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Novel Biomarkers of Necrotizing Enterocolitis in Neonates Undergoing Congenital Heart Disease Surgery: A Pilot Cohort Study

2023; Wiley; Volume: 12; Issue: 15 Linguagem: Inglês

10.1161/jaha.123.030712

2047-9980

Eiman A. Ali, Aneesa Syed, Ludmila Khailová, John Iguidbashian, Alejandro Suarez‐Pierre, Jelena Klawitter, Matthew L. Stone, Christopher A Mancuso, Benjamin S. Frank, Jesse Davidson,

Neonatal Respiratory Health Research

HomeJournal of the American Heart AssociationAhead of PrintNovel Biomarkers of Necrotizing Enterocolitis in Neonates Undergoing Congenital Heart Disease Surgery: A Pilot Cohort Study Open AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessLetterPDF/EPUBNovel Biomarkers of Necrotizing Enterocolitis in Neonates Undergoing Congenital Heart Disease Surgery: A Pilot Cohort Study Eiman A. Ali, Aneesa Syed, Ludmila Khailova, John P. Iguidbashian, Alejandro Suarez‐Pierre, Jelena Klawitter, Matthew Stone, Christopher A. Mancuso, Benjamin S. Frank and Jesse A. Davidson Eiman A. AliEiman A. Ali , Children's Hospital Colorado, , Research Institute, , Aurora, , CO, , USA, , Aneesa SyedAneesa Syed https://orcid.org/0009-0006-8925-9319 , Children's Hospital Colorado, , Research Institute, , Aurora, , CO, , USA, , Ludmila KhailovaLudmila Khailova , University of Colorado‐School of Medicine, , Department of Pediatrics, , Aurora, , CO, , USA, , John P. IguidbashianJohn P. Iguidbashian https://orcid.org/0000-0002-5974-7876 , University of Colorado‐School of Medicine, , Department of Surgery, , Aurora, , CO, , USA, , Alejandro Suarez‐PierreAlejandro Suarez‐Pierre https://orcid.org/0000-0003-1821-6240 , University of Colorado‐School of Medicine, , Department of Surgery, , Aurora, , CO, , USA, , Jelena KlawitterJelena Klawitter , University of Colorado‐School of Medicine, , Department of Anesthesiology, , Aurora, , CO, , USA, , Matthew StoneMatthew Stone , University of Colorado‐School of Medicine, , Department of Surgery, , Aurora, , CO, , USA, , Christopher A. MancusoChristopher A. Mancuso https://orcid.org/0000-0003-3081-2758 , University of Colorado‐School of Public Health, , Department of Biostatistics and Informatics, , Aurora, , CO, , USA, , Benjamin S. FrankBenjamin S. Frank https://orcid.org/0000-0002-2227-6265 , University of Colorado‐School of Medicine, , Department of Pediatrics, , Aurora, , CO, , USA, and Jesse A. DavidsonJesse A. Davidson *Correspondence to: Jesse Davidson, MD, Children's Hospital Colorado, 13123 E 16th Ave, ‐Box 100, Aurora, CO 80045. Email: E-mail Address: [email protected] https://orcid.org/0000-0001-9193-9803 , University of Colorado‐School of Medicine, , Department of Pediatrics, , Aurora, , CO, , USA, Originally published25 Jul 2023https://doi.org/10.1161/JAHA.123.030712Journal of the American Heart Association. 2023;0:e030712Congenital heart disease (CHD) is a risk factor for the development of necrotizing enterocolitis (NEC).1 NEC has an incidence of 3% to 5% in the population with CHD and is associated with a 2‐fold increase in mortality (24.4% versus 11.8% in neonates with CHD without NEC), and a 3‐fold increase in length of stay (54 versus 18 days).1, 2 Although poorly understood, cardiac NEC appears to be a distinct entity from NEC of prematurity.1, 3 Biomarkers capable of predicting NEC in patients with CHD are lacking. One candidate is intestinal fatty acid binding protein (iFABP/FABP2). iFABP is highly expressed in the intestinal epithelium and is involved in lipid metabolism, control of inflammation, cell proliferation, and barrier function.1, 3 Circulating iFABP increases immediately after infant cardiac surgery and higher immediate postoperative levels are associated with increased risk of subsequent NEC.4 iFABP's utility as a biomarker for the detection of acute NEC or the response to treatment has not been established. Furthermore, the systemic response to NEC is poorly defined. This pilot study sought to evaluate changes in iFABP levels and the circulating proteome associated with the onset of NEC and the natural history of iFABP levels during treatment.This pilot study is part of a parent study on the metabolic response to CHD surgery (R01HL156936). The data supporting this study are available from the corresponding author upon reasonable request. The Colorado Multiple Institution Review Board approved this study. Written informed consent was obtained from the subjects' parents. Neonates undergoing cardiothoracic surgery with cardiopulmonary bypass were prospectively enrolled. NEC was defined as Bell's staging ≥1A/B. Plasma samples (10–40 μL) were collected from routine clinical chemistries on the first day of acute NEC symptoms and on day 5 of treatment (NEC days 1 and 5 respectively). Treatment was based on local NEC clinical protocols. For patients without a NEC diagnosis, remnant plasma samples from postoperative day 7 served as controls. Samples were stored at –80 °C for batch analysis. Proteomic analysis of 1536 proteins (including iFABP) was performed using Olink Explore (Olink, Uppsala, Sweden). Proteins were log2 transformed for analysis. The primary outcome was the difference in iFABP levels between patients with NEC on NEC day 1 and controls on postoperative day 7 (t test; significance exact P<0.05). Secondary outcomes included change in iFABP from NEC days 1 to 5 and exploratory proteomic analysis to identify proteins demonstrating at least a 2‐fold difference between patients with and without NEC at an exact P value<0.1. Proteins meeting the outlined statistical significance were then expanded using GenePlexus,5 a network‐based protein discovery tool. The resulting proteins were entered into Metascape for exploration by tissue of origin (enrichment analysis via PaGenBase), and STRING for protein–protein network analysis (significance=false discovery rate<0.05, Benjamini–Hochberg procedure).Ten neonates with comparable high‐risk cardiac surgeries were included (characteristics shown in Figure [A]). Three subjects developed NEC (postoperative days 20, 33, and 34), with 1 subject experiencing a second episode of NEC (postoperative day 50). Seven subjects did not develop NEC. iFABP levels at the onset of NEC were significantly higher compared with the non‐NEC group (Figure [B]). iFABP levels then trended down between NEC day 1 and day 5, decreasing significantly in survivors (P=0.02) but increasing in a single subject who died 10 days later. Proteomic analysis identified 246 proteins that were significantly dysregulated (Figure [C]). Of these proteins, 229 were upregulated in NEC patients and 17 were downregulated. The primary tissues of origin were the colon and small intestine (P<1.6×10−6). Protein–protein interaction analysis demonstrated three predominant protein clusters (Figure [D]) centered around (1) innate immune response/cytokine production (false discovery rate =8.76×10−20), (2) maintenance of gastrointestinal epithelium (false discovery rate =0.002), and (3) angiogenesis and endothelial migration (false discovery rate =0.00042).Download figureDownload PowerPointFigure 1. Protein biomarkers of cardiac NEC (necrotizing enterocolitis).A, Subject characteristics. B, Comparison of iFABP levels on NEC day 1 (green) vs controls (red). Comparison of iFABP levels on NEC day 1 (green) vs day 5 (blue). Red arrow=trajectory of iFABP in the single nonsurvivor. C, Volcano plot analysis of differentially expressed proteins between NEC patients (NEC day 1) and controls; red: ≥2‐fold increase (P<0.1) in NEC cases vs controls, blue: ≥50% decrease (P<0.1) in NEC cases compared with controls. Significant proteins are annotated with their HUGO Gene Nomenclature. D, Protein clusters showing extensive immune system upregulation (blue), angiogenesis and endothelial migration (green), and disruption in proteins involved in maintaining the gastrointestinal epithelium (red). AVSD indicates atrioventricular septal defect; DORV, double outlet right ventricle; HLHS, hypoplastic left heart syndrome; iFABP, intestinal fatty acid binding protein; NPX, normalized protein expression (log2 scale); TAPVR, total anomalous pulmonary venous return; TGA, transposition of the great arteries; and VSD, ventricular septal defect.This pilot analysis provides the first systematic exploration of the widespread changes in circulating proteins associated with cardiac NEC. iFABP shows promise as a diagnostic biomarker in this population, along with its potential for treatment monitoring and prognosis. In addition, this study provides preliminary biomarker evidence for systemic immune upregulation, gastrointestinal epithelial disruption, angiogenesis, and endothelial migration in neonates who develop cardiac NEC. Future studies will focus on (1) evaluating iFABP as a diagnostic/therapeutic monitoring biomarker in a large validation cohort, (2) assessing if a panel of our top performing biomarkers can improve diagnostic accuracy over iFABP alone, and (3) determining if NEC phenotypes (eg, ischemic versus allergic) demonstrate distinct proteomic signatures that can be used to personalize therapy and inform mechanistic studies.Several limitations should be considered. First, the study is underpowered to assess biomarker changes associated with severity of illness or response to therapy. Second, biomarker sampling began on day 1 of clinical NEC so we cannot determine if biomarker changes preceded clinical symptoms. These limitations will be addressed in future studies by expanding the cohort size and frequency of longitudinal sampling with the goal of developing a clinical biomarker panel for cardiac NEC and advancing our understanding of the pathogenesis of this complex disease.Sources of FundingThis work was supported by the National Institutes of Health/National, Heart, Lung, and Blood Institute R01HL156936 (PI Davidson). No relationship with industry exists for this study.DisclosuresNone.Footnotes*Correspondence to: Jesse Davidson, MD, Children's Hospital Colorado, 13123 E 16th Ave, ‐Box 100, Aurora, CO 80045. Email: jesse.[email protected]orgThis article was sent to Amgad Mentias, MD, Associate Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 3.References1 Roychaudhuri S, Grewal G, Vijayashankar SS, Lavoie P, Maheshwari A. Necrotizing enterocolitis associated with congenital heart disease‐a review article. Newborn (Clarksville).2022; 1:170–176. doi: 10.5005/jp-journals-11002-0016CrossrefMedlineGoogle Scholar2 Spinner JA, Morris SA, Nandi D, Costarino AT, Marino BS, Rossano JW, Shamszad P. Necrotizing enterocolitis and associated mortality in neonates with congenital heart disease: a multi‐institutional study. Pediatr Crit Care Med. 2020; 21:228–234. doi: 10.1097/PCC.0000000000002133CrossrefMedlineGoogle Scholar3 Burge KY, Gunasekaran A, Makoni MM, Mir AM, Burkhart HM, Chaaban H. Clinical characteristics and potential pathogenesis of cardiac necrotizing enterocolitis in neonates with congenital heart disease: a narrative review. J Clin Med. 2022; 11:3987. doi: 10.3390/jcm11143987CrossrefMedlineGoogle Scholar4 Watson JD, Urban TT, Tong SS, Zenge J, Khailova L, Wischmeyer PE, Davidson JA. Immediate post‐operative enterocyte injury, as determined by increased circulating intestinal fatty acid binding protein, is associated with subsequent development of necrotizing enterocolitis after infant cardiothoracic surgery. Front Pediatr. 2020; 8:267. doi: 10.3389/fped.2020.00267CrossrefMedlineGoogle Scholar5 Liu R, Mancuso CA, Yannakopoulos A, Johnson KA, Krishnan A. Supervised learning is an accurate method for network‐based gene classification. Bioinformatics. 2020; 36:3457–3465. doi: 10.1093/bioinformatics/btaa150CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails Article InformationMetrics Copyright © 2023 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.https://doi.org/10.1161/JAHA.123.030712PMID: 37489765 Manuscript receivedApril 21, 2023Manuscript acceptedJune 29, 2023Originally publishedJuly 25, 2023 Keywordsintestinal fatty acid binding proteininflammationproteomicsNECsingle ventricle heart diseasecardiopulmonary bypassPDF download SubjectsBiomarkersClinical StudiesCongenital Heart DiseaseInflammationProteomics