of HLH in patients with HM (HM-HLH) is confounded by a number of factors: the most commonly used HLH-2004 diagnostic criteria are derived from studies in infants while the Hscore used in adults is not specific for HMs; moreover, most parameters in these scoring systems may reflect features of the underlying HM rather than HLH associated inflammation; and finally specific diagnostic cutoff values for laboratory abnormalities in HM-HLH have not been defined. We therefore conducted a study to optimize the HLH-2004 laboratory thresholds for the diagnosis of HM-HLH. Methods: A multi-center retrospective study in adult patients with HM in whom testing for HLH was performed. HM-HLH was defined as fulfillment of 5/8 HLH-2004 diagnostic criteria. We established the optimal diagnostic cutoff levels for HLH-2004 laboratory parameters using receiver operating curves (ROC) and combined the best performing parameters into a combined index, using binary logistic regression. We then created a clinical decision tree using a Classification and Regression Tree (CART) analysis with all available parameters, using cross validation. We also determined the prognostic value of our combined diagnostic tool. Results: 225 adults were analyzed (112 with HM-HLH per HLH-2004 and 113 with HM only). 35% of patients were evaluated for HLH routinely upon HM diagnosis. Soluble CD25 (sCD25) and ferritin best discriminated HM-HLH from HM, with an area under the curve (AUC) of 0.83 for each. ROC analysis demonstrated an optimal cutoff of > 4190 U/mL for sCD25 (sensitivity/specificity 91%/69%) and an optimal cutoff of > 2636 ng/ml for ferritin (sensitivity/specificity 64%/86%) for HM-HLH. We term the combination of elevated sCD25 and ferritin using optimized cutoff levels the ‘optimized HLH inflammatory’ (OHI) index. This OHI index was highly specific for the diagnosis of HM-HLH (specificity of 92%, sensitivity 79%). CART analysis demonstrated that OHI index positivity was sufficient to diagnose HM-HLH. In patients without a positive OHI index an Hscore > 168 and either splenomegaly or triglycerides > 279 ng/dL can still diagnose HM-HLH. By following this decision pathway, approximately 92% of patients were accurately classified based on HLH-2004. Furthermore, the OHI was better (odds ratio (OR) 7.9; 95% confidence interval (CI) 4.2-14.6) than Hscore >169 (OR 5.5; CI 3.9-9.6) and > 5/8 HLH-2004 (OR 5.3; CI 3-9.3) at predicting mortality at 1 year. Conclusions: The OHI index derived here is a simple tool that can accurately diagnose HLH and predict mortality in patients with hematologic malignancies. Some patients may not need full HLH workup before intervening with therapy that is HLH directed and not only malignancy directed.

e20072 Background: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyper-inflammatory syndrome with distinct clinical and laboratory features. In adults, HLH is often associated with underlying malignancy, most commonly hematologic malignancies (HM). HLH occurs in 1% of adults with HM and overall survival can be low as 10-20%. Abnormal serum levels of the inflammatory markers sCD25 and ferritin are diagnostic criteria for familial HLH. However, because these reactants are often elevated in malignancy, appropriate levels for diagnosis in HM-HLH are unknown. In this study, we establish optimal sCD25 and ferritin levels for the diagnosis of HM-HLH in adults. Methods: Patients from three centers in Israel and Japan with HM-HLH and HM in whom sCD25 testing was performed were studied. The diagnosis of HLH was according to the HLH 2004 diagnostic criteria. Initial (at HLH presentation) and the maximum ferritin levels were analyzed. Sensitivity, specificity, and optimal cutoffs were calculated by receiver operating characteristic (ROC). Results: 62 patients with HM's without HLH and 40 patients with HM-HLH were included. The distribution of ages and HM subtypes was similar between groups (mostly B cell, T/NK cell, and Hodgkin's lymphoma). The median sCD25 concentration in HM was 1776 U/ml versus 8077 U/ml in HM-HLH. The median initial/ maximum ferritin levels were 190/202 ng/ml for the HM group and 2267/4515 ng/ml for the HM-HLH group. Both sCD25 and ferritin were very sensitive but nonspecific. sCD25 > 2400 U/ml had a sensitivity/specificity of 95%/65%, while initial ferritin > 500 ng/ml had a sensitivity/specificity of 95%/75%. ROC analysis demonstrated optimal confirmatory cutoff values (maximizing specificity) of > 10,056 ng/ml for sCD25 (sensitivity/specificity 47%/95%). While initial ferritin demonstrated a cutoff of > 5231 ng/ml (sensitivity/specificity 22.5%/95%, AUC = 0.88) the maximum ferritin performed better with the cutoff of > 5748 ng/ml (sensitivity/specificity 45%/95%, AUC = 0.92). Conclusions: Our data suggest that the current HLH 2004 criteria of ferritin > 500 ng/ml and sCD25 > 2400 U/ml are effective screening criteria for the complication of HLH in HM patients. sCD25 > 10,000 U/ml and initial ferritin > 5,200 ng/ml are highly specific. Patients with suspected HM- HLH should have serial ferritin testing which increases specificity of this test. Future prospective studies are needed to confirm these findings.

BACKGROUND Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes involved in rare anemias and the similarities in the clinical presentation of the different syndromes. OBJECTIVE We aimed to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. METHODS Genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. RESULTS Genetic diagnosis was achieved in 13 out of 21 patients (62%). Six patients were diagnosed with pyruvate kinase deficiency, 4 with dehydrated hereditary stomatocytosis, 2 with sideroblastic anemia, and 1 with CDA type IV. Eight novel mutations were found. In 7 patients, the genetic diagnosis differed from the pretest presumed diagnosis. The mean lag time from presentation to diagnosis was over 13 years. CONCLUSIONS Targeted next-generation sequencing led to an accurate diagnosis in over 60% of patients with rare anemias. These patients do not need further diagnostic workup. Earlier incorporation of this method into the workup of patients with congenital anemia may improve patients' care and enable genetic counseling.

The purpose of this study was to assess the efficacy and side effect profile of the repeated use of anti-D for the treatment of pediatric immune thrombocytopenia (ITP) in a large pediatric hematology center. We performed a retrospective analysis of patient records for children (aged 4 months-18 years) treated for ITP at Schneider Children's Medical Center of Israel from 1995-2015. Demographic and clinical data, reported adverse events, and therapy response were extracted from written and electronic files for all patients having received anti-D. Therapy response was defined as time to platelet count >30 x 10(9)/L. Thirty-six patients received 170 treatments of anti-D at a dose of 75 μg/kg. The majority were previously treated with corticosteroids and/or intravenous immunoglobulin (IVIG). Minimal adverse events were recorded including fever (3.5%), vomiting (2.9%), and headaches (1.7%). Notably only 1/170 treatments required blood transfusion and no life-threatening events occurred. The average time to platelets >30 x 10(9)/L was 2.3 days, with a median of 1 day, range 1-12 days. Despite the reported severe adverse events in mainly elderly patients, the use of anti-D can be safe and effective in carefully chosen, low-risk pediatric patients with ITP.

Platelet function disorders (PFDs) are a common cause of mild bleeding tendency. However, they cannot be recognized by standard screening studies. The gold standard test for PFD is platelet aggregation, performed by light transmission aggregometry (LTA). A newer and less validated method is the closure time (CT), performed by the platelet function Analyzer 100 (PFA-100). Data regarding the validity of these tests in children are limited. The aim of this study was to evaluate the usefulness of LTA and PFA-100 for the diagnosis of pediatric patients with bleeding tendency. This retrospective study included patients one month-18 year old that had LTA tests performed at the coagulation laboratory of Rabin Medical Center between the years 2006-2015. Bleeding severity was assessed using a pediatric bleeding score. Patients were excluded from analysis if they had thrombocytopenia, thrombocytosis or coagulation factors deficiencies. One hundred and thirty-seven (137) patients were included in the analysis. The median age was 7.5 years (range one month-18 years). Most patients (93%) had a bleeding score of 2 or more. Abnormal LTA was found in 40% and prolonged CT in 23% of the patients. Abnormal LTA was significantly more common in patients with a bleeding score of 2 or more compared to patients with a lower bleeding scores (P = 0.04). No significant correlation was found between the bleeding severity and the number of agonists which induced abnormal responses (p = 0.52) or the CT (p = 0.35). Furthermore, no correlation was found between abnormal LTA and prolonged CT. To conclude, we were able to diagnose 40% of children who presented with bleeding tendency with platelet aggregation defects by LTA. Abnormal LTA was significantly more prevalent in patients with a bleeding score of 2 and above. In contrast, CT was not found to be sensitive as a screening tool for PFD. Therefore, our data extend the validity of the use of LTA for the evaluation of pediatric patients with bleeding tendency.

Background The use of central venous catheters (CVCs) has greatly improved the quality of care in children receiving chemotherapy, yet these catheters may cause serious infectious complications. The aim of this prospective registry study was to assess the host and CVC-related risk factors for blood stream infections (BSIs). Procedure Patients undergoing CVC insertion for chemotherapy were followed prospectively for CVC complications. At the time of enrollment demographic-, clinical- and CVC-related data were collected. Survival and Cox-regression analysis were performed. Results A total of 423 CVCs were inserted into 262 patients for a total of 76,540 catheter-days. The incidence of BSIs was 1.95 per 1,000 patient-days (95% CI 1.66–2.29). Myeloid leukemia and younger age were associated with higher risk for BSI. At least one BSI occurred in 187 CVCs with an incidence of 2.84 per 1,000 catheter-days (95% CI 2.47–3.24). Externalized CVCs, that is, tunneled externalized catheters and peripheral inserted central catheters, were associated with higher risk for BSI in the group of diseases with relatively lower rate of infection. However, in diseases with high rate of infection no such association was found. The type of BSI was associated with the diagnosis and the CVC type. CVC occlusion was associated with higher risk for recurrent BSI and for coagulase negative staph BSI. Conclusions Both patient and CVC-related factors are associated with higher risk of BSI in children receiving chemotherapy. The results of this study could be used in developing studies aiming to reduce the rate of BSIs in children with cancer. Pediatr Blood Cancer 2015;62:471–476. © 2014 Wiley Periodicals, Inc.

Background The relative frequency of mutated genes among patients with severe congenital neutropenia (SCN) may differ between various ethnic groups. To date, few population-based genetic studies have been reported. This study describes the genetic analysis of 32 Israeli patients with SCN. Procedures Clinical data were retrieved from the prospective Israeli Inherited Bone Marrow Failure Registry. Recruitment included living and deceased patients who were diagnosed between 1982 and 2012, for whom molecular diagnosis was performed. ELANE, HAX1 and G6PC3 genes were sequenced in all patients, and GFI-1 and WAS genes were sequenced if other genes were wildtype. Results Eleven patients (34%) had heterozygous mutations in ELANE (10 kindreds), eight (25%) had homozygous mutations in G6PC3 (5 kindreds) and 13 (41%) had no detected mutations. No patients had mutations in HAX1 or WAS. Four of the eight patients with G6PC3 mutations had congenital anomalies. The probability of survival for all patients was 50% at age of 18. Deaths were mainly due to sepsis (5 patients, 4/5 not responding to G-CSF, none with G6PC3 mutation). Two patients developed acute myelogenous leukemia (AML) and one myelodysplastic syndrome (MDS), none with G6PC3 mutation. Conclusions We found a unique pattern of SCN mutations in Israel with homozygous G6PC3 mutations in eight (25%) patients, the highest frequency described so far. HAX1 mutations, reported mainly in Sweden and Iran, were absent. Patients with G6PC3 mutations had congenital anomalies, appeared to have a better response to G-CSF, and so far have not developed AML or MDS. Pediatr Blood Cancer 2015;62:103–108. © 2014 Wiley Periodicals, Inc.

Objectives Otogenic sigmoid sinus thrombosis is a rare complication of acute otitis media. Treatment remains controversial particularly regarding extent of surgical intervention. The aim of the study was to review the 12-year experience of a major medical center with the treatment of sigmoid sinus thrombosis in children. Methods Retrospective case series identified by database review in a tertiary university-affiliated pediatric medical center. Twenty-four children aged 7–155 months were treated for sigmoid sinus thrombosis from 2000 through 2011. Results The transverse sinus was also involved in 10 patients, and the jugular vein, in 4. Acute otitis media with mastoiditis was the causative factor in all cases. Subperiosteal abscess was diagnosed in 21 patients, 11 with epidural involvement. Treatment in all cases consisted of broad-spectrum antibiotics and ventilation tube insertion. Twenty-one children (87.5%) underwent mastoidectomy with removal of bone covering the sigmoid sinus to drain pus and remove granulations from the epidural cavity, without aspiration or sinus drainage. Twenty-two patients received low-molecular-weight heparin for 3–6 months postoperatively. Children infected with Fusobacterium necrophorum had a longer and more severe course with coexisting osteomyelitis. There were no neurologic sequelae or hematologic complications. Follow-up imaging, performed in 15 children, revealed partial or full recanalization in 87%. Conclusions Relatively conservative surgical intervention appears to yield good results in children with sigmoid sinus thrombosis consequent to acute otitis media. Anticoagulants are safe if correctly administered and may prevent extension of the thrombus.