CD34+ Donor Chimerism and Wilms Tumor Gene 1 (WT1) Expression Provide an Early Indication of Relapse in Patients with Acute Leukemias and MDS after Hematopoietic Cell Transplantation (HCT) with Reduced-Intensity Conditioning.
Authors: Hubmann Max , Burkhardt Ralph , Franke Georg , Cross Michael , Scholz Markus , Leiblein Sabine , Thiery Joachim , Niederwieser Dietger W. , Lange Thoralf 
Published: October 2019

Abstract
HCT following reduced intensity conditioning (RIC) relies mainly on immunological effects for disease control. The early detection and quantification of minimal residual disease and the timely adjustment of immune suppression are therefore particularly important in this setting. Since appropriate disease-specific gene markers are available only in a minority of patients with acute leukemias or MDS, the potential of both donor chimerism and Wilms Tumor gene 1 (WT1) expression to provide quantification of MRD was investigated.
Patients and Methods: Ninety-five consecutive patients with AML (n=68), ALL (n=7) and intermediate/high-risk MDS (n=20) were analyzed. Patients were 60 (median; range 21–74) years old and in CR1 (n=45), CR2 (n=25), or more advanced disease status (n=25). Grafts were obtained either from related (n=24) or unrelated (n=71) donors. Conditioning regimens consisted of fludarabine 30mg/kg BW day -4 to -2 (n=90) and total body irradiation with 2 Gy at day 0 (n=95), and post-transplant immunosuppression employed cyclosporin A and mycophenolate mofetil. Total donor chimerism (TDC, n=93 patients, 236 samples), CD34+− chimerism (n=89, 219 samples) and disease-specific molecular markers detected by FISH (DSM, n=39, 77 samples) were all determined prospectively from bone marrow (BM) samples at baseline and on days +28, +56 and +84 post-transplant. WT1 expression was analyzed retrospectively by RT-PCR from stored peripheral blood (PB) samples (n=95, 321) from the same time points.
Results: With a median follow-up of 11.7 (range 2–61) months, 34 (36%) patients relapsed (defined by BM blasts >5%). Since complete results from all techniques were available up to day 84, we analyzed the diagnostic power of all methods to predict hematological relapse one month in advance up until the fourth month after HCT (n=21 patients, 22%). First, we estimated the value of the three different prospective MRD techniques (DSM, TDC and CD34+ chimerism) using Receiver Operating Curves (ROC). Relapse was predicted 1 month in advance by CD34+−chimerism [p= 0.001, area under the curve (AUC) = 0.875], but not by TDC or DSM, (n=30). The cut-off value of 5% decrease in CD34+ chimerism in a one month period achieved a sensitivity of 71% and specificity of 91%. In comparison, WT1 expression was similarly associated with a pending relapse (p< 0.0001, AUC = 0.861). The optimal cut-off value of 24 WT-1 copies per 10000 ABL copies was assessed by ROC and resulted in a sensitivity of 79% and specificity of 91%. In a logistic regression model, we estimated the ratios of the odds of relapsing within the next month. WT1 achieved a higher odds ratio (36/1) than CD34+ chimerism (25/1). Combining both techniques yielded a specificity of 98% and an odds ratio of 72/1
Conclusion: WT1 expression in PB and CD34+ chimerism in BM are superior to full donor chimerism and disease-specific markers determined by FISH in predicting relapse in all patients with acute leukemia and MDS. Both markers are suitable to develop effective post-transplant treatment strategies to further decrease the relapse rate using immunological or cytotoxic approaches.