Enterocytes Of Patients With Uncontrolled Acute Graft Versus Host Disease Of The Gut Undergo Massive Telomere Shortening Compared To Unaffected Controls
Authors: Hummel Sebastian , Heudobler Daniel , Huber Elisabeth , Ventura Ferreira Monica S , Fahrenkamp Dirk , Schmid Karin , Gremse Felix , Müller-Newen Gerhard , Ziegler Patrick , Jost Edgar , Brümmendorf Tim H. , Holler Ernst , Beier Fabian 
Published: October 2019

Introduction
Acute graft versus host disease of the gut (agGVHD) is caused by a T-cell mediated attack against the gut epithelia and remains one of the complications with the highest mortality after allogenic stem cell transplantation (allo-SCT). In particular, patients not responding to the initial immunosuppressive therapy during the first weeks have a poor outcome and frequently do not show complete recovery of their symptoms. The underlying mechanisms of refractory agGVHD are not fully understood. Telomere length (TL) reflects the replicative history of a cell. Reaching a critically short TL, cells stop dividing and enter cellular senescence. The aim of the study was to elucidate the role of telomere-mediated replicative exhaustion of enterocytes in affected patients with agGVHD compared to unaffected control patients after allo-SCT.

Methods
The study was carried out in a single-blinded fashion including 13 patients with refractory agGVHD and 7 patients undergoing allo-SCT, but without clinical history of GVHD. Proliferation score of gut enterocytes was assessed in a semi quantitative manner using MIB-1 immunohistochemistry staining. The TL of the enterocytes obtained from paraffin-embedded diagnostic colonoscopy/sigmoidoskopy biopsies were analyzed using confocal quantitative telomere FISH (cqFISH). Patients were matched according to age, clinical characteristics and onset of GVHD.

Results
Patients with agGVHD showed a significantly increased proliferation score (median: 2.0, range: 0.5-3.0, n= y, p=0.04) compared to the control group (median: 1.0, range: 0.5-2.0, n=y). TL of initial samples obtained during the first 100 days after transplantation did not differ significantly between patients with (mean±SE: 8.1±0.8 kb, n=13) and without GVHD (7.4±1.4 kb, n=7, p=0.65). Of 10 patients, longitudinal analysis was carried out. Five out of six patients with acute GVHD showed massive telomere shortening during the first weeks after development of GVHD (-2.9 kb, p=0.03), whereas no changes were observed in unaffected controls (-0.1 kb, n=3, p=0.75). This translates into a loss of 0.209 kb per week in patients with acute GVHD, compared to 0.004 kb in the control group.

Conclusion
In this preliminary study, we found that enterocytes of patients with refractory acute gut GVHD show increased proliferation and dramatically shortened TL in affected gut endothelium during the course of the disease. Increased cellular turnover due to T-cell mediated attack on the enteral stem cell pool and subsequent replicative exhaustion might provide a mechanism explaining eventual refractoriness of gut GVHD to immunosuppressive therapy. Based on our findings, TL measurement should be further explored prospectively as a prognostic biomarker in patients with agGVHD.

Disclosures:
No relevant conflicts of interest to declare.