A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytomerty Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Beckerich Florence , Sobh Mohamad , Morisset Stephane , Plesa Adriana , Dubois Valerie , Mollet Isabelle , Labussiere Helene , Gilis Lila , Ducastelle Sophie , Le Borgne Oriane , Monfray Jeremy , Barraco Fiorenza , Tigaud Isabelle , Thomas Xavier , Nicolini Franck E. , Michallet Mauricette 
Published: October 2019

Background
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative strategy for acute myeloid leukemia (AML) patients in complete remission (CR) presenting poor prognostic factors or with relapsed/refractory disease. However, the risk for disease recurrence following allo-HSCT remains significant and associated with poor outcomes. After transplantation, the early detection of minimal residual disease (MRD) using immunophenotyping combined to chimerism documentation before morphological relapse may allow for immediate interventions and can lead to better results. Immunophenotyping using Multiparameter Flow Cytometry (MFC) and chimerism documentation by PCR have been widely used to track disease recurrence, although a validated consensus on the use of these techniques in the post-allo-HSCT follow-up has not been established yet. The aim of our study was to evaluate the impact of positive MRD by MFC associated to chimerism documentation at 3 months after allo-HSCT on patients overall and progression-free survival (OS, PFS).

Patients and Methods
We evaluated 137 AML patients who received allo-HSCT in a single center between January 2005 and October 2012 at our department and for whom a 3 months MRD evaluation and chimerism documentation has been performed using MFC, and PCR analysis. There were 71 (52%) males and 66 females with a median age of 47 years (range: 19-66), 77% had de novo AML, 20% had secondary AML and 3% had biphenotypic AML. According to cytogenetics, 40% were normal, 51% were unfavorable (9% classified as failure). According to molecular markers, 9% were favorable, 31% intermediate, 44% unfavourable and 16% had no molecular markers. At allo-HSCT, 46% of patients were in first complete remission (CR1), 25% were in CR 2 and 29% had active disease; 40% received a full intensity conditioning and 60% got reduced intensity one. As cell source, 35% were bone marrow, 53% peripheral blood and 12% cord blood cells. Donors were related in 53% of the cases (45% were 10/10 HLA matched) and unrelated in 47% of cases (20% were 10/10 HLA matched). MFC was performed using BM samples with a sensitivity of 0.01%. Chimerism analysis was performed on marrow and/or blood samples using polymerase chain reaction (PCR) based on informative polymorphic short tandem repeat with an accuracy of ± 5%, a mixed chimerismwas defined by having 5% or more of recipient cells.

Results
After transplantation, all patients engrafted, the cumulative incidence of acute GVHD at 3 months was 19.9% (95% CI: 16.2-20.6) while the cumulative incidence of chronic GVHD reached 26.7% (95% CI: 22.9-30.5) at 1 year. After a median follow-up of 16 months (range: 3-77), the median OS was 66 months (65-NR) with a 3 years probability of 64% (95% CI: 56-73), the median PFS was 32 months (13-NR) with a 3 years probability of 50% (95% CI: 37-58) while the transplant related mortality rate reached 13.6% (95% CI: 10-16) at 2 years. The 3 months chimerism evaluation (n=137) showed a mixed chimerism in 12 (9%) patients, while the MFC (n= 62) detected 15 patients with leukemic cells. Sixty eight patients showed morphological relapse after a median time of 4.8 months (1-34.7); the correlation study between MRD positivity, mixed chimerism detection and morphological relapse showed a higher correlation for both chimerism and MFC (correlation=0.69, p<0.001) than if we consider chimerism or MFC alone. Multivariate analysis showed a significant worse OS for patients with 3 months positive MFC [1 year OS of 20% vs. 80%, HR= 4 (95% CI: 1.4-11.7), p=0.01] and patients with mixed chimerism [1 year OS of 21% vs. 70%, HR= 4 (95%CI: 1.3-12.1), p=0.01]; these results were still valid even after stratification on disease status at transplantation. These results applied also in terms of PFS for positive MFC [1 year PFS of 13% vs. 76%, HR= 3.6, p=0.02], and mixed chimerism[1 year PFS of 0% vs. 70%, HR= 7, p=0.001], Figure 1.

Conclusion
The 3 months MRD evaluation using MFC combined to chimerism documentation seems to be an independent prognostic factor on overall and progression-free survival for AML patients undergoing allo-HSCT. The standardisationof this evaluation may lead to the identification of patients with high relapse risk suggesting the need of early therapeutic intervention.

Disclosures:
No relevant conflicts of interest to declare.