Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) Is Followed by Epithelial Chimerism of the Oral Mucosal.
Authors: Spyridonidis Alexandros , Metaxas Yannis , Zeiser Robert , Waterhouse Miguel , Faber Phillip , Finke Jurgen 
Published: April 2020

Abstract
Several study groups have reported on a non-hematopoietic chimerism following allogeneic hematopoietic cell transplantation (aHCT). However, most studies were performed in animal models. Therefore, we investigated this phenomenon in humans and described the pitfalls of identification of epithelial chimerism
The purpose of the present study was to examine epithelial chimerism in the oral mucosa. Buccal scrapings obtained from 14 female patients (pts) who underwent a sex-mismatched aHCT were employed to prepare cytospins of buccal cells. The examination was performed 56 days to 1226 days after HSCT when the pts visited the ambulance for regular examination. At this time point no patient had signs of mucositis or oral graft versus host disease (GvHD). Cytospin preparations were examined with a combination of fluorescence in situ hybridization (FISH) for the Y-chromosome, fluorescent stain for the epithelial specific marker cytokeratin (CK), CD45, and Drag cell nuclei stain; with APAAP immunocytochemistry using either CD45, CD68 or CD3 specific antibodies; and with hematoxylin/eosin (H/E) stain. Evaluation of epithelial chimerism was performed with laser scanning confocal microscopy from examiners who were not aware about the past medical history of the patients regarding mucositis or oral GvHD. Pts who received sex matched HSCT were used as controls and showed hybridization specificity and ]95% FISH-Y detection sensitivity. H/E stain of the cytospins revealed the presence of cells with an epithelial morphology. No CD68 positive cells were ever detected indicating the absence of macrophages having down-regulated CD45. CD45 or CD3 positive cells were found in 3 pts with a frequency of less than 1%. In the FISH-Y combined stains we detected Y+/CK+/CD45− cells in 9/12 pts (75%), with a median of 8.3 Y+/CK+/CD45− cells/200 total cells analyzed (range 1–121). Screening of 5 pts with XY stain demonstrated all of them to have XY+/CK+/CD45− epithelial cells, making fusion as the underlying mechanism unlikely. We retrospectively reviewed the transplantation documents of every pt and found a significant correlation (p-value=0.0163) between the severity of mucositis in the early post-transplant period (absent 3pts, mild mucositis 4pts, moderate 5pts and severe 2 pts) and the degree of epithelial chimerism found at later time points (d+56 to d+1226) where no signs of mucositis were present. Further clarification in the possible clinical implications of the epithelial chimerism could be given only by material examination of a great number of transplanted patients. Oral scrapings are easily and non-invasive obtained from pts and can be evaluated for degree of epithelial chimerism with the method described here. In order to further elucidate the mechanisms of this biological phenomenon we recently established PCR-based methods of laser-microdissected nuclei. Evaluation of whole cell samples with microsatellite STR markers in 4 patients with Y+/CK+/CD45− cells, disclosed a mixed epithelial chimerism in 4/4.
Our data suggest that epithelial chimerism in the oral cavity after aHSCT is a real phenomenon, however further investigation on a single nucleus level is necessary to understand the underlying mechanism, responsible for the donor derived chimeric cells.