ALBERT

Description: 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.

Description: Hematopoietic chimerism has been demonstrated to be relevant for donor cell engraftment and detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). In the light of increasing numbers of aHCT as a treatment modality, rapid, reliable and cost effective chimerism monitoring techniques acquire novel relevance. We evaluated the informativeness of five microsatellite markers in 376 donor/recipient pairs and compared the ability of polyacrylamide gel electrophoresis (GE) and capillary electrophoresis (CE) to detect mixed chimerism (MC) after aHCT. The sensitivity for GE and CE with respect to different markers was determined by limiting dilution assays with MC samples containing defined amounts of cells or DNA. Furthermore, CE was applied in 37 retrospectively selected patients with a MC detected by GE, having undergone aHCT for different hematological diseases and initially achieving a complete donor chimerism. The sensitivity and accuracy quantified by limiting dilution was higher CE as compared to GE with all microsatellites. Potential pitfalls with the combined application of CE was preferential amplification, leading to the misdiagnosis of CC in the presence of MC and the occurrence of stutter peaks in the representative area. In case of preferential amplification with the initially applied marker D1S80, evaluation with a second microsatellite was beneficial. Investigation of the selected pts samples demonstrated that detection of an increasing MC was earlier with CE as compared to GE. The detected recipient genotype by CE examination, despite a negative GE result, ranged from 0.7% to 7.1%. We conclude that chimerism assessment with our five microsatellites identified informative alleles in 99% of all donor/recipient pairs. CE displayed a higher sensitivity and accuracy when compared to GE and additionally allowed for detection of an increasing MC suggestive for relapse. Prospective clinical investigation with CE for the predictive value of an increasing MC for graft failure or relapse is necessary. Informativeness of the institutional microsatellite marker panel No of pairs/ markers D1S80 THO1 D14S120 YNZ22 SE33 Numbers indicate patients were the marker was informative (ratio in %). rel: related donor; unrel: unrelated donor; n.t.: not tested; non inf.: non-informative. 165 rel/211 unrel 87 (53%)/145 (69%) n.t. n.t. n.t. n.t. 78 rel/66 unrel non inf. 22 (22%)/20 (30%) n.t. n.t. n.t. 56 rel/46 unrel non inf. non inf. 18 (32%)/16 (35%) n.t. n.t. 38 rel/20unrel non inf. non inf. non inf. 17 (45%)/15 (75%) n.t. 21 rel/5 unrel non inf. non inf. non inf. non inf. 19 (90%)/4 (80%) rel vs unrel 163/165 (98%) vs 210/211 (99%)

Description: In order to detect minimal residual disease (MRD) or relapse after allogeneic hematopoietic cell transplantation (aHCT) the immunophenotype of the original leukemic clone can be used for separation of distinct cell populations, expected to harbor malignant cells. Since conventional (CCA) and lineage-specific (LCA) chimerism analysis as diagnostic means have potential pitfalls, the present study assesses the results of both methods, to determine how predictive the evaluations prior to a hematological relapse were with respect to the risk that the patient would develop a relapse during the course of his disease. 118 individuals with acute myeloid leukemia (n=98) and myelodysplastic syndrome (n=20), investigated with CCA and LCA, were evaluated with a median follow-up of 26.6 months (range: 8–47). Subset chimerism samples were cell-separated by using immunomagnetic beads with respect to the patient’s leukemia phenotype, expressed at diagnosis. Mean purity of the positive-selected CD34 fractions were 97% (93–99%). PCR products were analyzed and quantified by capillary electrophoresis. 33 patients experienced a clinical relapse after aHCT. CCA and LCA detected a mixed chimerism (MC) in 27/118 and 25/118 patients, respectively. MC was detected at a median of 27 (12–54) and 34 (10–71) days before clinical relapse by CCA and LCA, respectively. LCA reached a significantly higher sensitivitiy than CCA with 91% (CI:54–98%) as compared to 83% (CI: 69–91%) and was also more sensitive as compared to bone marrow morphology examination (BME) (61%; CI:44–75%). Specificity was also higher with LCA (98%, CI: 89–100%) as compared to CCA (92%; CI: 75–99%) and BME (91%; CI: 82–96%). Our data suggest a higher sensitivity and specificity of LCA as compared to CCA and BME with respect to the clinical follow-up. We conclude from these data that due to the high predictive value subset chimerism monitoring may be employed for clinical decision making concerning adoptive immunotherapy after myeloablative aHCT for patients with AML and MDS, especially when lacking other molecular markers for detection of MRD. Differential accuracy of Lineage-specific versus Conventional Chimerism analysis Lineage-specific Chimerism analysis (LCA) Conventional Chimerism analysis (CCA) Bone marrow examination (BME) Sensitivity and specificity of lineage specific (LCA), conventional (CCA) chimerism analysis and bone marrow examination (BME) with respect to the event that the patient would develop a relapse in the clinical follow-up. Sensitivity in % (Confidence interval) 91 (54–98) 83 (69–91) 61 (44–75) Specificity in %(Confidence interval) 98 (89–100) 92 (75–99) 91 (82–96)