Development and Validation of a Bronchoalveolar Lavage Genomic Classifier for Acute Cellular Rejection

Methods

We performed RNA-seq on 806 BAL-cp from 181 lung transplant recipients enrolled in CTOT-20. Differential expression was based on fold difference >2.0 and False Discovery adjusted p-value <0.05. Samples were randomly split 80:20 into training and testing sets.

A Random Forest model was optimized for area under the curve (AUC), and the threshold for genomic ACR was selected for classification accuracy. We validated performance in an independent single-center cohort. Cox models evaluated risk for CLAD.

Findings: From 37 cases and 151 controls, we identified 62 ACR genes, indicating upregulation of T-cell receptor signaling, and downregulation of CTLA4 signaling in cytotoxic lymphocytes, among other enriched pathways. A 31-gene Random Forest model’s AUC was 0.99 (SE 0.0053) in the training set, and 0.72 (SE 0.0874) in the test set. At a probability threshold of 0.396, accuracy for distinguishing clinically significant ACR cases from stable controls was 93.1% (specificity 95.4%, sensitivity 83.8%).

In the independent validation cohort, accuracy was 82.1% (specificity 87.5%, sensitivity 73.3%). The model classified 138 (17.1%) CTOT-20 samples as genomic ACR. Late genomic ACR (≥90 days posttransplant) associated with increased CLAD risk (HR 2.52, 95% CI 1.47 - 4.34, p<0.001).

Interpretation: A BAL-cp genomic classifier can identify ACR, predict CLAD risk, and may be a less invasive alternative to TBB after lung transplant.