Proteomic Analysis of Saliva from Patients with Oral Chronic Graft-Versus-Host Disease

Abstract

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa including the salivary glands is affected in the majority of cGVHD patients; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from oral cGVHD(+) and oral cGVHD(-) patients using iTRAQ (isobaric Tags for Relative and Absolute Quantification) labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 proteins exhibited altered expression in oral cGVHD patients compared to allo-HSCT patients without oral cGVHD. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that two of these proteins, IL-1 receptor antagonist and Cystatin B, showed decreased expression in patients with active oral cGVHD (P < 0.003). Receiver Operator Characteristic analysis revealed that these two markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients studied within 12 months of allo-HSCT transplantation (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, anti-microbial defense and tissue protection in oral cGVHD that may also reflect changes in salivary gland function and damage to the oral mucosa.

NIH Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. The 2014 Pathology Working Group Report

“…Involvement of the oral mucosa did not correlate with salivary dysfunction [107]. In the future, quantitative proteomic analysis of saliva may be added as a biomarker to identify active oral chronic GVHD, especially in newly diagnosed patients within the first 12 months after HCT [108]. Quantitative proteomic analysis of saliva from patients with and without oral chronic GVHD demonstrated altered expression in the chronic GVHD group with decreased IL-1 antagonist receptor and Cystatin B compared to the non-GVHD group.…”

Biology of chronic graft-vs-host disease: Immune mechanisms and progress in biomarker discovery

“…Two recent studies utilizing mass spectrometry approaches identified a total of 82 and 102 salivary proteins, respectively, that showed altered expression in oral cGVHD[80,81]. IL-1 receptor antagonist (IL-1ra) exhibited reduced expression in patients with oral cGVHD[80].…”

“…IL-1 receptor antagonist (IL-1ra) exhibited reduced expression in patients with oral cGVHD[80]. The changes in IL-1ra expression coupled with higher levels of IL-1 family cytokines[16] in saliva likely enhance oral inflammation and subsequent tissue damage.…”

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

“…The study used LC-MS/MS labeled free quantification for achieving the results [15]. Alteration of proteins associated with immune response was also found in another study performed with isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass spectrometry (MS/MS) [16]. Additionally, two downregulated proteins were proposed as potential biomarkers: IL-1 receptor antagonist and cystatin B.…”