Quantitative salivary proteomic differences in oral chronic graft-versus-host disease

Abstract

Purpose: Chronic graft-versus-host disease (cGVHD) is a severe immunological complication that occurs after allogeneic hematopoietic stem cell transplantation (HSCT). Although oral cGVHD occurs in >25% of cGVHD patients and leads to decreased quality of life, its etiology is poorly understood. The present retrospective cross-sectional analysis of oral cGVHD patients sought to (1) test the feasibility of liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify protein biomarkers of oral cGVHD and (2) to gain a clearer understanding of salivary proteins impacted by oral cGVHD.

Methods: Using unstimulated whole saliva, we compared pooled saliva from five patients with a diagnosis of moderate or severe oral cGVHD, with a gender-and age- matched pool of five cGVHD patients with no oral mucosal findings. LC-MS/MS was used to identify salivary proteins, followed by Ingenuity Pathway Analysis (IPA). Selected mass spectrometric findings, including lactotransferrin, lactoperoxidase, and albumin, were confirmed by targeted label-free quantification.

Results: LC-MS/MS led to confident identification of 180 proteins. Of these proteins, 102 changed in abundance at least 2 fold, including 12 proteins identified only in the No oral cGVHD group. Downregulation of ~0.4 fold was confirmed for both lactotransferrin and lactoperoxidase in Oral cGVHD saliva using targeted label-free quantification. IPA analysis implicated pathways involved in cellular metabolism and immunoregulation.

Conclusions: Reduction of salivary lactoperoxidase, lactotransferrin, and several cysteine proteinase inhibitor family proteins suggests impaired oral antimicrobial host immunity in cGVHD patients. This shotgun proteomic analysis of oral cGVHD saliva using targeted label-free quantification of select proteins supports the use of mass spectrometry for future validation in a large patient population as noninvasive tests for screening, early detection, and monitoring of cGVHD.

Fig. 1

Clinical and histological presentation of no oral cGVHD (a–d) versus oral cGVHD (e–h). Clinical presentation of oral cGVHD includes atrophy, erythema, hyperkeratosis, lichenoid reactions, ulcerations, and edema, here seen on the lower lip (e), maxillary anterior gingiva (f), and buccal mucosa (g) of a patient with oral cGVHD, as compared with the normal intraoral mucosa of a patient with no oral cGVHD (a–c). H&E staining of the oral mucosa from the above-matched patients demonstrates characteristic GVHD-like changes in the buccal mucosa compared with No oral cGVHD tissue (d), including prevalent lymphocytic infiltrate and apoptotic bodies (h). Buccal biopsy sections are shown in similar orientation at 10x magnification

Fig. 2

Study Workflow. The diagram overviews the procedures used for the identification and quantification of differentially expressed salivary proteins for No oral cGVHD and Oral cGVHD subjects

Fig. 3

Distribution of differentially expressed proteins. Differentially expressed proteins (n = 180) were categorized using an arbitrary fold ratio change cut-off of ≤ 0.5 or ≥ 2 into proteins that did not change in abundance (n = 78), proteins that were identified only in the No oral cGVHD group (n = 12), and proteins that were down-regulated in the Oral cGVHD samples (n = 90). The number of proteins in each pathway is given above each bar

Fig. 4

Canonical Pathways identified by Ingenuity Pathway Analyses (IPA). Significant pathways (n = 12) were mapped via identified down-regulated proteins. Values on each the y-axis indicate the number of involved proteins in each pathway

Fig. 5

Label-Free Quantification of Selected Differentially Expressed Proteins between No oral cGVHD and Oral cGVHD groups. Shown are the extracted ion chromatograms showing “peptide elution plotted against intenity” for (a) lactotransferrin, and (b) lactoperoxidase by oral cGVHD classification to illustrate downregulation of each protein in the Oral cGVHD group