Early-life liver cirrhosis and variable clinical presentation in telomere disease.

AIM: Telomeres are DNA sequences of tandem TTAGGG repeats that protect chromosome ends from degradation and instability. Constitutional loss-of-function telomerase mutations result in rapid telomere shortening, premature senescence and cell death. Liver cirrhosis is rare and has only been reported in adults. We present five family members of Bedouin-Muslim origin, all of which carry the same mutation, and yet demonstrate an extremely variable phenotypical presentation, including liver cirrhosis during early childhood. METHODS: A multidisciplinary long-term follow-up of two healthy and three affected patients was analysed. The mutation (r.95G>C) was identified in all patients using Sanger sequencing. Telomere length samples were obtained and analysed. RESULTS: Clinical phenotypes were extremely variable, including age at first symptoms, organ involvement, disease severity and patient prognosis. The most prominent clinical phenotype is liver involvement, including end-stage liver disease early in life, which affects three members of the family. Affected patients had markedly shorter telomeres. CONCLUSION: We describe an unusual presentation of early liver failure in telomere disease patients. Little, if any, is known about the association between the genotype and phenotype among children with telomere disease and whether the mutation we have described (r.95G>C) is predisposed to early severe hepatic involvement.

Syndromes predisposing to leukemia are a major cause of inherited cytopenias in children.

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.

Pediatric myelodysplastic syndrome with inflammatory manifestations: Diagnosis, genetics, treatment, and outcome.

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.

Inhaled corticosteroids increase blood neutrophil count by decreasing the expression of neutrophil adhesion molecules Mac-1 and L-selectin.

OBJECTIVE: The objective was to investigate the effect of commonly used inhaled corticosteroids on white blood cell count (WBC) and to examine the mechanisms involved. METHODS: This randomized comparative study comprised 60 healthy adults. We measured the effects of budesonide (by face mask inhalation or aerosol inhaler), fluticasone (by inhaler), and saline inhalation (control) on WBC and the differential leukocyte count, especially the absolute neutrophil count (ANC). To elucidate the mechanisms involved, we measured the expression of the adhesion neutrophil ligands Mac-1 (CD11b) and L-selectin (CD62L), and granulocyte colony-stimulating factor serum levels. RESULTS: Six hours after a single-dose inhalation of budesonide, mean increases of 23.4% in WBC (95% confidence interval [CI], 11.3-35.4) and 30.1% in ANC (95% CI, 7.2-53.0) were noted. The percentage of neutrophils increased from 54.6% to 58.1% (P< .001). Inhaled fluticasone increased WBC and ANC by 12.6% (95% CI, 1.5-23.7) and 22.7% (95% CI, 6.2-39.2), respectively (P< .01 for both). The absolute lymphocyte and eosinophil counts did not change significantly from baseline. The expression of Mac-1 and L-selectin decreased by 51.0% (P< .01) and 30.9% (P= .02), respectively, following face mask inhalation of budesonide and by 39.8% (P= .01) and 17.4% (P= .17), respectively, following inhalation of fluticasone. No significant changes in granulocyte colony-stimulating factor levels were noted. CONCLUSIONS: Glucocorticoid inhalation increases WBC by increasing ANC. Reduced neutrophil adhesion to the endothelial surface, mediated by decreased adhesion molecule expression on neutrophils, is a plausible mechanism. Physicians should be aware of the effect of inhaled corticosteroids on WBC, as it may influence clinical decisions, especially in the emergency department.

miR expression profiling at diagnosis predicts relapse in pediatric precursor B-cell acute lymphoblastic leukemia.

Our aim was to identify miRNAs that can predict risk of relapse in pediatric patients with acute lymphoblastic leukemia (ALL). Following high-throughput miRNA expression analysis (48 samples), five miRs were selected for further confirmation performed by real time quantitative PCR on a cohort of precursor B-cell ALL patients (n = 138). The results were correlated with clinical parameters and outcome. Low expression of miR-151-5p, and miR-451, and high expression of miR-1290 or a combination of all three predicted inferior relapse free survival (P = 0.007, 0.042, 0.025, and <0.0001, respectively). Cox regression analysis identified aberrant expression of the three miRs as an independent prognostic marker with a 10.5-fold increased risk of relapse (P = 0.041) in PCR-MRD non-high risk patients. Furthermore, following exclusion of patients harboring IKZF1 deletion, the aberrant expression of all three miRs could identify patients with a 24.5-fold increased risk to relapse (P < 0.0001). The prognostic relevance of the three miRNAs was evaluated in a non-BFM treated precursor B-cell ALL cohort (n = 33). A significant correlation between an aberrant expression of at least one of the three miRs and poor outcome was maintained (P < 0.0001). Our results identify an expression profile of miR-151-5p, miR-451, and miR-1290 as a novel biomarker for outcome in pediatric precursor B-cell ALL patients, regardless of treatment protocol. The use of these markers may lead to improved risk stratification at diagnosis and allow early therapeutic interventions in an attempt to improve survival of high risk patients.

The association between let-7, RAS and HIF-1α in Ewing Sarcoma tumor growth.

Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family.We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment.Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.

Neuroimmunological function in parents of children suffering from cancer.

The main aim of this study is to evaluate the relationship between depression and immunological function in parents of children with cancer. Thirty-two parents participated in the study. The parents completed the following assessments: a list of major stressful events in a Hemato-Oncology ward, beck depression inventory II (BDI-II), posttraumatic diagnostic scale (PDS) and quality of life (QOL) questionnaire. A single blood sample was drawn from parents for evaluation of cortisol levels and lymphocyte cell subgroups. The parents were divided into two groups: Those who suffered from depression as defined by BDI-II cutoff score of 14 (depressed parents (DP), n = 7), and non-depressed parents (non-DP, n = 25). In parents of children with cancer the DP group had statistically significantly higher stressful event scores, dysfunction scores (from the PDS) and CD8 percentage compared to the non-DP group. QOL, CD4 percentage and CD4/CD8 ratio were significantly lower in the DP group. The BDI scores significantly positively correlated with events and dysfunctional scores, and significantly negatively correlated with QOL scores and CD4/CD8 ratio. High psychiatric morbidity was found in parents of children with cancer. The findings of altered immunity in DP provide further evidence that the physiological response to stress and depression may alter immune functions.

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

Peripheral blood stem-cell harvest using percutaneous arterial lines in children.

BACKGROUND: Autologous peripheral blood stem-cell collection (PBSCC) in children has become an integral part of contemporary treatment protocols, but the procedure is often complicated due to technical issues related to vascular access. Central line placement is often implemented to surmount this problem, but is associated with complications such as bleeding, thrombosis and pneumothorax. As an alternative we have introduced the use of radial arterial lines for PBSCC in children. PROCEDURE: Data from autologous stem cell collections performed from October 2002 to December 2011 using a radial arterial line were collected. RESULTS: A total of 372 PBSCC procedures were performed during the study period; an arterial line was used in 311 PBSCC's in 208 children. The average patient age and weight were 7.9 years (SD 5.4) and 28.3 kg (SD 20.4), respectively. The smallest patient was 9 months old and weighed 7 kg. The mean total volume processed was 8,593 cm(3) (SD 4,854), and the mean number of blood volumes processed was 4.3. Mean collection time for a single blood volume was 55 minutes (SD 15.5). The mean number of CD34+ cells collected per donation was 5.8 × 10(6) /kg. Ninety-seven patients (46%) required more than one collection to meet the requested CD34+ cell target. No serious adverse effects associated with vascular access occurred in this cohort. CONCLUSION: Percutaneous placement of radial artery catheters can be rapidly and safely performed in very small infants and in children with difficult venous access. This technique provides a reliable platform for efficient PBSCC.

Prospective comparison of two flow cytometry methodologies for monitoring minimal residual disease in a multicenter treatment protocol of childhood acute lymphoblastic leukemia.

BACKGROUND: Minimal residual disease (MRD) is a powerful prognostic indicator in childhood acute lymphoblastic leukemia (ALL). Multiparametric flow cytometry (FC) is a rapid and sensitive methodology for detection of MRD, applicable for most patients and is being incorporated in multicenter treatment protocols. The influence of different techniques and of individual interpretation of data on the interlaboratory variability in FC-MRD determinations has not been described. METHODS: We compared FC-MRD of identical bone marrow samples processed as either Ficoll separated mononuclear cells or lyse and wash nucleated cells (NC) in two central laboratories of a national multicenter childhood ALL study. A total of 290 samples at diagnosis and 494 follow-up samples (Day-15 n = 261; Day-33 n = 233) were analyzed. A group of 52 paired list mode data (LMD) of D-15 and D-33 samples was blindly reanalyzed by both laboratories. RESULTS: Pearson correlations for all samples of D-15 (n = 261) and D-33 (n = 233) were 0.875 and 0.82, respectively (P < 0.001), being lower for T-ALL 0.716 and 0.719, respectively. Quantitative concordance defined as less than 0.5 log difference in MRD measured by the two methodologies was 80.8% at D-15 but only in 57.9% at D-33. Reanalysis of LMD revealed that data interpretation explained half of the discordance. CONCLUSIONS: FC-MRD analysis of childhood ALL is a robust method during the earliest phases of induction therapy in a multicentric setting. Standardization of data analysis could improve about half of the discordance between different technical approaches.

Presence of D8/17 B-cell marker in patients with poststreptococcal reactive arthritis.

An elevated expression of the alloantigen D8/17 on B lymphocytes has been previously proposed as a susceptibility marker in rheumatic fever. The aim of the study was to investigate the presence of the D8/17 marker on B lymphocytes in poststreptococcal reactive arthritis (PSRA). The study sample included 19 patients (15 boys, 4 girls; mean age 11.7 +/- 5.4 years) who were diagnosed with PSRA (mean age at diagnosis, 9.4 +/- 5.6 years) and 18 healthy controls (10 boys and 8 girls) matched for ethnic background. B-cell D8/17 expression was tested by flow cytometry assay using monoclonal antibodies. Laboratory results showed a higher expression of D8/17 in the patient group (23.1 +/- 10.4%, range 11.6-51.9%) than in the control group (17.1 +/- 8.2%, range 7.1-35.7) in controls; this difference was statistically significant after log transformation of the data (P = 0.035). The high rate of expression of the D8/17 marker in patients with PSRA suggests that RF and PSRA share the same genetic susceptibility.

Prolonged course of familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis is usually diagnosed in the first 2 years of life and, if untreated, is rapidly fatal. We describe a 10-year-old boy with a 9-year history of prolonged fever and progressive hepatosplenomegaly who was diagnosed as having hemophagocytic lymphohistiocytosis 2, being homozygote to a previously described mutation in the PRF1 gene, and cured by the HLH-2004 protocol and allogenic bone marrow transplantation. This unique case emphasizes the heterogeneity of this disease and the diversity of its clinical presentations.

Treatment of human bone marrow with recombinant placenta immunoregulator ferritin results in myelopoiesis and T-cell suppression through modulation of the cytokine-chemokine networks.

OBJECTIVE: Placenta immunomodulator ferritin (PLIF) is a cloned human chimeric ferritin H chain with a novel non-ferritin C-terminal 48 amino acid sequence (C48). Recombinant PLIF-C48 exhibited cell-mediated immunosuppression. The aim of the current study was to investigate the regulatory effects of native placental ferritin (PLF), recombinant PLIF, and C48 on hematopoiesis of human bone marrow (BM). METHODS: BM mononuclear cells (BM-MNCs) and CD34(+) selected cells were treated in vitro with either PLF, PLIF, or C48 without and in combination with granulocyte (G)-monocyte (M) colony-stimulating factor (GM-CSF) and subjected to hematopoietic progenitor cell assay. Cytokines and chemokines secreted by the treated cells were evaluated in culture supernatant using antibody array assays to determine mechanism of action. RESULTS: In vitro treatment of BM-MNCs with PLF, PLIF, or C48 induced significant growth of myeloid colonies and when mixed with GM-CSF or Granulocyte-Colony Stimulating Factor (G-CSF) exhibited additive enhanced colony forming units-granulocyte monocyte growth. Yet, C48 treatment of selected CD34(+) cells did not yield colony formation and did not affect their response to GM-CSF. Treatment of BM-MNCs with C48 for 48 hours induced secretion of marked levels of GM-CSF, interleukin (IL)-6, IL-1, and IL-10. These cytokines were secreted primarily by C48-treated BM adherent cells and partly by nonadherent cells, whereas the CD34(+) selected cells secreted IL-6 only. CONCLUSION: C48-PLIF enhancement of myelopoiesis resulted from cross talk between BM accessory cells and progenitor cells. The differential PLIF-C48 effects (i.e., myeloid progenitor cell growth and T-cell suppression) are due to their effect on the cytokine-chemokine networks.