Molecular Assessment (RNA-sequencing) of CLAD in Lung Transplant Biopsies: A Pilot Study

Abstract

Purpose Lung transplantation (LTx) is the only effective therapeutic option for end-stage lung diseases. Chronic lung allograft dysfunction (CLAD) has been recognized as a heterogeneous condition. The understanding of its pathophysiology has evolved over time and the exact pathogenetic mechanisms of CLAD are still unclear. During the last decade, different high-throughput approaches have occasionally been used to unveil CLAD characteristics, and the majority have investigated transcripts in blood or bronchoalveolar lavage. The main goal of the study was to identify crucial gene transcripts in the development of CLAD. Methods Formalin-fixed paraffin-embedded (FFPE) transbronchial biopsies (TBBs) obtained from 18 cystic fibrosis patients undergoing LTx from 2001 to 2016 were analyzed. RNA sequencing (RNA-seq) was performed in 40 scheduled FFPE TBBs with the diagnosis: no-rejection (13 cases), acute cellular rejection (6 cases) and CLAD (11 cases); 10 cadaveric lung donors were used as normal tissue. Validation using real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) was also carried out using larger numbers of cases (45 samples for real-time PCR and 38 for IHC) to confirm emerging markers detected by RNA-seq. Results In all cases RNA showed adequate quantity and quality for RNA-seq. The gene expression analysis of CLAD vs acute rejection samples revealed three different networks with 33, 26, and 36 differentially expressed genes (DEGs), of which 18, 12, and 18 genes, respectively, were up-regulated. Among these, eight genes (CCL11, LCN2, MT-CO2, SCARA3, CX3CL1, CXCL12, MUC4 and MUC13) were selected being biologically plausible that they may be linked to CLAD. Their overexpression was confirmed by real-time PCR and IHC. Conclusion This is the first study that demonstrates the feasibility of omic technology RNA-seq in archival FFPE TBBs coming from a homogeneous LTx population. Our data revealed different molecular profiles of CLAD in lung tissue, routinely evaluated for histology. An ongoing analysis of additional cases, also including other LTx centers, to verify the present preliminary study will be presented at an upcoming scientific conference. These results along with standardized clinical and pathologic information will allow us to implement sophisticated bioinformatics aimed at more appropriate patient clustering.