Chronic oral Graft-versus-Host Disease: clinical risk factors and biochemical markers of disease activity
AuthorHull, Katrusha Mihalyna
AffiliationMelbourne Dental School
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Katrusha Mihalyna Hull
Purpose: Allogeneic hematopoietic stem cell transplantation (HSCT), along with the associated immunosuppression required post transplant, is associated with several potentially debilitating long-term complications, one of the most significant being Graft-versus-Host Disease (GvHD). The oral cavity is one of the most commonly affected sites in the GvHD spectrum causing a detrimental impact on oral health, function and quality of life. As outlined by the National Institutes of Health (NIH), clinical manifestations are the mainstay in the diagnosis of oral chronic GvHD, however clinical manifestations are often non-specific and diagnosis is frequently complicated by both inter and intra-examiner variability. Objective, validated methods for diagnosis and monitoring are limited. Oral incisional biopsy is still the most ideal method for diagnosis and monitoring of disease progression, however repetitive oral tissue biopsies during an often protracted clinical course are also not practical. Although candidate serum biomarkers are being explored, limited information is available on the expression of cytokines in the tissues involved in oral chronic GvHD. Identification of reliable local biomarkers that reflect disease presence and are readily available by non-invasive means, would facilitate both the management and understanding of oral chronic GvHD. Oral chronic GvHD is the focus of this thesis, specifically its clinical presentation, the risk factors for development and the identification of potential locally measurable biomarkers which differentiate oral chronic GvHD from other, similar presenting, oral conditions. Materials and Methods: Forty-eight patients, eighteen years of age or older, planned for allogeneic HSCT were recruited from June to December 2013 across two bone marrow transplant units. Patients were examined prior to transplantation as well as at Days 100, 180 and 270. At each study time point patients were required to complete a series of mouth specific questionnaires including visual analogue scales for oral pain, dryness and sensitivity, a Xerostomia Inventory (XI) and Oral Health Impact Profile. Oral examination included visual assessment of the oral and peri-oral tissues and periodontal assessment via CPITN scoring. Location and extent of oral mucosal pathology was recorded diagrammatically; each feature scored mild, moderate or severe utilising the modified Schubert Oral Mucositis Rating Scale (Pavletic, Martin et al. 2006). Patients were given 10 mls of deionized water and asked to vigorously swish the solution for 45 seconds. Mouth rinse samples were centrifuged to obtain a supernatant and cell pellet. The BD™ cytometric bead array (CBA) human Th1/Th2/Th17 cytokine kit (Catalogue 5600484) was used to measure the concentration of chronic inflammatory cytokines in salivary supernatant, specifically IL-2, IL-4, IL-6, IL-10, INF-γ, TNF and IL-17A. The cellular phase of saliva was re-suspended with RNA fixative (TRIzol) and total RNA was isolated using the PureLink® RNA mini kit. Gene expression of IL-6 in the cellular phase of saliva was established via real-time polymerase chain reaction (RT-PCR). Eleven patients with established oral chronic GvHD along with five patients of each of the following diagnoses, oral lichen planus, Sjögrens Syndrome and temporomandibular disorders, were recruited from the Oral Medicine Department of the Royal Dental Hospital of Melbourne. Salivary rinse samples were used to measure the concentration of the same panel of chronic inflammatory cytokines in oral chronic GvHD patients with comparison to those measured in the other, common oral mucosal conditions. Results: Over the 9 month post-transplant study period six patients developed oral chronic GvHD and a further ten non-oral chronic GvHD. Elevated pre-transplant XI scores were identified in patients who would go on to develop oral chronic GvHD, patients on average scoring 8 points higher than those who would develop non-oral chronic GvHD and six points higher those who would not develop any chronic GvHD. Analysis of the oral rinse samples from patients with oral chronic GvHD patients demonstrated a positive association between disease presence and the concentration of both IL-6 and TNF (p=0.007, p=0.034 respectively) and a negative association with IL-17 and IFN (p=0.001, p=0.015 respectively). IL-6 was also found to be associated with the clinical presentation of ulceration and erythema (p=0.003, p<0.001 respectively), oral symptom scores and oral-health related quality of life. RNA of sufficient quantity and quality was able to be extracted from the cellular phase of saliva. A significant positive correlation was shown between IL-6 gene expression in the cellular phase of oral rinse samples and the level measured within the supernatant (p=0.035). This significant association was not replicated when disease status was considered. Significant differences were also shown between the cytokine profile measured in the saliva of oral chronic GvHD patients and those measured in other, common mucosal diseases. Specifically raised IL-6, IFN- γ and TNF combined with a marked absence of IL-4 and IL-2. Conclusions: Findings support the hypothesis that a unique cytokine profile is measurable in the local environment (saliva) of oral chronic GvHD patients. No one cytokine was a clear marker for oral chronic GvHD and accurately distinguished between all other conditions tested, however when changes across the entire panel of cytokines were assessed a unique combination of changes was reflective of the presence of oral chronic GvHD disease. A positive association was seen between oral chronic GvHD and salivary IL-6 and TNF and a negative trend with salivary IL-17 and IFN. IL-6 was shown to be of specific interest reflective of both the symptom profile and clinical features of oral chronic GvHD. The outcomes of this study also showed that the cellular phase of saliva can be used to extract RNA of suitable quality for downstream application and that IL-6 gene expression within the cellular phase of saliva is correlated with IL-6 concentration within the associated salivary supernantant suggesting local production. Generalisation of these results is however limited by both the small sample size and reduced observation period. Findings provide the basis for further study. Should these findings be replicated this may provide the basis for investigation of the utility of specific therapies targeted at IL-6 in the management of oral chronic GvHD as an adjunct or alternative to the use of systemic immunosuppression.
Keywordsoral; graft-versus-host disease; transplantation
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