Prognostic Value of Minimal Residual Disease Assessed By WT1 Expression Level and Flow Cytometry in Acute Myeloid Leukemia Patients Undergoing Allogeneic Marrow Transplantation
Authors: Giannoni Livia , Guolo Fabio , Minetto Paola , Galaverna Federica , Ghiggi Chiara , Clavio Marino , Bagnasco Samuele , Di Grazia Carmen , Grasso Raffaella , Colombo Nicoletta , Avenoso Daniele , Pastori Giordana , Ballerini Filippo , Kunkl Annalisa , Miglino Maurizio , Lemoli Roberto , Gobbi Marco , Bacigalupo Andrea 
Published: October 2019

Abstract
Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients.
Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse.
Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression.
Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p <0.001) since no relapses occurred in the eleven MFC-MRD negative patients. Among MFC-MRD positive patients a further stratification of relapse risk is obtained by the evaluation of WT1. Patients with double positive MRD had a significantly worse DFS compared with patients who were MRD positive by MFC but MRD negative by WT1 (p <0.01). The predictive value of MRD was independent from different induction schedules; furthermore the favorable prognostic value of achieving a negative MRD status was not affected by undergoing BMT in second or subsequent remission. Median OS was 26 months and was significantly influenced by disease status and MRD status at transplantation and by relapse after BMT. Cumulative non relapse mortality was 23% at 36 months and was not associated with pre-BMT status.
Conclusion: pre BMT MRD evaluation by WT1 and MFC on bone marrow samples is a reliable tool to predict relapse risk. Patients with negative pre-BMT MRD have a significantly longer DFS and OS, while MRD positive patients by both methods display a higher risk of relapse. Patients at higher risk of poor outcome should undergo a more stringent program of post BMT evaluations, in order to detect disease relapse earlier and might be candidate for pre-emptive therapeutic interventions aimed at delaying or avoiding AML reoccurrence.

Disclosures
No relevant conflicts of interest to declare.