INTEGRATIVE TRANSCRIPTOMIC AND METABOLOMIC STUDY OF PATIENTS WITH CHRONIC LUNG ALLOGRAFT DYSFUNCTION (CLAD)

Background Chronic lung allograft dysfunction (CLAD) represents the principal barrier to long-term survival after lung transplantation, developing in approximately half of recipients within five years. Despite extensive clinical monitoring, diagnosis often occurs only after irreversible graft injury has developed. Over the past decade, high-throughput omics technologies have provided new insights into the molecular networks driving CLAD, particularly from studies on blood and bronchoalveolar lavage (BAL) samples. Yet, the heterogeneity of patient cohorts, methodological variability, and limited sample numbers have constrained the discovery of reproducible and clinically useful biomarkers. The present study employs an integrative multi-omics framework applied to transbronchial biopsies (TBB) to uncover signatures of CLAD, thereby contributing to a deeper understanding of disease pathogenesis and providing insights potentially predictive of response to the target therapy. Materials and Methods We conducted a bicentric retrospective study including 107 TBB from 67 lung recipients transplanted for cystic fibrosis (2006-2020). RNA-sequencing was performed on 47 TBB showing chronic rejection (Obliterative bronchiolitis ,histological signs of restrictive allograft syndrome, or mixed) and compared with 10 without any post-transplant complications (NR) and 10 with acute cellular rejection (ACR). Differentially expressed genes (DEG) were identified and candidate genes were validated by Real Time PCR and immunohistochemistry (IHC) and evaluated longitudinally in 21 CLAD patients. Untargeted metabolomics was conducted on BAL from 33 patients by high-resolution mass spectrometry, followed by integrative multi-omics analysis. Results RNA-seq revealed 309 and 285 DEGs in CLAD vs NR and CLAD vs ACR, respectively, mainly involved in immune regulation and fibrogenesis. Four genes (P3H1, CDH2, CXCR2, TREML1) were identified as crucial and validated by Real Time PCR and IHC. Metabolomics identified >50 deregulated metabolites, with higher acetyl-L-carnitine and carnitine, and lower tryptophan and indole-3-acrylic acid in CLAD, indicating altered fatty acid oxidation and kynurenine pathway activity. No shared transcriptomic-metabolomic pathways were observed. Conclusions This multi-omics study identified and validated transcriptomic and metabolic biomarkers of CLAD, underscoring its biological and morphological heterogeneity. The overexpression of genes involved in EMT and fibrogenesis, together with the dysregulation of genes related to the innate immune response, are crucial in disease development, and some of these changes appear to occur early in the course of the disease. Validation in a larger longitudinal cohort could confirm the presence of molecular alterations acting as precursors of tissue injury and facilitate an earlier diagnosis of the disease.