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Proteomic analysis of saliva from patients with oral chronic graft-versus-host disease.

Devic I1, Shi M1, Schubert MM2, Lloid M3, Izutsu KT4, Pan C1, Missaghi M4, Morton TH4, Mancl LA4, Zhang J1, Presland RB5.

Biol Blood Marrow Transplant. 2014 Jul;20(7):1048-55.

1Department of Pathology, University of Washington, Seattle, Washington and

2Department of Oral Medicine, University of Washington, Seattle, Washington; Seattle Cancer Care Alliance, Seattle, Washington; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington and

3Department of Oral Medicine, University of Washington, Seattle, Washington; Seattle Cancer Care Alliance, Seattle, Washington and

4Department of Oral Health Sciences, University of Washington, Seattle, Washington and

5Department of Oral Health Sciences, University of Washington, Seattle, Washington; Division of Dermatology, Department of Medicine, University of Washington, Seattle, Washington. Electronic address: [email protected]

 

 

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 patients with cGVHD; 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 patients with and without oral cGVHD-cGVHD(+) and cGVHD(-), respectively-using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in theoral cGVHD(+) group compared with the cGVHD(-) group. 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 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 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 evaluated within 12 months of allo-HSCT (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, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa.

Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

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Significance Statement:

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), affecting 30-70% of patients who survive more than 100 days.  Typically, allo-HSCT is used as a curative treatment for blood diseases such as leukemia, but this stem cell-based procedure has also been used in clinical trials for other chronic diseases such as multiple sclerosis.  Chronic GVHD (cGVHD) is a long term, often life-threatening inflammatory disease that commonly affects a number of tissues particularly the skin, mouth, liver and eyes.  In order to develop a molecular profile of oral cGVHD, we used a quantitative mass spectrometry method to compare the proteomic profile of saliva pooled from allo-HSCT patients with or without oral cGVHD. We identified a total of 82 salivary proteins that changed significantly in expression as a result of oral cGVHD.  Two proteins with known roles in immunity and tissue protection, IL-1 receptor antagonist and cystatin B, were further validated by ELISA immunoassay as being significantly downregulated in oral cGVHD patients compared to controls.  Our studies provide further insight into the molecular pathways involved in chronic GVHD, and add new proteins to the list of potential salivary and serum biomarkers that have been identified for this chronic inflammatory disease.

Figure Legend

The Figure shows a biopsy of a minor salivary gland from a patient with Graft-versus-Host Disease. The immunolabeled cells are CD4-positive T-lymphocytes that have infiltrated the tissue (image courtesy of Dr. Thomas Morton).

 

 

 

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