Real-Time Quantitative Y Chromosome Specific PCR vs. XY-FISH vs. Fluorescent STR-PCR for Chimerism Analysis after Allogeneic Sex-Mismatched PBSCT.
Authors: Baak Ulrike , Marinets Olga , Rieger Kathrin , Blau Igor Wolfgang , Uharek Lutz , Burmeister Thomas , Thiel Eckhard 
Published: October 2019

Abstract
Chimerism analysis after allogeneic stem cell transplantation (SCT) is of major importance to monitor engraftment, graft rejection and disease relapse. Besides the routine application of short tandem repeat (STR)-based methods, XY-FISH comprises a well established method in sex-mismatched transplantation. While the sensitivity levels of these methods are sufficient for monitoring engraftment, a higher sensitivity seems desirable for detection of minimal residual disease (MRD). In this context highly sensitive quantitative Real-Time PCR (RQ-PCR) assays appear to be of great utility, among them a Real-Time quantitative Y chromosome-specific PCR (QYCS-PCR). QYCS-PCR allows chimerism analysis in male patients with female donors, accounting for about 25% of all transplantations. Here we have evaluated the clinical utility of this method compared to the routine methods of STR-PCR and XY-FISH in 36 samples of BM or PB derived from 11 patients at various time points after myeloablative (n=3) and nonmyeloablative (n=8) allogeneic PBSCT. Patients were transplanted for MM (5), AML (3), ALL (2) and NHL (1). Follow-up time ranged from 138 days to 1776 days post PBSCT. The majority of patients (n=8) showed a complete remission, whereas three patients experienced a relapse after PBSCT. Routine STR-analysis was performed applying 4 highly polymorphic markers in a multiplex PCR on an A.L.F. sequencer (sensitivity 5%). In the standardized XY-FISH analysis 200 nuclei were evaluated (sensitivity 1%). RQ-PCR was performed on a Rotor-Gene 3000 cycler. Serial dilutions of male mononuclear blood cells in female cells (100%, 50%, 25%, 10%, 1%, 0,1% and 0,01%) were prepared. The DFFRY gene and the HCK gene (control) were amplified in a duplex Real Time PCR (sensitivity 0,01%). Results of STR-analysis ranged from 40% to 100% donor chimerism. In the lower range of donor chimerism STR-PCR and XY-FISH showed a good correlation and a higher reproducibility than QYCS-PCR. In the majority of samples with complete chimerism in STR-PCR (n=20), compared to 13 samples with complete chimerism in XY-FISH, QYCS-PCR was able to detect residual host cells in 19 samples. Patients in complete remission showed a stable low level of persisting host cells (0,01-0,1%). In one patient with complete donor chimerism in STR-PCR and XY-FISH, QYCS-PCR was able to detect a rising level of host cells before clinical apparent relapse. Thus our data indicate that real-time quantitative Y chromosome-specific PCR based on the DFFRY gene allows highly sensitive and reliable detection of MRD in patients after sex-mismatched allogeneic transplantation. QYCS-PCR seems to be a valuable complementary tool complementing STR-PCR and XY-FISH. In some patients with complete donor chimerism in STR-PCR and XY-FISH analysis, it might allow earlier diagnosis of imminent relapse and offer more time for therapeutic intervention.