Salivary Zymogen Granule Protein 16B Drops at Onset of Chronic Graft-Versus-Host Disease: A Possible Salivary Biomarker for Oral Chronic Graft-Versus-Host Disease

Objective Chronic graft-versus-host disease (cGVHD) targets the oral mucosa and salivary glands in 30-70% of cGVHD patients after allogeneic hematopoietic stem cell transplant (HSCT). In the absence of an incisional biopsy, no diagnostic test exists to confirm clinical suspicion of cGVHD processes in oral tissues. Consequently, therapy is often withheld until severe manifestations, such as ulceration, develop. This proteomic study examined whole saliva for the presence of a diagnostic biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Methods Patients from NIH protocols (NCT00331968, NCT00520130, or NCT01851382) were sampled at oral cGVHD onset. All cases had biopsy-proven oral GVHD. Five-minute unstimulated whole saliva from 3 individual oral cGVHD patients, 3 post-transplant controls and 2 healthy volunteers was processed, iTRAQ-tagged, HPLC-fractionated, and analyzed via liquid chromatography coupled tandem mass spectrometry. High confidence protein identifications were tested using a Kruskal-Wallis test with Bonferroni correction. Selected proteins were validated by Western blot (WB) using two independent cohorts.  Cohort 1:  post-HSCT patients with (n=10) and without (n=10) oral cGVHD; Cohort 2: post-HSCT patients with (n=12) and without (n=12) oral cGVHD, controlled for recipient sex and prior total-body irradiation. Fluorescent immunohistochemistry (IHC) was used to further analyze zymogen granule protein 16B (ZG16B) in patient labial minor salivary glands (MSG). Results The discovery dataset contained 569 confidently-identified proteins, with 77 significantly differentially expressed at onset of cGVHD. A 6-protein panel was selected for WB validation in two independent sample cohorts; proteins were selected based on Ingenuity Pathway Analysis, biological function, and protein level. In WB analysis, MMP9, Ezrin, PIP and alpha-1-antichymotrypsin displayed similar abundance levels as in the iTRAQ screen but were not significantly different. ZG16B, a secretory lectin protein associated with the tear release in the lacrimal gland, was reduced 2-fold in saliva of post-HSCT patients with oral cGVHD (898.1 ± 371.8 AU) compared with post-HSCT patients without oral cGVHD (1905 ± 598.6 AU; p  Conclusion Taken together, results indicate that ZG16B is present in lower amounts in unstimulated whole saliva and within the salivary gland acinar units at oral cGVHD onset. Reduction in this protein may indicate cGVHD activity and/or salivary gland damage and further investigation of its biological function and potential utility as a clinical biomarker of oral cGVHD onset is needed.