[THE BENEFIT OF MOLECULAR ANALYSIS USING NEXT GENERATION SEQUENCING METHODS IN PEDIATRIC "HARD TO TREAT" TUMORS - EXPERIENCE IN A TERTIARY MEDICAL CENTER].

INTRODUCTION: While survival rates among children with cancer are high, a significant proportion of the solid tumors are considered as hard to treat (HTT). Next generation sequencing (NGS) offers the ability to detect molecular changes in tumors. Its implementation may allow usage of targeted therapy for tumors that fail to respond to acceptable oncological treatment. Furthermore, these therapies are characterized by milder side effects than chemotherapy. NGS may also aid in establishing pathological diagnoses and occasionally, identifying cancer-predisposition syndromes. However, the benefit of NGS in the pediatric population is not clear. AIMS: Evaluating the benefit of NGS in children with 'HTT' Tumors. METHODS: A retrospective study of the usage of NGS in pediatric 'HTT' in the Department of Pediatric Hemato-Oncology at the Hadassah Medical Center. Patients' demographic and clinical characteristics, molecular changes in tumor, their influence on medical decisions and disease course - were all documented. RESULTS: Forty-seven NGS tests from 'HTT' tumors were completed between January 2018 to August 2020. The results of these tests dictated medical decisions in 18 cases (38.3%) while it proved utility in 10 cases (21.3%). Clinical response to targeted therapy, clarification of diagnosis and identification of germline changes were documented in 3 (6.4%), 4 (8.5%) and 3 (6.4%) cases, respectively. CONCLUSIONS: The usage of NGS may benefit children with 'HTT' and tumors with difficult diagnoses and in some cases may be life-saving. DISCUSSION: Cost-benefit considerations presumably prevent the assimilation of NGS tests in the standard care of pediatric oncology. It is possible that the current results will strengthen the more accurate usage of theses genomic techniques in children with 'HTT'.

Bacteremia in Children with Solid Tumors: Etiology, Antimicrobial Susceptibility, Factors Associated with Multidrug Resistance, and Mortality.

This retrospective study aims to describe the etiology and resistance patterns of pathogens causing bacteremia in children with solid tumors in a tertiary pediatric hematology-oncology center in Jerusalem, Israel (2011-2019). Factors associated with multidrug-resistant (MDR) bacteremia and mortality were analyzed. A total of 228 pathogens were isolated in 126 patients; 61.0% were gram-negative rods (GNR) and 38.2% were gram-positive cocci (GPC). The most common pathogens were Klebsiella pneumoniae (19.3%), Escherichia coli (17.5%), and coagulase-negative staphylococci (16.2%). The proportion of MDR-GNR was 18.2%, while the proportion of MDR-GPC was 55.2%. In logistic regression analysis, breakthrough bacteremia on a penicillin-group antibiotic (odds ratio [OR] 5.69, [95% confidence interval 1.42-22.76], p-value = 0.014) was associated and underlying diagnosis of neuroblastoma was inversely associated (OR 0.17, [0.04-0.81], p-value = 0.026) with MDR-GNR bacteremia; while the previous hospitalizations' duration (OR 1.032/day, [1.01-1.06], p-value = 0.007) and oncologic treatment intensity (OR 2.19, [1.08-4.45, p-value = 0.03) were associated with MDR-GPC bacteremia. Shock, prolonged profound neutropenia, and pediatric intensive care unit (PICU) admission were associated with 7-day mortality; and relapsed disease, oncologic treatment intensity, prolonged profound neutropenia, and PICU admission-with 30-day mortality in the univariate analyses. Empirical antibiotic choice should be based on factors associated with MDR infections in this specific population.

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.

Thrombophilia screening and thromboprophylaxis may benefit specific ethnic subgroups with paediatric acute lymphoblastic leukaemia.

This study investigated the prevalence of inherited thrombophilia, risk of venous thromboembolism (VTE) and benefit of low molecular weight heparin prophylaxis in 476 Israeli children with acute lymphoblastic leukaemia (ALL) treated between 2004 and 2016. Thrombophilia was found in 15·5%. Arab children had a higher prevalence of F5 R506Q (factor V Leiden) than Jewish children (19·4% vs. 2·9%, P < 0·01). Patients with thrombophilia had higher VTE rates VTE (26·5% vs. 5·6%, P < 0·001). None of the thrombophilic children given prophylaxis had severe VTE. Routine evaluation for inherited thrombophilia followed by thromboprophylaxis when findings are positive may benefit at-risk patients with ALL.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds.

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.

Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease.

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20-30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease.

Prophylactic therapy with enoxaparin in children with acute lymphoblastic leukemia and inherited thrombophilia during L-asparaginase treatment.

INTRODUCTION: Thrombotic events (TE) are well documented in patients with acute lymphoblastic leukemia (ALL). They occur due to a combination of disease, host and treatment-related risk factors. Low molecular weight heparin (LMWH) has been found to be effective and safe in children with ALL during L-asparaginase treatment. At present, whether or not to give primary anticoagulant prophylaxis for TE during induction or reinduction courses to children with ALL is controversial. Our group investigated the use of LMWH as a prophylactic treatment for ALL children with a genetic prothrombotic predisposition. METHODS: Eighty consecutive children with ALL treated between the years 1999 and 2008 were studied. Genetic analysis of factor V Leiden (G1691A) and prothrombin (G20210A) gene mutations were done at diagnosis. LMWH was given once daily subcutaneously at a dose of 1mg/kg, starting with the first dose of L-asparaginase (day 12 of induction, day 8 of consolidation) until one week after the last dose (day 40 of induction, day 25 of consolidation), to patients with inherited thrombophilia stemming from either factor V Leiden or prothrombin gene mutation. RESULTS: Eighteen patients were found to have a genetic predisposition for TE, all of them received prophylactic LMWH. Six of the 80 (7.5%) patients developed thromboembolic events. Three of these six had a prothrombin (PT) gene mutation and received prophylactic LMWH. No TE event occurred in patients with factor V Leiden mutation receiving prophylactic LMWH. CONCLUSION: It is suggested that patients with ALL and PT gene mutation may have a higher risk of clotting complications in comparison to patients with factor V Leiden mutation. A randomized trial of LMWH should be performed to assess its safety and efficacy in preventing venous TE.

Difficult-to-control asthma and obstructive sleep apnea.

This study tested the hypothesis that asthma can promote obstructive sleep apnea (OSA) by looking at the prevalence of OSA among patients with difficult-to-control asthma receiving long-term oral corticosteroid (CS) therapy and examined some possible etiological factors. The study design was a prospective cohort study and was conducted in the pulmonary outpatient clinic of a tertiary care center in Haifa, Israel. Twenty-two consecutive patients with severe unstable asthma, 14 on continuous and 8 on bursts of oral CS, in addition to their standard therapy for a mean of 8.9 +/- 3.3 years, underwent a night polysomnography in a sleep laboratory regardless of sleep complaints. A standard questionnaire was completed upon attending the sleep laboratory. The OSA was defined as respiratory disturbance index (RDI) of > or = 5 and typical complaints. The correlation between RDI to asthma and morphometric parameters was tested. All but one patient had OSA [95.5% prevalence], with mean RDI of 17.7 +/- 2.5. The RDI values were significantly higher in the continuous CS therapy subgroup (21.4 +/- 3.4 vs. 11.1 +/- 1.6, p < 0.05]. The study group had above normal neck circumferences and body mass index. The former increased by 12.1% +/- 3.1% % to 29.8% +/- 1% during the oral CS therapy interval but had no significant effect on RDI as a covariant. This study showed an unexpectedly high prevalence of OSA among patients with unstable asthma receiving long-term chronic or frequent burst of oral CS therapy. It may be assumed that prolonged and especially continuous oral CS therapy in asthma increases airway collapsibility.