Comparison Between Wilms' Tumor 1 (WT1) Expression Using a Standardized European Leukemia Net (ELN)-Certified Assay and Other Methods for Detection of Minimal Residual Disease (MRD) in MDS and AML Patients after Allogeneic Blood Stem Cell Transplantation
Authors: Rautenberg Christina , Pechtel Sabrina , Hildebrandt Barbara , Betz Beate , Dienst Ariane , Nachtkamp Kathrin , Geyh Stefanie , Schmidt Pia Verena , Heyn Claudia , Kondakci Mustafa , Gattermann Norbert , Haas Rainer , Germing Ulrich , Kobbe Guido , Schroeder Thomas 
Published: October 2019

Abstract
Introduction
An "ideal" marker to monitor MRD after allo-SCT should be informative in the majority of pts and facilitate the use of a method with high sensitivity and specificity in a standardized manner. In addition, to allow repeated monitoring in timely tight intervals but also ensuring patients' comfort such marker should ideally be measurable in peripheral blood (PB).
Despite recent identification of several molecular aberrations AML and MDS, many of these do not fulfil the above-mentioned requirements, as they are present only in small patient groups, their potential instability during disease course, the absence of standardized assays and the need for BM as optimal sample source.
A molecular marker which might provide these properties is the WT1 gene, as it is overexpressed in the majority of AML pts and in about 50% MDS pts and is measurable in PB by a standardized assay. To estimate its value after allo-SCT we compared serial WT1 measurement with other methods used to monitor MRD in a real-life situation.
Patients and Methods
For this retrospective analysis all AML and MDS pts who underwent allo-SCT at our center between 2012 and 2016 were screened for PB WT1 mRNA overexpression using the ELN certified Ipsogen® WT1 ProfileQuant® Kit. Pts with WT1 overexpression, as defined by an validated cut-off level of 50 copies/104 ABL copies, were routinely monitored after transplant. In addition, in all pts STR-based chimerism analysis was performed. In pts with chromosomal aberrations existing prior allo-SCT metaphase and FISH analysis was performed, while XY FISH was additionally conducted in pts with sex-mismatched donor-recipient constellation. Furthermore, pts with molecular markers were regularly monitored by NGS or qPCR. Results of WT1 monitoring were correlated with clinical course and compared with results obtained from the other methods.
Results
Of 104 screened pts 59 (57%) showed an WT1 overexpression at diagnosis and underwent an allo-SCT. This included 40 AML pts (WT1 overexpressed in 66%) and 19 MDS pts (WT1 overexpressed in 44%).
Chimerism analysis was accessible in all 59 pts (100%), while 20 pts (34%) could also be monitored by XY-FISH. Additionally, in 40 pts (68%) cytogenetics and FISH were applicable, while 22 pts (37%) could be investigated by NGS or qPCR. Overall, in 5 pts MRD could be monitored by WT1 and chimerism only, while in 29 pts MRD could be monitored by 1, in 22 pts by 2 and in 3 pts by 3 additional methods.
With a median follow up of 13 months (2 - 51) we analyzed a total of 472 WT1 samples reflecting a median of 8 samples per patient (2 - 19). One month after allo-SCT 57 pts (97%) showed complete remission and a rapid decrease of WT1 expression below threshold. Only 2 pts had persistant hematological disease with sustained WT1 overexpression.
Twenty-four pts (41%) experienced hematologic (62%) or molecular (38%) relapse at a median of 126 d (28 - 938 ) after allo-SCT. In 20 (83%) of these at least one WT1 value wasabove the cut-off before relapse. Median time from first elevated WT1 to relapse was 2 weeks (0 - 27). Only 4 pts (17%) with molecular relapse showed WT1 expression below cut-off.
In contrast, known molecular aberrations were found again in 63% and loss of complete donor-chimerism or a positive signal in XY-FISH analyses were only seen in 46% and 57% before relapse. Furthermore, cytogenetics or FISH detected known or new aberrations in 39% before relapse.
From 35 pts remaining in CR for a median of 13 months, only 1 (3%) had a transient increase of WT1 expression above the cut-off, whereas WT1 levels of the other 34 pts persisted below. Three pts (9%) with ongoing remission showed a transient decrease of donor-chimerism, whereas even 31% of pts accessible for XY-FISH showed temporary conspicuous results. Conventional cytogenetics and FISH in pts with CR showed transient abnormalities in 18%, whereas in 14% with molecular aberrations these were temporary detectable. Conclusion
Measurement of PB WT1 overexpression is an easy accessible method to monitor MRD after allo-SCT that can be employed by a standardized assay in the majority of AML and MDS pts independent from their individual leukemic genotype. Besides these advantages, the results from our real-life experience also suggest that WT1 overexpression allows sensitive detection of imminent relapse after allo-SCT. Taking into account the methodical restrictions of this retrospective analysis, our data requires confirmation in a prospective trial.

Disclosures
Gattermann: Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.