Gene expression-based predictive models of Graft versus Host Disease-associated Dry Eye

Abstract

Purpose: To develop a predictive model based on mRNA expression of inflammatory molecules in conjunctival cells of Graft versus Host Disease (GvHD)-associated Dry Eye (DE) patients, as well as to find meaningful correlations between gene signals and clinical signs.

Methods: 20 GvHD-DE patients and 14 healthy controls (similar age and gender distribution) were recruited. Patients had discontinued medications for one week before examination. DE-related symptoms and signs were recorded, and conjunctival epithelial cells were collected by impression cytology after spending 20 min under standard indoor conditions within The Controlled Environmental Research Laboratory (CERLab). Gene expression of inflammatory molecules was determined in the extracted epithelial cells by array-PCR (Polymerase Chain Reaction) and results correlated with clinical signs. Shrinkage discriminant analysis (SDA), Support Vector Machine (SVM) and k-Nearest neighbor classifier (kNN) methods were used to develop predictive models, which were validated considering accuracy, calibration, and discrimination capability.

Results: Out of the 84 genes analyzed, 34 showed significant differences in expression. IL-6, IL-9, CCL24, CCL18, IL-10, IFN-γ, and CCL2 were highly increased (>6 fold), 26 genes were moderately up-regulated (2-6 fold), whereas EGFR was down-regulated (2.63 fold) in GvHD-DE samples compared to controls. A panel based on EGFR, IL-6, IL-9, and NAMPT obtained an area under the ROC curve of 0.994, a sensitivity of 100%, and a specificity of 92.86%. EGFR expression correlated negatively with ocular surface damage markers, while IL-6, IL-9 and NAMPT correlated positively.

Conclusions: Several genes encoding for inflammation-related molecules were differently expressed in conjunctival epithelial cells of GvHD-DE. Particularly EGFR, IL-6, IL-9 and NAMPT had the greater potential as diagnostic biomarkers, as they showed not only excellent sensitivity and specificity but a clinically relevant association with the lacrimal functional unit status of GvHD patients and healthy individuals.