Genetic backgrounds and clinical characteristics of congenital neutropenias in Israel.

BACKGROUND: Congenital neutropenias are characterized by severe infections and a high risk of myeloid transformation; the causative genes vary across ethnicities. The Israeli population is characterized by an ethnically diverse population with a high rate of consanguinity. OBJECTIVE: To evaluate the clinical and genetic spectrum of congenital neutropenias in Israel. METHODS: We included individuals with congenital neutropenias listed in the Israeli Inherited Bone Marrow Failure Registry. Sanger sequencing was performed for ELANE or G6PC3, and patients with wild-type ELANE/G6PC3 were referred for next-generation sequencing. RESULTS: Sixty-five patients with neutropenia were included. Of 51 patients with severe congenital neutropenia, 34 were genetically diagnosed, most commonly with variants in ELANE (15 patients). Nine patients had biallelic variants in G6PC3, all of consanguineous Muslim Arab origin. Other genes involved were SRP54, JAGN1, TAZ, and SLC37A4. Seven patients had cyclic neutropenia, all with pathogenic variants in ELANE, and seven had Shwachman-Diamond syndrome caused by biallelic SBDS variants. Eight patients (12%) developed myeloid transformation, including six patients with an unknown underlying genetic cause. Nineteen (29%) patients underwent hematopoietic stem cell transplantation, mostly due to insufficient response to treatment with granulocyte-colony stimulating factor or due to myeloid transformation. CONCLUSIONS: The genetic spectrum of congenital neutropenias in Israel is characterized by a high prevalence of G6PC3 variants and an absence of HAX1 mutations. Similar to other registries, for 26% of the patients, a molecular diagnosis was not achieved. However, myeloid transformation was common in this group, emphasizing the need for close follow-up.

Alloimmunization and autoimmunization in adult transfusion-dependent thalassemia patients: a report from a comprehensive center in Israel.

Patients with beta thalassemia major (TM) are transfusion-dependent (TD) since early childhood and for life. Development of alloantibodies and autoantibodies against red blood cell (RBC) antigens is increasingly recognized as a significant transfusion hazard, especially among heavily transfused patients. The aim of this study is to assess RBC alloimmunization and autoimmunization rates in TD TM patients treated in our Comprehensive Center of Adult Thalassemia, Hemoglobinopathies and Rare Anemias. TD TM patients, regularly transfused every 2-3 weeks, were included in the study. Clinical and RBC transfusion records, including RBC antibodies, since diagnosis in early childhood, were retrieved from patients' files and from the blood bank database. Forty TD TM patients, > 18 years of age, were included in the study. Alloimmunization was demonstrated in 17 (42.5%) patients. Thirty-four alloantibodies were detected, with the most frequent being RH related (12 of 34, 35.3%) followed by those of the Kell system (8 of 34, 23.5%). Age at first transfusion was positively related to the probability of developing alloantibodies (p = 0.02). Splenectomy was found to be correlated with developing alloantibodies (p = 0.016). Logistic regression analysis of the lifelong probability of developing alloantibodies on the age at first transfusion and splenectomy demonstrates a strong positive relationship (p = 0.002). A substantially high rate of alloimmunization was found among adult TD TM patients. Early initiation of RBC transfusions, avoidance of splenectomy and extended Rh and K antigen matching, can reduce the incidence of alloimmunization in TD TM patients.

Splenectomy in childhood for non-malignant haematologic disorders - long-term follow-up shows minimal adverse effects.

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.

Targeted next generation sequencing for the diagnosis of patients with rare congenital anemias.

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.

High incidence of silent cerebral infarcts in adult patients with beta thalassemia major.

OBJECTIVES: Survival of beta thalassemia major (TM) patients has improved significantly over the past few decades. Consequently, less commonly reported complications are now being recognized. An incidence as high as 60% of silent cerebral infarcts (SCI) has been demonstrated by brain Magnetic Resonance Imaging (MRI) studies in beta thalassemia intermedia (TI). The aim of this study was to determine whether regularly transfused TM adult patients experience less SCI, as compared to the incidence described in TI. METHODS: In this observational study, 28 transfusion dependent TM patients, >18years of age underwent brain MRI studies. RESULTS: Focal bright foci in the cerebral white matter were demonstrated in 17 (60.7%) patients; most of them had multiple lesions. Elevated serum ferritin (SF), primarily 5years Area Under the Curve, was found to have a significant association with the presence of SCI (p<0.031). Similar results were found when 4 patients with intact spleen and 2 patients with splenules were excluded (p=0.027). There was no significant association between number of SCI and clinical or other laboratory parameter evaluated. CONCLUSIONS: The present study demonstrates a high rate of SCI in regularly transfused TM adult patients. Effective continuous iron chelation, preventive low dose aspirin and routine periodical brain MRI are recommended.

GFR in Patients with ß-Thalassemia Major.

BACKGROUND AND OBJECTIVES: Patients with ß-thalassemia major (TM) may have tubular dysfunction and glomerular dysfunction, primarily hyperfiltration, based on eGFR. Assessment of GFR based on serum creatinine concentration may overestimate GFR in these patients. This study sought to determine GFR by using inulin clearance and compare it with measured creatinine clearance (Ccr) and eGFR. DESIGN, SETTING, PARTICIPANTS & MEASUREMENTS: Patients followed up in an Israeli thalassemia clinic who had been regularly transfused for years and treated with deferasirox were included in the study. They were studied by inulin clearance, Ccr, the CKD Epidemiology Collaboration and the Modification of Diet in Renal Disease equations for eGFR, and the Cockcroft-Gault estimation for Ccr. Expected creatinine excretion rate and tubular creatinine secretion rate were calculated. RESULTS: Nine white patients were studied. Results, given as medians, were as follows: serum creatinine was 0.59 mg/dl (below normal limits); GFR was low (76.6 ml/min per 1.73 m(2)) and reached the level of CKD; Ccr was 134.9 ml/min per 1.73 m(2), higher than the GFR because of a tubular creatinine secretion rate of 30.3 ml/min per 1.73 m(2) (this accounted for 40% of the Ccr); and eGFR calculated by the CKD Epidemiology Collaboration and Modification of Diet in Renal Disease equations and Cockcroft-Gault-estimated Ccr were 133, 141, and 168 ml/min per 1.73 m(2), respectively. These latter values were significantly higher than the GFR, reaching the hyperfiltration range, and indicated that the estimation techniques were clinically unacceptable as a method for measuring kidney function compared with the GFR according to Bland and Altman analyses. CONCLUSIONS: Contrary to previous reports, patients in this study with TM had normal or reduced GFR. The estimating methods showed erroneous overestimation of GFR and were clinically unacceptable for GFR measurements in patients with TM by Bland and Altman analysis. Therefore, more accurate methods should be used for early detection of reduced GFR and prevention of its further decline toward CKD in these patients.

Acquired proximal renal tubular dysfunction in ß-thalassemia patients treated with deferasirox.

Deferasirox is a recently approved oral iron chelator for treatment of patients with transfusion-related iron overload. Although renal function disturbances were recognized, proximal renal tubulopathy was not addressed in published safety reports for deferasirox. Although subclinical proximal tubulopathy was described in ß-thalassemia homozygotes, overt Fanconi kidney is not an established disease complication. We describe 4 cases out of 50 children and adults with transfusion-dependent ß-thalassemia, treated with deferasirox for iron overload, who developed clinically significant Fanconi syndrome. Three had concomitant infectious events; the fourth case was entirely spontaneous. In addition, all 4 patients were moderately to well chelated. Cessation of deferasirox resulted in prompt recovery. We propose the necessity for diligent monitoring for proximal tubule nephropathy, possibly related to infectious events, during treatment with deferasirox.

Gelatinous transformation of bone marrow in chronic myeloid leukemia during treatment with imatinib mesylate: a disease or a drug effect?

Gelatinous marrow transformation (GMT) is an unusual pathological manifestation of progressive malignant diseases and severe malnutrition states. GMT has been associated with various accelerated hematological malignancies, but has never been described in patients with chronic myelogenous leukemia (CML) treated with imatinib mesylate (IM), a novel tyrosine kinase inhibitor. Herein we report 2 patients with stable chronic phase CML who developed GMT during the course of treatment with IM. A comprehensive review of the relevant published data, several possible mechanisms and therapeutic alternatives are suggested.

Expression of VEGF-C, VEGF-D and their receptor VEGFR-3 in diffuse large B-cell lymphomas.

Lymphangiogenesis-the new growth of lymphatic vessels is an important route for the metastatic spread of human cancer. The receptor tyrosine kinase VEGFR-3 is expressed predominantly on lymphatic endothelium, and activation by its ligands VEGF-C and VEGF-D induces lymhpangiogenesis. VEGF-C, VEGF-D and VEGFR-3 have been found to play an important role in the lymphangiogenesis of several cancers. The present study investigated the expression of these factors by immunohistochemical staining of diagnosis specimens from 38 patients with diffuse large B-cell lymphoma (DLBCL). VEGF-C, VEGF-D and VEGFR-3 were expressed in both lymphoma cells and endothelial cells of blood and lymphatic tissue in all but one patient (who was negative for VEGF-D in lymphoma). There was a significant correlation in the intensity of staining between VEGF-C and -D in lymphoma and blood vessels (P < 0.001), and between the intensity of staining of VEGF-D and the patient International Prognostic Index score (P = 0.049) and borderline significance with overall survival (P = 0.051). Mean microvessel count was 58 (range 23-120), and it increased in association with high-intensity VEGF-C staining in lymphoma cells. Our findings indicate the importance of lymphangiogenic factors in the pathogenesis of DLBCL and suggest a potential therapeutic role for antilymphangiogenesis agents.

Antiphospholipid antibodies may be a new prognostic parameter in aggressive non-Hodgkin's lymphoma.

OBJECTIVES: Patients with malignancies have an increased prevalence of antiphospholipid antibodies (APA). The aim of this study was to determine the prevalence of IgG, IgM, and IgA anticardiolipin antibodies (aCL) and anti-beta-2 glycoprotein I antibodies (anti-beta2-GPI) in patients with non-Hodgkin's lymphoma (NHL), and to investigate their clinical and prognostic significance. METHODS: The study group included 86 patients with NHL. Enzyme-linked immunosorbent assay kits were used to measure the concentrations of aCL and anti-beta2-GPI, and coagulation tests, to measure lupus anticoagulant (LAC) activity. Blood was collected at diagnosis in all patients and at follow-up in 15. Median follow-up time was 1.9 yr. RESULTS: Elevated APA levels were found in 35 patients (41%) at diagnosis: one patient aCL IgG, five patients aCL IgM, five aCL IgA, one anti-beta2-GPI IgG, 14 anti-beta2-GPI IgM, and 19 anti-beta2-GPI IgA; LAC activity was found in three of 67 patients (4.5%). There was no significant correlation between elevated APA levels and patient's age or sex, disease stage or grade, bone marrow involvement, B symptoms, serum lactate dehydrogenase levels, serum beta2 microglobulin levels, International Prognostic Index (IPI) score, performance status, type of treatment, or response to treatment. There was a correlation between elevated APA and absence of extranodal disease (P = 0.045). A strong negative correlation was found between elevated APA at diagnosis and survival time. Two-year survival was 90 +/- 5% for patients without APA at diagnosis compared with 63 +/- 11% for patients with an elevated APA levels (P = 0.0025). APA added to the predictive value of IPI for event-free and overall survival. CONCLUSIONS: APA are elevated in 41% of NHL patients at diagnosis and are correlated with shortened survival. Their level may serve as an independent prognostic variable in aggressive NHL.

Lack of HER-2/neu expression in Hodgkin and non-Hodgkin lymphoma.

OBJECTIVE: Overexpression of HER-2/neu oncoprotein, a tyrosine kinase receptor, occurs in a variety of human cancers and has been shown to play a critical role in their development. This overexpression is usually associated with poor clinical outcome. The significance of HER-2/neu in lymphomas is unknown. The aim of this study was to evaluate the expression of HER-2/neu in the malignant lymphomas: non-Hodgkin and Hodgkin lymphomas. METHODS: We studied formalin-fixed, paraffin-embedded tissue from 50 patients with lymphoma. Forty-two specimens were from patients with various types of non-Hodgkin lymphoma, and 8 were from patients with Hodgkin lymphoma. HER-2/neu expression was examined by an immunohistochemical technique using the HercepTest. RESULTS: None of the specimens demonstrated overexpression or even any expression of HER-2/neu. Reactive plasma cells showed cytoplasmic staining, which was not found in malignant plasma cells from patients with multiple myeloma. CONCLUSIONS: Non-Hodgkin and Hodgkin lymphomas do not express the HER-2/neu oncoprotein. This finding suggests that HER-2/neu does not play a role in these diseases.