BackgroundCirculating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death.MethodsWe sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR-sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease.ResultsLoci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover likely releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients.ConclusionsUniversal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

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Background We previously reported the effectiveness of vedolizumab (VDZ) to induce remission in children with CD and UC enrolled in the prospective,multicenter VEDOKIDS study.In this extended analysis,we aimed to explore the effectiveness and safety of VDZ to maintain remission through week 30 Methods Children with CD or UC commenced on VDZ at any stage of the disease were followed at baseline and 2, 6, 14 and 30 weeks thereafter.Explicit demographic,clinical and safety data were prospectively recorded.The primary outcome was steroid-and EEN-free clinical remission (SFR) at 30 weeks,analyzed under the ITT principle with non-response imputation.Response was defined as change of ≥ 20 points in PUCAI or >20 points in wPCDAI and clinical remission as PUCAI<10 or wPCDAI<12.5. Adverse events (AE's) were classified as severe or non-severe and related or unrelated to VDZ Results A total of 142 children were enrolled (65 [46%] CD,77 [54%] UC;Table).At the end of the induction period,the rates of response were 75% in UC vs 64% in CD (p=0.2),of remission 52% vs 38% (p=0.1),and of SFR 47% vs 32% (p=0.08),respectively.At week 30,SFR rates were 47% in UC and 34% in CD (p=0.1);outcomes were also numerically higher in UC,but without statistical significance(Figure) Response to induction therapy predicted week 30 remission.Of the responders,19 (59%) children with CD and 31 (60%) with UC achieved SFR at week 30,while the corresponding figures for those not achieving response were 3 (12%; p=0.004) in CD and 5 (29%; p=0.03) in UC.Similarly,in multivariable model,response to induction treatment was highly associated with SFR at week 30,in both CD (OR 11.2 [95%CI 2.3-73]) and UC (OR 4.8 [95%CI 1.4-19]).Of the 22 patients who were responders but not remitters at week 14,four eventually achieved SFR at week 30 (1/10 [10%] in CD and 3/12 [25%] in UC) By week 30,159 AE's were reported by 72 children including one case of Hodgkin's lymphoma (VDZ was resumed after completing the chemotherapy);the patient was never exposed to thiopurines.No cases of progressive multifocal leukoencephalopathy or deaths were reported.Thirty-six AEs in 23 (16%) children were classified as possibly related to VDZ,11 of which (31%) were moderate and the others mild.Four children (1.4%) discontinued treatment due to AEs (one due to infusion reaction,one leukocytoclastic vasculitis,one azotemia and one due to fever and malaise Conclusion In this prospective multicenter study,VDZ was effective for maintaining remission in children with CD, and more so in UC.Contraty to common notions,children with CD not achieving complete remission by week 14 had a very low likelihood of entering remission thereafter.Safety profile was generally good except for one lymphoma case with questionable relation to VDZ

Background Mucosal healing (MH) has become a treatment goal for inflammatory bowel disease (IBD), especially for ulcerative colitis. However, MH is less feasible to assess frequently in patients with Crohn’s disease (CD). In this study, we investigated Protein Fingerprint Assays (PFA) of collagen degradation and formation, and neutrophil and macrophage activity markers in the ImageKids study as non-invasive markers of MH. Methods This is a proteomics planned sub-study of the multi-center prospective ImageKids study which included serum samples from children with CD (n=178) undergoing MRE and ileocolonoscopy concurrently (ImageKids study: NCT01881490). Serum from healthy age and gender-matched controls were obtained from BioIVT (n=83). PFA panel for mucosal damage (C1M, C3M, C4M, C6M, ELP-3), Immune cell activity panel (CPa9-HNE, VICM, C4G), and tissue fibrosis panel (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6, PRO-C11, PRO-C22) were determined. MH was classified as having an SES-CD score of 0 and global radiologist assessment of inflammation in the MRE of 0, using a visual analogue scale (VAS) 0-100mm, and mucosal damage (MD) was defined as having SES-CD or MRE VAS>0. Spearman rho correlations, one-way ANOVA with Tukey correction, and receiver operator characteristics (ROC) curves were applied for statistical analysis. Results Some of the previous biomarkers demonstrated to correlate with the SES-CD score and ulcer size (C1M, C3M, C6M, PRO-C4, VICM, PRO-C1, PRO-C3). CPa9-HNE, ELP-3, and PRO-C22 also correlate with the SES-CD score (table 1). The combined SES-CD and MRE VAS score of MH correlated positively with CPa9-HNE (r=0.30, p<0.0001), VICM (r=0.39, p<0.0001), C1M (r=0.29, p=0.0001), C3M (r=0.22, p=0.0022), C6M (r=0.35 p<0.0001), PRO-C4 (r=0.2, p<0.0001), PRO-C22 (r=0.32, p<0.0001), (figure 1 and 2) and showed significantly lower serum levels in pCD patients with MH compared to patients with mild, moderate, or severe mucosal damage (p<0.001) (figure 2). PRO-C3 correlated negatively with the combined SES-CD and MRE VAS score (r=-0.17, p=0.0211). These PFA biomarkers were combined in a multivariate regression model, demonstrating increased accuracy to identify patients in MH healing compared to the individual biomarkers (p<0.001, MH vs. MD: AUC=0.82 [CI:0.69-0.95), MH+Mild MD vs. moderate+severe MD: AUC=0.77 [0.70-0.84], (figure 3). Conclusion PFA biomarkers of Neutrophil activity (CPa9-HNE), Macrophage activity (VICM), mucosal damage (C1M, C3M, C4M, C6M, ELP-3), and tissue fibrosis (PRO-C3, PRO-C22) are potential surrogate biomarkers for identifying pCD patients with mucosal healing and for monitoring of mucosal damage and stenosis.