Combining Flow Cytometry and Molecular Assessment Improves the Prognostic Value of Pre-Transplant Minimal Residual Disease in Acute Myeloid Leukemia
Authors: Guolo Fabio , Minetto Paola , Galaverna Federica , Guardo Daniela , Colombo Nicoletta , Kunkl Annalisa , Clavio Marino , Di Grazia Carmen , Raiola Anna Maria , Ballerini Filippo , Miglino Maurizio , Lemoli Roberto Massimo , Gobbi Marco , Bacigalupo Andrea 
Published: October 2019

Abstract
BACKGROUND AND AIMS
Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for high-risk acute myeloid leukemia (AML) patients. Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. Multicolor flow cytometry (MFC) is the most common tool to evaluate MRD, whereas WT1 gene expression is the most widely applicable and standardized molecular MRD marker in AML. The aim of this study was to analyze the role of pre-BMT combined MFC-molecular MRD assessment as predictor for the post-transplant relapse risk.
MATERIALS AND METHODS
We retrospectively analyzed the outcome of 224 consecutive AML patients receiving allo-BMT in 1st or 2nd CR. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation. Median age at transplant was 44 years. Disease phase was CR1 in 161 (72%) and CR2 in 63 patients (28%). BMT conditioning wasmyeloablative in 163 (73%), whereas 61 patients (27%) received reduced intensity conditioning. Stem cell source was HLA-identical sibling in 79 (35%),haploidentical in 59 (27%) and alternative donor in 86 (38%).Median follow-up was 64 months (95% CI 50.3 - 77.3 months). Cumulative Incidence of Relapse (CI of relapse) at various time-points was calculated in competing risk analysis by takingin account non relapse mortality as competing event. Overall Survival (OS) was calculated from the time of BMT until death by any cause or last follow-up.
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. WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression.
RESULTS
Relapse occurred in 62 patients (27.7%). Three-year estimate of CI of relapse was 25.9% (median not reached). The CI of relapse was significantly affected by occurrence of acute GVHD (lower for grade ≥2, p <0.05) and MRD status before BMT, measured with any method (p <0.001 for WT1-based MRD, p<0.03 for MFC based MRD, p<0.0001 for combined MRD). In multivariate analysis the combined MRD evaluation and the onset ofaGVHDwere the only independent predictor of CI of relapse (p <0.001 and 0.04, respectively, Table I).
Specifically, patients with double positive molecular and MFC MRD had a very poor outcome, with a 3-year CI of relapse of 51.9% (Figure 1). On the contrary, patients with negative combined MRD had a very low CI of relapse, regardless of the conditioning regimen received, whereas a trend toward benefit frommyeloablativeconditioning was observed in patients with MFC positive/WT1 negative MRD and double positive patients.
Patients with negative MFC-MRD undergoing BMT in 1st or 2nd CR had a very similar outcome (3-year CI of relapse 8.3 and 5.6%, respectively). Similarly, MFC positive/WT1 negative MRD patients had a 3-year CI of relapse of 17.3 and 18.% for CR1 and CR2 patients, respectively. Patients who were MRD positive by both methods undergoing BMT in CR1 or CR2 had a similar, very high CI of relapse (Figure 2).
MRD evaluation was also a strong predictor of long term survival: 3- years OS was 73.5% for MFC negative and 36.7% for double WT1 and MFC MRD positive patients, respectively, (p <0.001). Multivariate OS analysis showed that conditioning intensity and combined MRD evaluation significantly influenced OS duration (p <0.005 and <0.001, respectively)
CONCLUSIONS
Pre transplant MRD evaluation by combined molecular and MFC assessment on bone marrow samples is a reliable predictor of relapse risk. Patients with both negative pre-BMT MRD markers have a significantly lower CI of relapse, while patients with both positive MRD markers display an higher risk of relapse. Given the high prognostic value, combined molecular/MFC pre-transplant MRD status may be taken in account when considering the most appropriate stem cell source or conditioning for each patient. Furthermore, identifying patient in morphological CR at BMT but with a very high risk of relapse, such as double positive patients, could open the way to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches.
Figure 1. CI of Relapse and Overall Survival according to risk group Figure 1. CI of Relapse and Overall Survival according to risk group Figure 2. a: OS according to MRD in patients transplanted in CR1 b: OS according to MRD in patients transplanted in CR2 Figure 2. a: OS according to MRD in patients transplanted in CR1. / b: OS according to MRD in patients transplanted in CR2

Disclosures
Gobbi: Janssen: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Celgene: Consultancy; Roche: Honoraria; Mundipharma: Consultancy, Research Funding; Takeda: Consultancy; Gilead: Honoraria.