Activity In Pediatric Patients Research Articles

SERS spectroscopy as a tool for the study of thiopurine drug pharmacokinetics in a model of human B leukemia cells

Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT).Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs.The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed.SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS).A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques.Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments.A strong positive correlation (Spearman's rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm−1).In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients’ leukocytes.

Evaluation of bowel wall flow by color Doppler ultrasound in the assessment of inflammatory bowel disease activity in pediatric patients.

To assess inflammatory bowel disease (IBD) activity with Doppler ultrasound in pediatric patients, comparing the accuracy of the ultrasound findings with that of the concentrations of fecal calprotectin (FC). In a consecutive series, we evaluated 53 examinations of 44 pediatric patients seen between 2014 and 2020: 28 with Crohn's disease, 15 with ulcerative colitis, and one with IBD unclassified. The diagnosis of IBD was made in accordance with the Porto criteria. The alteration studied in the greatest detail was bowel wall flow, which was classified by the lead investigator and two pediatric radiologists, all of whom were blinded to the FC concentrations and the other ultrasound findings. Bowel wall flow was categorized as low if there were up to 2 Doppler ultrasound signals/cm2, moderate if there were 3-5 signals/cm2, and high if there were more than 5 signals/cm2. The agreement among the radiologists was substantial (kappa = 0.73). In cases in which ultrasound showed low bowel wall flow, the median FC concentration was 92 µg/g (interquartile range, 33-661 µg/g), whereas it was 2,286 µg/g (interquartile range, 1,728-5,612 µg/g) in those in which ultrasound showed high bowel wall flow. In the sample as a whole, the sensitivity and specificity of ultrasound was 89.7% and 92.0%, respectively, for the detection of inflammatory activity; 95.5% and 90.9%, respectively, for the detection of Crohn's disease; and 81.3% and 100.0%, respectively, for the detection of ulcerative colitis. Ultrasound of the bowel wall showed a strong correlation with FC concentrations in the assessment of inflammatory activity in pediatric patients with IBD.

Evaluation of bowel wall flow by color Doppler ultrasound in the assessment of inflammatory bowel disease activity in pediatric patients

Resumo Objetivo: Avaliar a atividade da doença inflamatória intestinal (DII) por ultrassonografia (US) com Doppler em cores, comparada à concentração de calprotectina fecal (CF) em pacientes pediátricos. Materiais e Métodos: Em uma série consecutiva, no período entre 2014 e 2020, foram avaliados 53 exames de 44 pacientes pediátricos: 28 casos de doença de Crohn, 15 de colite ulcerativa e um de colite indeterminada. O diagnóstico da DII foi feito pelos critérios de Porto. O fluxo parietal foi a alteração estudada mais detalhadamente e classificada pelo pesquisador principal e por dois radiologistas pediátricos cegados aos valores de CF e de US Doppler. Baixo fluxo parietal foi definido pela captação de até 2 sinais de US Doppler/cm2, fluxo moderado entre 3 e 5 sinais/cm2 e alto fluxo mais de 5 sinais/cm2. Resultados: Houve concordância substancial entre os radiologistas (kappa = 0,73). Nos exames com baixo fluxo parietal a CF média foi 92 μg/g (intervalo interquartil: 33-661 μg/g) e nos exames com alto fluxo a CF média foi 2.286 μg/g (intervalo interquartil: 1.728-5.612 μg/g). Na amostra total, a US demonstrou sensibilidade de 89,7% e especificidade de 92,0% para detecção da atividade inflamatória, 95,5% e 90,9% na doença de Crohn e 81,3% e 100,0% na colite ulcerativa, respectivamente. Conclusão: Houve forte correlação entre a US da parede intestinal e os valores da concentração de CF na avaliação da atividade inflamatória na DII de pacientes pediátricos.

Echographic characterization of extraocular muscles in pediatric patients with thyroid dysfunction

ABSTRACT Purpose To characterize the size of extraocular muscles (EOMs) in a pediatric population with thyroid dysfunction using orbital echography. Methods Patients under age 18 with thyroid dysfunction who presented to an academic ophthalmology department from 2009 to 2020 and received orbital echography were included in this IRB-approved retrospective study. Data collected included age, clinical activity score (CAS), thyroid stimulating immunoglobulin (TSI), and extraocular recti muscle thickness on echography. Patients were organized into three age cohorts, after which statistical analysis compared recti measurements to previously reported normal ranges. Results Twenty patients with thyroid dysfunction were included. When comparing average recti muscle thicknesses of study patients to those of previously published normal children in similar age ranges, the levator-superior rectus complex was significantly increased in all age groups of children with thyroid dysfunction (p-values = <.004), and the levator-superior rectus complex was most frequently enlarged compared to published normal values (78% of eyes). CAS was not correlated with EOM size in the youngest group (5–10 years old, p-values >.315) but was significantly correlated in older groups (11–17 years old, p-values <.027). TSI was not correlated with EOM size in any group (p-values >.206). Conclusions Echographic reference ranges for EOMs in children with thyroid dysfunction were established. There are increased rates of levator-superior rectus complex enlargement in children with TED compared to adults with TED, and EOM size is correlated with CAS in children older than 10 years. Though limited, these findings may serve as an additional tool for ophthalmologists to ascertain disease activity in pediatric patients with thyroid dysfunction.

A phase 2 study of sirolimus in combination with metronomic chemotherapy (CHOAnome) in children with recurrent and/or refractory solid and CNS tumors.

10024 Background: Multiple low-dose, anti-angiogenic (metronomic) chemotherapy regimens have demonstrated activity in pediatric solid and central nervous system (CNS) tumors. The mTOR pathway is activated in many human cancers and provides an attractive therapeutic target. We previously conducted a phase 1 trial establishing the dose (2 mg/m2), safety and tolerability of sirolimus in combination with metronomic chemotherapy. In the current study, we conducted a prospective, multi-institutional phase 2 trial to determine the objective response rate (ORR) in pediatric patients with recurrent and/or refractory (R/R) solid and CNS tumors treated with this regimen (NCT02574728); herein we report the results of the CNS tumor stratum. Methods: Patients aged 12 months to 30 years with R/R CNS tumors of all world health organization (WHO) grades were eligible. After completion of the trial’s first stage, patients with diffuse intrinsic pontine glioma (DIPG) were excluded due to lack of efficacy. Patients were required to have measurable disease and no known curative therapeutic options. Treatment consisted of continuous sirolimus (2 mg/m2/dose PO daily), celecoxib (100 mg PO BID), and oral etoposide (50 mg/m2/day; max dose: 100 mg) alternating every 21 days with oral cyclophosphamide (2.5 mg/kg/day; max dose: 100 mg) in 42-day cycles. Sirolimus was dose-adjusted to maintain a serum trough concentration of 10-15 ng/ml. Response was determined using a modified Response Assessment in Neuro-Oncology (RANO) criteria. Enrollment proceeded using a Fleming’s two-stage design based on best overall response (BOR), which required two objective disease status determinations. Results: Twenty-two CNS tumor patients were enrolled; 20/22 were evaluable for response. Median age was 11.2 years (range: 5.9 – 18.1); 6 (30%) were female. Diagnoses included DIPG (N = 5), medulloblastoma (N = 3), high-grade (HG) glioma (N = 5), HG/malignant neuroepithelial tumor (N = 3) and one each of diffuse midline glioma, atypical teratoid/rhabdoid tumor (ATRT), choroid plexus carcinoma, and pineoblastoma. Median number of cycles was 2 (range: 1-6). Best response after any cycle was partial response (PR) in 4 [medulloblastoma (N = 3); pineoblastoma (N = 1)], stable disease (SD) in 4, and progressive disease (PD) in 12 for an ORR of 20%; all patients with DIPG had PD at or before their first disease evaluation. BOR was PR in 3 (15%), SD in 2 (10%), PD in 14 (70%), and unknown in 1 (5%). Four patients had SD or response for ≥ 6 months [medulloblastoma (n = 1), pineoblastoma (N = 1), and HG glioma (N = 2)]. Conclusions: The combination of sirolimus with metronomic chemotherapy showed activity in pediatric patients with some R/R CNS tumors, producing objective responses in medulloblastoma and pineoblastoma. Patients with DIPG did not benefit from this regimen and should be offered alternative treatments. Clinical trial information: NCT02574728 .

Clinico-Pathogenic Similarities and Differences between Infection-Related Glomerulonephritis and C3 Glomerulopathy.

Recently, the comprehensive concept of "infection-related glomerulonephritis (IRGN)" has replaced that of postinfectious glomerulonephritis (PIGN) because of the diverse infection patterns, epidemiology, clinical features, and pathogenesis. In addition to evidence of infection, hypocomplementemia particularly depresses serum complement 3 (C3), with endocapillary proliferative and exudative GN developing into membranoproliferative glomerulonephritis (MPGN); also, C3-dominant or co-dominant glomerular immunofluorescence staining is central for diagnosing IRGN. Moreover, nephritis-associated plasmin receptor (NAPlr), originally isolated from the cytoplasmic fraction of group A Streptococci, is vital as an essential inducer of C3-dominant glomerular injury and is a key diagnostic biomarker for IRGN. Meanwhile, "C3 glomerulopathy (C3G)", also showing a histological pattern of MPGN due to acquired or genetic dysregulation of the complement alternative pathway (AP), mimics C3-dominant IRGN. Initially, C3G was characterized by intensive "isolated C3" deposition on glomeruli. However, updated definitions allow for glomerular deposition of other complement factors or immunoglobulins if C3 positivity is dominant and at least two orders of magnitude greater than any other immunoreactant, which makes it challenging to quickly distinguish pathomorphological findings between IRGN and C3G. As for NAPlr, it was demonstrated to induce complement AP activation directly in vitro, and it aggravates glomerular injury in the development of IRGN. A recent report identified anti-factor B autoantibodies as a contributing factor for complement AP activation in pediatric patients with PIGN. Moreover, C3G with glomerular NAPlr deposition without evidence of infection was reported. Taken together, the clinico-pathogenic features of IRGN overlap considerably with those of C3G. In this review, similarities and differences between the two diseases are highlighted.

Digital Health Nudging to increase physical activity in pediatric patients with congenital heart disease: A randomized controlled trial

Digital nudging is a modern e-health approach to increase physical activity (PA) in younger age groups. As activity promotion is particularly important in adolescents with congenital heart disease (CHD) this randomized-controlled trail examines if Digital Health Nudging via daily smartphone messages increases PA, activity-related self-efficacy (ArSE) and health-related quality of life (HrQoL) in adolescents with CHD. From May 2021 to April 2022, 97 patients (15.1 ± 2.0 years, 50% girls) with moderate or severe CHD were randomly allocated 1:1 to intervention (IG) or control group (CG). Daily PA was objectively assessed in minutes of moderate-to-vigorous PA (MVPA) by the wearable "Garmin Vivofit jr. 2®" over the entire study period. The IG received daily smartphone messages based on Bandura's social cognitive theory on the subject of PA over a period of 12-weeks. According to the linear mixed model, the change of MVPA over the study period did not significantly differ between IG and CG when taking baseline MVPA into account (b=0.136, 95%-CI [-0.355; 0.627], p=0.587). Activity level was comparably high and showed only minor variability in both groups with 73.7 [62.3; 78.8] min/day in IG and 78.4 [66.6; 93.9] min/day in CG throughout the whole 12-weeks. Emotional well-being significantly increased over the study period in the IG (IG: Δ1.60 [-0.2; 6.3] vs. CG: Δ0.0 [-12.5; 6.3], p=0.043), but not total HrQoL (p=0.518) and ArSE (p=0.305). 12-weeks of Digital Health Nudging did not increase PA, but improved feelings of emotional well-being in adolescents with CHD. Clinical Trials Identifier NCT04933786.

A14 TRANSABDOMINAL BOWEL ULTRASOUND AND CLINICAL OUTCOMES OVER ONE YEAR IN CHILDREN WITH NEWLY DIAGNOSED CROHN’S DISEASE

Abstract Background Transabdominal bowel ultrasound (TABUS) is an emerging non-invasive tool for monitoring inflammatory bowel disease (IBD). Its use is particularly increasing in pediatric IBD, given the need for anesthesia during endoscopy. The assessment of TABUS in pediatric IBD has been limited to small numbers of patients with no long-term follow-up. Purpose To describe TABUS findings and its relationship with clinical, biochemical, and endoscopic assessments in pediatric patients with Crohn’s disease up to one year post-diagnosis. Method Patients (0-18 years) with suspected IBD were prospectively enrolled through the Edmonton Pediatric IBD Clinic. Those with Crohn’s disease were included. Patients underwent repeated TABUS, clinical assessments, blood work, fecal calprotectin (FCP) (baseline, 1-, 3-, 6-, 12-months), and endoscopy (baseline and 6-12 months). The weighted Pediatric Crohn’s Disease Activity Index (wPCDAI), Simple Endoscopic Score for Crohn’s Disease (SES-CD; rectum excluded), and Simple Ultrasound Activity Score for Crohn’s Disease (SUS-CD; rectum excluded) were used. Remission was defined as FCP&amp;lt;250mg/kg, CRP&amp;lt;4mg/L, wPCDAI&amp;lt;12, no upcoming surgery, and SES-CD score ≤2 for any bowel segment. Result(s) Fifty-six patients (68% male), median age 12.5 years (range 6-17), were followed for 6 months. Forty (71%) were followed up to 12 months. Median TABUS bowel wall thickness (BWT) and SUS-CD total scores improved in all bowel segments over time. SUS-CD total scores significantly correlated with SES-CD (baseline, 6-, 12-months), wPCDAI (baseline, 1-, 6-, 12-months), CRP (baseline, 1-, 3-, 6-months), ESR (baseline, 1-, 3-, 6-, 12-months), and FCP (baseline, 1-, 6-, 12-months) (rho ranged 0.302-0.732, p&amp;lt;0.05). Patients in remission had sustained significantly thinner BWT of their most affected bowel segment (Figure 1) starting at 1-month (median 3.1mm (IQR 2.7-3.7) vs. 4.1mm (IQR 2.9-5.6; p&amp;lt;0.05), and sustained significantly lower SUS-CD total scores starting at 6 months (median 0 (IQR 0-1) vs. median 2 (IQR 1-3); p&amp;lt;0.05). Seven patients had surgery (n=7/7 ileocecal, n=2/7 jejunal resection). All 7 patients had complex TI disease (n=6 strictures, n=1 long-segment disease &amp;gt;25cm) and proximal small bowel disease (n=2/2) on TABUS. Those with baseline ultrasound findings of a stricture with upstream bowel dilatation (n=7/56) had increased odds (OR=288, p&amp;lt;0.01) and relative risk (RR=42, p&amp;lt;0.01) of needing surgery (n=6/7 with baseline obstructive findings, n=1/49 without) within the first year. Image Conclusion(s) TABUS had significant correlations with clinical, biochemical, and endoscopic markers of Crohn’s disease activity in pediatric patients over one year. Bowel wall thickness of the most affected bowel segment is a helpful measurement, becoming significantly thinner as soon as 1-month post-diagnosis in those who obtain remission. Baseline findings of bowel narrowing and upstream dilation increase the odds and relative risk of needing surgery in the first year. Please acknowledge all funding agencies by checking the applicable boxes below None Disclosure of Interest None Declared

Exploratory Longitudinal Analysis of the Circulating CHIT1 Activity in Pediatric Patients with Obesity.

Macrophage activation and cytokine release play a pivotal role in inflammation-mediated metabolic disturbances in obesity. The proinflammatory macrophage secretes human chitotriosidase (CHIT1). The expression of the CHIT1 in visceral adipose tissue is associated with cytokine production. Our study aimed to assess whether the CHIT1 circulating activity, as a macrophage activation indicator, reflects the change of the adiposity level and the insulin resistance (IR) in children with obesity. We longitudinally (median follow-up period of 7 months; IQR [5 to 8.5] and {2 to 13} months) evaluated the CHIT1 circulating activity, the adiposity level (waist circumference (WC), waist-to-hip ratio (WHR), waist-to-height ratio (WtHR), and body mass index (BMI)-for-age z score), and two surrogate markers of IR (Homeostatic Model Assessment for Insulin Resistance, HOMA-IR and the triglycerides-to-high density lipoprotein cholesterol ratio, TG/HDLc) in 29 pediatric patients (16 girls and 13 boys) with obesity. We found a significant reduction in CHIT1 circulating activity (Wilcoxon test, p = 0.015) and a decrease in TG/HDLc at the follow-up evaluation (Wilcoxon test, p &lt; 0.001). Indicators of adiposity were positively correlated with HOMA-IR at baseline, among which WC was the sole indicator associated with HOMA-IR (Spearman's rank correlation coefficients, p &lt; 0.05) at follow-up. Human chitotriosidase has the potential to be a valuable measure of the progression of subclinical inflammation in children with obesity. Subclinical inflammation, as expressed by the circulating CHIT1 activity, progresses independently of the abdominal adiposity, as measured by the clinical indicators, and is associated with a change in insulin resistance.

Abstract A089: Antithrombin III and fibrinogen activity as surrogate markers in predicting therapeutic asparaginase activity levels in children with acute lymphoblastic leukemia

Abstract Background. The incorporation of asparaginase has played an important role in improving survival rates in pediatric patients treated for acute lymphoblastic leukemia (ALL). Measuring asparaginase levels is one method to determine the efficacy of this chemotherapy. Determination of antithrombin III (ATIII) and/or fibrinogen (FIB) activity can be clinically useful as indirect asparaginase (ASP) level assessments since serum assays of ASP levels are not widely available, can be costly, and are performed at specialized labs. Previous studies have identified ATIII and FIB as potential markers in characterizing low or undetectable asparaginase activity in pediatric patients with ALL. The objective of this study was to determine whether FIB and ATIII levels can be used as surrogate markers in predicting therapeutic ASP activity in a predominantly Hispanic ALL population. Methods. FIB and ATIII levels were collected prior and 7 days post PEG-asparaginase (PEG) infusion. ASP levels were collected 7 days post-PEG. Mean differences in levels of FIB and ATIII activity were compared using t-tests for means and Mann-Whitney tests for medians. The Spearman correlation was used to evaluate the association between asparaginase activity, FIB, and ATIII levels. Unadjusted odds ratios were calculated to assess the predictive value of differences from baseline to Day 7 in ATIII and FIB levels associated with therapeutic levels (≥ 0.1 IU/mL). Results. Twenty patients were enrolled. The cohort was predominantly Hispanic (60%) and male (68%) with a median age of 8 years [range 2-19]. The median FIB level post-PEG was 155 mg/dL [nl 212-466] and the median ATIII activity level was 61% [nl 84-125] in those with therapeutic ASP levels. A median decrease in FIB and ATIII activity levels of 52% were seen with therapeutic ASP levels. By contrast in those with non-therapeutic levels, the difference was 7% and 28%, respectively. The bivariate correlation between ATIII and 7-day asparaginase levels was significant when controlling for BMI (Spearman’s ρ=0.62, p=0.001); the 7-day association between ASP and FIB was not influenced by BMI (Spearman’s ρ=-0.46, p=0.05). FIB and ATIII were correlated (Spearman’s ρ =.47;p=0.03). Both FIB (OR=0.004[0.0003-0.44]; p=0.02) and ATIII (OR=0.004[0.0002-1.0]; p=0.04) demonstrated reduced odds of higher therapeutic levels compared to lower therapeutic levels when mediated by Hispanic ethnicity. ATIII mean changes baseline to Day 7 were significantly smaller for Hispanic ethnicity [(Hispanic =-0.45(0.18) vs. Non-Hispanic =0.52(0.05); p=0.05)]. There were no significant differences in FIB by Hispanic ethnicity. Conclusions. Decreases in ATIII levels from baseline are associated with reduced odds of therapeutic asparaginase levels while decreased FIB levels may also indicate subtherapeutic levels. Hispanic ethnicity appears to mediate the observed effects from ATIII. ATIII and FIB levels may be important indirect markers in ASP activity monitoring when serum asparaginase assays are unavailable. Citation Format: Sonia Morales, Edith Williams, Kirsten Kasper, Keri Zabokrtsky, Lois Sayrs, Diane Nugent, Van Huynh. Antithrombin III and fibrinogen activity as surrogate markers in predicting therapeutic asparaginase activity levels in children with acute lymphoblastic leukemia [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A089.

EXTH-75. INTEGRATED STRESS RESPONSE ACTIVATORS SYNERGISTICALLY INDUCE APOPTOSIS AND SUPPRESS PROLIFERATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. Activation of the ISR is indicated by increased expression of activating transcription factor 4 (ATF4). Intermediate levels of ATF4 protect cells from stress, while persistent high levels result in cell death. The imipridone drug ONC201 increases mitochondrial stress and ATF4 expression by binding to and activating the mitochondrial protease ClpP. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. After determining the IC50 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO and single treatment (p&amp;lt; 0.0001, ANOVA). Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. The combination also increased ATF4 expression. Since Sal003 is not clinically available, we treated DIPG cells with ONC201 and fenretinide. Synergy testing in JHH-DIPG1 showed an overall ZIP synergy score of 14 with inhibitory concentrations of both drugs in the nanomolar range. CC3 immunofluorescence indicated elevated apoptosis in the combination vs. DMSO (p&amp;lt; 0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, p-eIF2α, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in brain organoids and orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.

High levels of cerebrospinal fluid soluble triggering receptor expressed on myeloid cells 2 might be a biomarker of activity in pediatric patients with MOG-AD.

Myelin oligodendrocyte glycoprotein antibody disease (MOG-AD) is characterized by its monophasic or relapsing course and inflammatory demyelinating condition which is unable to be classified in typical multiple sclerosis (MS) or other known neuroinflammatory conditions. In the condition of neuroinflammatory, activated microglia are essential for demyelination. The secreted ectodomain of soluble triggering receptor expressed on myeloid cells 2 (sTREM2), expressed by microglial cells, is associated with abnormal biological pathways. It is known that the cerebrospinal fluid (CSF) sTREM2 concentration is much higher in neuroinflammatory and neurodegeneration diseases. However, the role of activated microglia has not been reported in MOG-AD pediatric patients. For the first time, the increased CSF and serum sTREM2 concentration in pediatric patients with MOG-AD is investigated in this work, showing evidence of microglia activation in MOG-AD. CSF sTREM2 levels significantly correlated with clinical inflammatory indexes and adapted modified Rankin Scale score, indicating the potential value of sTREM2 as a severity biomarker.

Association of Vitamin D with Inflammatory Bowel Diseases Activity in Pediatric Patients

Association of Vitamin D with Inflammatory Bowel Diseases Activity in Pediatric Patients

In-vivo Sino-Atrial Node Mapping in Children and Adults With Congenital Heart Disease.

Background:Sinus node dysfunction (SND) and atrial tachyarrhythmias frequently co-exist in the aging patient with congenital heart disease (CHD), even after surgical correction early in life. We examined differences in electrophysiological properties of the sino-atrial node (SAN) area between pediatric and adult patients with CHD.MethodsEpicardial mapping of the SAN was performed during sinus rhythm in 12 pediatric (0.6 [0.4–2.4] years) and 15 adult (47 [40–55] years) patients. Unipolar potentials were classified as single-, short or long double- and fractionated potentials. Unipolar voltage, relative R-to-S-amplitude ratio and duration of all potentials was calculated. Conduction velocity (CV) and the amount of conduction block (CB) was calculated.ResultsSAN activity in pediatric patients was solely observed near the junction of the superior caval vein and the right atrium, while in adults SAN activity was observed even up to the middle part of the right atrium. Compared to pediatric patients, the SAN region of adults was characterized by lower CV, lower voltages, more CB and a higher degree of fractionation. At the earliest site of activation, single potentials from pediatrics consisted of broad monophasic S-waves with high amplitudes, while adults had smaller rS-potentials with longer duration which were more often fractionated.ConclusionsCompared to pediatric patients, adults with uncorrected CHD have more inhomogeneous conduction and variations in preferential SAN exit site, which are presumable caused by aging related remodeling. Long-term follow-up of these patients is essential to demonstrate whether these changes are related to development of SND and also atrial tachyarrhythmias early in life.

Increased acid sphingomyelinase levels in pediatric patients with obesity

The level of secretory acid sphingomyelinase (S-ASM), a key enzyme in the sphingolipid metabolism, is elevated in a variety of human diseases, including in the serum of obese adults. Alterations in S-ASM were also found to induce morphological changes in erythrocytes. Consequently, the inhibition of S-ASM by functional Inhibitors of ASM (FIASMA) may have broad clinical implications. The purpose of this study was to assess S-ASM activity in pediatric patients with obesity and healthy matched controls, as well as to investigate the erythrocyte morphology using transmission electron microscopy. We recruited 46 obese patients (mean age 11 ± 2.9 years) and 44 controls (mean age 10.8 ± 2.9 years). S-ASM activity was significantly higher (Wilcoxon signed-rank test p-value: 0.004) in obese patients (mean 396.4 ± 49.7 pmol/ml/h) than in controls (mean 373.7 ± 23.1 pmol/ml/h). No evidence of morphological differences in erythrocytes was found between the two populations. We then carried out a case–control study based on the spontaneous reporting system database to compare FIASMAs with NON-FIASMAs in terms of weight gain risk. Children who received FIASMA had a significantly lower frequency of weight gain reports than patients who took NON-FIASMA agents (p < 0.001). Our findings suggest there is an intriguing possibility that S-ASM may play a role in pediatric obesity. This pilot study could serve as the basis for future studies in this interesting field of research.

DIPG-52. Activators of the integrated stress response synergize to kill DIPG

DIPG has elevated baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that allows cells to tolerate various forms of stress. Increased expression of activating transcription factor 4 (ATF4) indicates activation of the ISR. Intermediate levels of ATF4 protect cells from stress, while sustained high levels result in cell death. The imipridone drug ONC201 binds to and activates the mitochondrial protease ClpP, leading to increased mitochondrial stress and persistent ATF4 activation. Because DIPG has a high baseline level of ATF4, we hypothesized that the ISR activators Sal003, ONC201, and fenretinide would synergize to kill DIPG. Sal003 inhibits dephosphorylation of ATF4 upstream regulator, eIF2α. The retinoic acid derivative fenretinide induces ATF4, increases reactive oxygen species, and has clinical activity in pediatric patients with neuroblastoma. After determining the IC25 of Sal003, fenretinide, and ONC201, we treated patient-derived DIPG cell lines with low micromolar doses. The combination of Sal003 and ONC201 significantly increased apoptosis as measured by CC3 immunofluorescence in comparison to DMSO (p<0.0001, ANOVA). Combination therapy also significantly increased CC3 positivity compared to single treatment. Western blots for cleaved PARP expression detected induction of apoptosis in DIPG treated with both Sal003 and ONC201 over DMSO and monotherapy treated cells. In some cell lines, the combination increased ATF4 expression. Since Sal003 is not yet available for clinical testing in humans, we treated DIPG cells with ONC201 and fenretinide. CC3 immunofluorescence indicated synergistically elevated apoptosis in the combination of ONC201 and fenretinide vs. DMSO (p<0.0001, ANOVA). Western blots showed increased cleaved PARP, ATF4, and CHOP expression in DIPG treated with ONC201 and fenretinide. We are currently testing the efficacy of this combination in orthotopic DIPG xenografts. Our results suggest the combination of ONC201 with fenretinide could potentially serve as a therapy for DIPG.

Both fecal calprotectin and fecal immunochemical tests are useful in children with inflammatory bowel disease.

Noninvasive biomarkers of intestinal inflammation can reduce the number of endoscopies in children with inflammatory bowel disease (IBD). This study aimed to prospectively investigate the usefulness of fecal calprotectin (FCP) and fecal immunochemical test (FIT) in pediatric IBD. Patients aged 6-17years who underwent ileocolonoscopy for established or suspected IBD were eligible for this study. Fecal samples for FCP and FIT were collected before colonoscopy. A total of 251 samples were analyzed: 88 from ulcerative colitis (UC), 74 from Crohn's disease (CD), 75 from healthy controls (HC), and 14 from children with functional gastrointestinal disorders and normal colonoscopy (NC). At IBD diagnosis, both FCP and FIT were significantly higher in the newly diagnosed UC/CD group than in the HC/NC group (P < 0.001). The optimal cutoffs of FCP and FIT to predict IBD diagnosis were 217mg/kg and 87ng/mL, respectively. Patients without mucosal healing (MH) showed higher FCP and FIT than those with MH in both UC and CD (P < 0.001). The FCP increased exponentially as the endoscopic activity score increased. The optimal cutoff values of FCP and FIT for predicting MH were 161mg/kg and 106ng/mL for UC and 367mg/kg and 57ng/mL for CD, respectively. FCP showed better specificity than the FIT. Patients with CD and normal ileocolonoscopy had elevated FCP during active small intestinal inflammation. Both FCP and FIT correlate well with endoscopic activity in pediatric patients with IBD. The FCP is a superior marker for predicting MH.

Serum levels of soluble HLA-G correlate with disease activity in pediatric patients with Crohn's disease.

Background:Human leukocyte antigen (HLA)-G, a member of the HLA family, is crucial for fetomaternal tolerance. Transmembrane or circulating/soluble HLA-G (sHLA-G) is elevated in autoimmune conditions and the tumor microenvironment. Circulating sHLA-G levels and their association with disease activity have not yet been assessed in pediatric patients with inflammatory bowel disease (IBD). This study aimed to quantify the serum sHLA-G levels of pediatric patients with IBD and assess the association of serum sHLA-G with disease activity.Methods:We enrolled 24 pediatric IBD patients Crohn's disease (CD) and ulcerative colitis (UC), n = 12 each] and 24 healthy controls. Based on the disease activity index, five and seven of the CD patients had mild and moderate/severe disease, respectively, whereas six of the UC patients were in remission and six had mild disease. Serum was collected and sHLA-G levels were determined by enzyme-linked immunosorbent assay (ELISA).Results:Pediatric patients with CD had significantly higher sHLA-G levels compared with patients with UC and healthy controls. Notably, serum sHLA-G levels were significantly higher in patients with moderate/severe CD than in those with mild CD.Conclusions:Serum sHLA-G levels correlate with disease activity in pediatric patients with CD and are higher in CD patients than in UC patients. Thus, sHLA-G is a potential biomarker for disease activity in CD.

P099 Serum proteomic analysis reveals novel biomarkers associated with disease activity in pediatric patients with Crohn’s disease

Abstract Background Although Crohn’s disease (CD) is associated with a marked hyper-inflammatory response, identifying serum biomarkers associated with disease activity and outcomes is considered challenging, given lack of sensitivity of different assays. Our aim was to identify novel markers using Olink, a novel technology based on proximity extension assay, enabling detection of minute amounts of proteins. Methods Serum samples were obtained from pediatric patients with CD at the time of diagnosis and following induction therapy, and from control subjects, consisting of pediatric patients with normal endoscopic procedures, without past or present history of an immune-mediated disorder (e.g. celiac, diabetes). Serums were subjected to Olink (two panels used, consisting of 184 proteins), and analysis was performed on a Normalized Protein eXpression file by supervised, multivariate, principal component analysis and verification by univariate ANOVA with Benjamini-Hochberg and post-hoc Tukey analysis. Results Eighty-eight serum samples were collected: 30 from control subjects and 58 from 32 patients with CD (24 of them pre- and post-induction therapy). The median age of patients in the control and CD groups was 13.9 years (IQR 11.1–16.6) and 14.6 years (IQR 12.2–16.9), respectively (P=0.32). Twenty-four patients were treated with anti-TNFa agents and 8 with EEN. The median pediatric Crohn’s Disease Activity Index (PCDAI) decreased from 35 (22.5–42.5) to 5 (0–12.5, P&amp;lt;0.001) following induction therapy. We identified 72 proteins that significantly differed between patients and controls, and many of them were associated with CRP or ESR levels. String analysis identified several important nodes linking different proteins, including MMPs, MPO, EGFR and TNFRSF1A. Many proteins, such as resistin and different MMPs, showed a strong positive correlation with disease activity, based on PCDAI, while others, such as EGFR, showed a negative correlation. Several novel markers were identified to be highly expressed in the serum of patients with CD, including LRIG1, an intestinal stem cell marker, that was highly correlated with disease activity among the patients. Conclusion We identified novel serum markers that are associated with disease activity in pediatric patients with CD. These findings should be validated in future studies, and be complemented with expression and functional studies to define their role in mediating intestinal immune responses.

Results Of a Phase I Study Of Clofarabine and 2 Gy Total Body Irradiation (TBI) As a Non-Myeloablative Preparative Regimen For Hematopoietic Stem Cell Transplantation (HSCT) In High Risk Children With Hematologic Malignancies: A Multi-Center TACL Study

BackgroundReduced intensity conditioning (RIC) regimens for HSCT in hematologic malignancies are successful in reducing transplant related mortality (TRM) and making HSCT available for a select group of high risk patients. Despite HSCT and its associated graft-versus-leukemia (GVL) effect, the risk of relapse remains high with RIC regimens. Clofarabine is a second generation purine analogue with significant anti-leukemic activity in pediatric patients. We postulated that a non-myeloablative (NMA) preparative regimen with clofarabine and 2 Gy TBI may lead to sustained engraftment with low TRM in high-risk children with hematologic malignancies undergoing related and unrelated HSCT. Patient and MethodsThe primary aim of the Phase I study was to establish the maximum feasible dose (MFD) of clofarabine in conjunction with 2 Gy TBI that leads to durable engraftment with acceptable toxicity in a high-risk pediatric population. Patients between 1-21 years of age with leukemia and undergoing HSCT with significant co-morbidities or any condition that will make them at high risk of TRM with a myeloablative regimen were eligible. Patients had to be in CR or CRp (no morphologic disease, ANC >0.75 x 109/L but without platelet recovery) status prior to HSCT with a PS score of >50 with no active infection, a creatinine clearance of >70 ml/min/1.73m2 and liver enzymes <5X ULN. Patients were stratified according to donor source- related donor (RD) or unrelated donor (UD; 10/10 or 9/10 HLA match) arms. Peripheral blood stem cells (PBSC) were the preferred graft source (BM was allowed for RD only). Cyclosporine and mycophenolate mofetil were used as GVHD prophylaxis. A phase I modified 3+3 design was used with engraftment, severe toxicity and TRM as study end-points. Starting dose of clofarabine was 40 mg/m2 x 5 days with escalation to 52 mg/m2 in case of non-engraftment and acceptable toxicity in the first cohort or de-escalation to 30 mg/m2for dose limiting toxicity (DLT) or TRM. Patients completed the dose evaluation at 100 days post-HSCT, but were followed for 1 year post- transplant and were censored at relapse or death. ResultsSeventeen pediatric patients with a median age of 9 years (range 1-18) enrolled on the study between 7/2010 and 5/2013 and 16 have completed 100-day post-HSCT evaluations. Ten patients had ALL (CR2 =5; CR3 =4 and CR4=1) while 7 had AML (PIF=1; CR1= 1; CR2=3 and secondary AML=3); 9 received UD and 8 received RD grafts. All patients were heavily pre-treated and the indication for the NMA approach was: prior HSCT (n=6), prior liver transplant (n=2), trisomy 21 (n=2) or significant comorbidities (n=7).There was no observed TRM or DLT in the first cohort (clofarabine 40 mg/m2/dose x 5 days) in both the UD and RD strata. The dose was escalated to 52 mg/m2/dose in the next cohorts. All patients tolerated the maximum dose well and no DLT was reported. Significant non-hematologic toxicities (CTC ≥ grade 3) observed were- transient transaminitis (ALT increase in 30% and AST increase in 17%); anorexia (35%) and skin reactions in 16%.PBSC was the stem cell source for all patients, except 3 who had received BM from a RD. Median cell dose was 6.8 x 106 CD34+/kg (range: 2.2-11.5 x 106/kg). All patients achieved neutrophil and platelet engraftment at a median of 19 days (range: 9-25) and 11 days (range: 3-29), respectively. CD3 chimerism analysis at day+30 post-transplant showed donor engraftment in all patients; 15 were full donor (>95%) and 2 showed mixed chimerism (53% and 81% donor).Ten (58%) patients developed acute GVHD, 8 (47%) had ≤ Grade II and 2 (11%) had Grade III acute GVHD. Seven (41%) patients relapsed (4/8 RD and 3/9 in UD arms) at a median time of +106 days (range: 88-190) post-HSCT and all of them died due to disease. Ten patients (58%) are alive and disease free; six have completed 1 year evaluations and 4 are still in follow-up. ConclusionThe MFD of clofarabine in combination with 2 Gy TBI was determined to be 52 mg/m2. This regimen is well tolerated and leads to brisk and sustained donor engraftment in a pediatric population that is at high risk of TRM. Disclosures:Thompson:Sanofi Aventis: Speakers Bureau.