Benefits of early aggressive immunomodulatory therapy (tocilizumab and methylprednisolone) in COVID-19: Single center cohort study of 685 patients

Introduction: A growing evidence suggests that immune dysregulation and thrombotic phenomena are key features in the pathophysiology of COVID-19. Apart from antivirals and respiratory support, anticoagulants, corticoids and immunomodulators are increasingly being prescribed, especially for more severe cases. We describe the clinical outcome of a large cohort of patients preferentially treated with glucocorticoids and interleukin inhibitors. Methods: Single center and retrospective case series. Adult patients admitted with COVID-19 related respiratory insufficiency were included. Patients who died within 2 days after admission and those testing positive but asymptomatic were excluded. We defined two study periods: from March 3rd to March 31 st, 2020 (beginning of epidemic until peak of incidence) and April 1 st to May 7 th, 2020 (second half of epidemic). The majority of patients received respiratory support, combinations of antimicrobials, anticoagulants, corticoids and interleukin inhibitors. Antivirals were preferentially given in the first period. The clinical outcome (death and ventilator dependency) of both periods was compared. Results: From March 3 rd to May 7 th, 685 patients were included for analysis (58.4% males, mean age 68.9 years). Patients in the first period (n = 408) were younger (66.6 vs 71.1 years, p = 0.003), presented lower mean P a O2/FiO2 ratio at admission (256.5 vs 270.4 mm Hg,p = 0.0563), higher ferritin (1520 vs 1221 ng/ml, p = 0.01), higher IL-6 (679 vs 194 pg/ml, p < 0.0001) and similar D-dimer levels (3.59 vs 3.39 mu g/mL, p = 0.65) compared to the second period (n = 277). Lopinavir/ritonavir and interferon were preferentially given in the first period (23.8% and 32% vs 1.8% and 11.9%, p < 0.0001). Use of corticoids (88.2% vs 87.4%, p = 0,74) and tocilizumab (26.29 vs 20.22% p = 0.06) were similarly administered in both periods. Patients in the second period needed less mechanical ventilation (4.9% vs 16.9%, p < 0.0001), fewer ICU admission (6.1% vs 20.1%,p < 0.0001) and showed similar mortality (17.7% vs 15.4%, p = 0.43). Infectious and thrombotic complications were comparable in both periods (both around 8%, with no statistical difference). Patients treated with tocilizumab (n = 163) had lower mortality rate compared to those untreated under the same indication (7.9% vs 24.2%, p < 0.0001). Conclusions: In this large retrospective COVID-19 in-hospital cohort, lopinavir/ritonavir and interferon showed no significant impact on survival. Extensive use of corticosteroids and tocilizumab resulted in good overall outcome and showed acceptable complication rates

A Three-Protein Panel to Support the Diagnosis of Sepsis in Children

Sepsis is a syndrome without a standard validated diagnostic test. Early recognition is crucial. Serum proteome analysis in children with sepsis may identify new biomarkers. This study aimed to find suitable blood biomarkers for an early diagnosis of sepsis. An analytical observational case-control study was carried out in a single center. Children admitted to a Pediatric Intensive Care Unit with clinical diagnosed sepsis were eligible for study. A proteomic analysis conducted by mass spectrometry was performed. Forty patients with sepsis and 24 healthy donors were recruited. Proteomics results revealed 44 proteins differentially expressed between patients and healthy controls. Six proteins were selected to be validated: lactoferrin, serum amyloid-A1 (SAA-1), complement factor B, leucine-rich alpha-2 glycoprotein (LRG1), soluble interleukin-2 alpha chain receptor (sCD25) and soluble haptoglobin-hemoglobin receptor. Our results showed that sCD25, SAA-1, and LRG1 had high levels of specificity and sensitivity, as well as an excellent area under the ROC curve (>0.9). Our study provides a serum proteomic analysis that identifies new diagnostic biomarkers in sepsis. SAA-1, sCD25 and LRG1 were able to separate septic from healthy donor, so they could be used together with other clinical and analytical features to improve sepsis diagnosis in children.This work was funded by Research Projects from University of Basque Country (US10/02) and from the Basque Government (SAIO10-PE10BF02, SAIO12-PE12BF002, 2012111052, 2019111056). CICbioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County, the ProteoRed-ISCIII (Grant PRB3 IPT17/0019), CIBERehd Network and Severo Ochoa Grant (SEV-2016-0644)

Identification of a panel of serum protein markers in early stage of sepsis and its validation in a cohort of patients

Background: Sepsis is a life-threatening illness with a challenging diagnosis. Current serum biomarkers are not sensitive enough for diagnosis. With the aim of finding proteins associated with sepsis, serum protein profile was compared between patients and healthy donors and serum classical inflammatory proteins were analyzed in both groups. Methods: Serum protein profiles were characterized by two-dimensional electrophoresis (2DE). Identification of the proteins was carried out by mass spectrophotometry and their validation was performed by Enzyme-Linked-lmmunoSorbent Assay (ELISA) in a cohort of 85 patients and 67 healthy donors. Seven classical inflammatory proteins were analyzed in the same cohort by ELISA: interleukin-2 receptor alpha-chain (sCD25), scavenger receptor cysteine

Confirmation of involvement of new variants at CDKN2A/B in pediatric acute lymphoblastic leukemia susceptibility in the Spanish population

The locus CDKN2A/B (9p21.3), which comprises the tumor suppressors genes CDKN2A and CDKN2B and the long noncoding RNA (lncRNA) known as ANRIL (or CDKN2B-AS), was associated with childhood acute lymphoblastic leukemia (ALL) susceptibility in several genome wide association studies (GWAS). However, the variants associated in the diverse studies were different. Recently, new and independent SNPs deregulating the locus function were also identified in association with ALL risk. This diversity in the results may be explained because different variants in each population could alter CDKN2A/B locus function through diverse mechanisms. Therefore, the aim of this study was to determine whether the annotated risk variants in the CDKN2A/B locus affect the susceptibility of B cell precursor ALL (B-ALL) in our Spanish population and explore if other SNPs altering additional regulatory mechanisms could be also involved. We analyzed the four SNPs proposed by GWAs and two additional SNPs in miRNA binding sites in 217 pediatric patients with B-ALL and 330 healthy controls. The SNPs rs2811712, rs3731249, rs3217992 and rs2811709 were associated with B-ALL susceptibility in our Spanish population. ALL subtypes analyses showed that rs2811712 was associated with B-hyperdiploid ALL. These results provide evidence for the influence of genetic variants at CDKN2A/B locus with the risk of developing BALL.This study was funded by the Basque Government (IT661-13, IT989-16), UPV/EHU (UFI11/35). AGC was supported by a pre-doctoral grant from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Involvement of SNPs in miR-3117 and miR-3689d2 in Childhood Acute Lymphoblastic Leukemia Risk

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Numerous studies have shown that microRNAs (miRNAs) could play a role in this disease. Nowadays, more than 2500 miRNAs have been described, that regulate more than 50% of genes, including those involved in B-cell maturation, differentiation and proliferation. Genetic variants in miRNAs can alter their own levels or function, affecting their target gene expression, and then, may affect ALL risk. Therefore, the aim of this study was to determine the role of miRNA genetic variants in B-ALL susceptibility. We analyzed all variants in pre-miRNAs (MAF > 1%) in two independent cohorts from Spain and Slovenia and inferred their functional effect by in silico analysis. SNPs rs12402181 in miR-3117 and rs62571442 in miR-3689d2 were associated with ALL risk in both cohorts, possibly through their effect on MAPK signalling pathway. These SNPs could be novel markers for ALL susceptibility

Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro-osteogenic paracrine response: TERCELOI clinical trial

Background Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. Methods To overcome the short-term effect of MSCs therapy, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs, administered in a 2.5-year period, in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in nonimmunosuppressed OI patients. The host response to MSCs was studied by analyzing the sera from OI patients, collected before, during, and after the cell therapy. Results We first demonstrated that the sequential administration of MSCs was safe and improved the bone parameters and quality of life of OI patients along the cell treatment plus 2-year follow-up period. Moreover, the study of the mechanism of action indicated that MSCs therapy elicited a pro-osteogenic paracrine response in patients, especially noticeable in the patient affected by severe OI. Conclusions Our results demonstrate the feasibility and potential of reiterative MSCs infusion for two pediatric OI and highlight the paracrine response shown by patients as a consequence of MSCs treatment.We are grateful to the patients affected by OI and their families, and to the AHUCE Foundation, especially to its director Ms Julia Piniella, for the support during the clinical trial. We also thank Dr ME Fernandez-Santos (GM-Cell Production Unit, IiSGM) for her expertise in the cell production, Jose Ignacio Pijoan Zubizarreta (Clinical Epidemiology Unit of Cruces University Hospital) for the overall support provided to the project, and Natale Imaz (Biocruces Bizkaia Health Research Institute) for providing the data and safety monitoring of the clinical trial. We are indebted to all the health professionals from Cruces University Hospital, especially to the Pediatric Intensive Care Unit for their participation. This study was funded by the Spanish Ministry of Health through the call for independent clinical trials projects "EC10-219," Instituto de Salud Carlos III through the project "PI15/00820" (Co-funded by European Regional Development Fund; "A way to make Europe"), Bioef-EiTB maratoia (BIO14/TP/007), and the AHUCE Foundation

Identification of germline cancer predisposition variants in pediatric sarcoma patients from somatic tumor testing

Genetic predisposition is an important risk factor for cancer in children and adolescents but detailed associations of individual genetic mutations to childhood cancer are still under intense investigation. Among pediatric cancers, sarcomas can arise in the setting of cancer predisposition syndromes. The association of sarcomas with these syndromes is often missed, due to the rarity and heterogeneity of sarcomas and the limited search of cancer genetic syndromes. This study included 43 pediatric and young adult patients with different sarcoma subtypes. Tumor profiling was undertaken using the Oncomine Childhood Cancer Research Assay (Thermo Fisher Scientific). Sequencing results were reviewed for potential germline alterations in clinically relevant genes associated with cancer predisposition syndromes. Jongmans´ criteria were taken into consideration for the patient selection. Fifteen patients were selected as having potential pathogenic germline variants due to tumor sequencing that identified variants in the following genes: CDKN2A, NF1, NF2, RB1, SMARCA4, SMARCB1 and TP53. The variants found in NF1 and CDKN2A in two different patients were detected in the germline, confirming the diagnosis of a cancer predisposition syndrome. We have shown that the results of somatic testing can be used to identify those at risk of an underlying cancer predisposition syndrome.This work was funded by Research Projects from Navarra Government (Ref. 54/2018), the Jesús de Gangoiti Barrera Foundation (FJGB18/004 and FJGB19/001), Asociación Pablo Ugarte APU (APU-osteosarcoma), La Cuadri del Hospi (BC/A/17/008), EITB Media AND BIOEF, SAU (BIO20/CI/015/BCB and BIO20/CI/011/BCB), Basque Government (2021111030) and Fundación La Caixa with Niños Contra el Cáncer. P.A.-P. is supported by a Basque Government fellowship (PRE_2021_2_0048)

Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma

Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis.This work was funded by Asociación Pablo Ugarte APU (BC/A/14/015), Pequerropa (BC/A/15/010), and the childhood cancer support Platform from EITB Media, SAU (BIO13/CI/016/BC). R.P. was funded by Ministerio de Economía y Competitividad (Spain and Fondo Europeo de Desarrollo Regional, grant number SAF2016-79847-R). C.E.N.-X. was funded by Instituto de Salud Carlos III (Spain and the European Social Fund+, grant number: CP20/00008). P.A.-P. was supported by a Basque Government fellowship (PRE_2020_2_0116)

Importance of Timely Treatment Initiation in Infantile-Onset Pompe Disease, a Single-Centre Experience

Abstract Classic infantile Pompe disease (IPD) is a rare lysosomal storage disorder characterized by severe hypertrophic cardiomyopathy and profound muscle weakness. Without treatment, death occurs within the first 2 years of life. Although enzyme replacement therapy (ERT) with alglucosidase alfa has improved survival, treatment outcome is not good in many cases and is largely dependent on age at initiation. The objective of the study was (a) to analyse the different stages in the diagnosis and specific treatment initiation procedure in IPD patients, and (b) to compare clinical and biochemical outcomes depending on age at ERT initiation (<1 month of age vs. <3 months of age). Here, we show satisfactory clinical and biochemical outcomes in two IPD patients after early treatment initiation before 3 months of life with immunomodulatory therapy in the ERT-naïve setting, with a high ERT dose from the beginning. Despite the overall good evolution, the patient who initiated treatment <1 month of life presented even better outcomes than the patient who started treatment <3 months of life, with an earlier normalization of hypertrophic cardiomyopathy, along with CK normalization, highlighting the importance of early treatment initiation in this progressive disease before irreversible muscle damage has occurred.This work was partially funded by the Basque Department of Education (IT1281-19)

Results of A Phase 1 study of the oncolytic adenovirus DNX-2401 with radiotherapy for newly diagnosed diffuse intrinsic pontine glioma (DIPG)

Background A Phase 1, single center study is ongoing to evaluate the conditionally replicative oncolytic adenovirus, DNX-2401 (tasadenoturev), followed by radiotherapy (RT) in pediatric patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG). Methods Patients 1–18 years with newly diagnosed DIPG with no prior treatment, Lansky/Karnofsky performance score ≥ 70, and adequate organ function were enrolled. A tumor biopsy was performed followed by a single intratumoral injection of 1e10-5e10 virus particles (vp) DNX-2401. Conventional radiotherapy was initiated within 1 month of DNX-2401 administration. Results Enrolled subjects (n=12) had a median age of 9 (range 3–18) and performance scores of 90–100 (n=4; 33%) or 70–80 (n=8; 67%). As part of a dose escalation design, subjects were treated with 1e10 vp (n=4) or 5e10 vp DNX-2401 (n=8), which was then followed by standard RT in 11 of 12 subjects (92%). No dose-limiting toxicities were observed and the treatment regimen was well-tolerated. Adverse events (AEs) have been primarily mild to moderate and consistent with underlying disease. The most commonly reported AEs (≥ 5 subjects), regardless of study drug relationship, include headache, asthenia, vomiting, anemia, leukocytosis, and fever. Two SAEs have been reported including grade 3 lymphopenia and grade 3 abdominal pain. Tumor reductions have been observed and efficacy evaluations are ongoing. As of 09Dec2020, 12-month survival (OS-12) was 71% and 4 of 12 patients had survived > 20 months. Four subjects continue to be followed for survival. Correlative analysis of tumor biopsy and peripheral samples is ongoing. Conclusions DNX-2401 followed by RT can be safely administered to pediatric subjects with newly diagnosed DIPG; clinical activity and preliminary survival are encouraging