Quantitative Real-Time PCR Detection of Wilms' Tumor Gene (WT1) Transcript in Autologous Peripheral Blood Stem Cell (PBSC) Products Predict the Risk of Acute Myeloid Leukaemia (AML) Relapse After Autologous Transplantation (ASCT).
Authors: Messina Carlo , Tresoldi Cristina , Crotta Alessandro , Tassara Michela , Malato Simona , Forno Barbara , Marcatti Magda , Peccatori Jacopo , Corti Consuelo , Fleischhauer Katharina , Bordignon Claudio , Ciceri Fabio , Bernardi Massimo 
Published: October 2019

Abstract
Abstract 4690

Introduction
when allogeneic transplantation is not feasible, ASCT is an alternative option as post-remission therapy for patients (pts) with AML, as it can prolong their disease-free survival (DFS). Relapse is the main cause of AML treatment failure after initial complete response; AML relapse after ASCT is partly due to contamination with leukemic blasts of the PBSC products, although neither morphological nor genetic evidence of disease are detected in the bone marrow before leukapheresis. Thus, identification and quantification of a reliable minimal residual disease (MRD) marker in the collected PBSC could be relevant in determining the relapse risk after ASCT. The WT1 gene is overexpressed in leukemic blasts of most AML cases; several studies have shown that quantification with RT-PCR of WT1 in the bone marrow and peripheral blood of AML pts in complete remission has prognostic value. We have determined the WT1 transcript levels in autologous PBSC of AML pts autografted for complete remission (CR) consolidation; preliminary results and data interpretation are here presented.

Aim
to evaluate quantitative WT1 transcript levels in autologous PBSC collections and to compare results with outcome in AML patients who received an ASCT as consolidation of CR, at our Institute.

Patients and Methods
9 pts, period 06/2006-03/2009, median age 68 years (range 41-76). At diagnosis cytogenetic prognostic risk was intermediate for all pts. All pts were in morphological and genetic CR at the time of PBSC collection and before ASCT. Eight patients were in first and 1 in second CR. PBSC collection by leukapheresis (COBE Spectra cell separator): median count of CD34+ 9.75 ×106/kg (3.79-32). Conditioning regimen: Treosulfan 30 mg/sqm, Fludarabine 150 mg/sqm and Cytarabine 5 g/sqm. Median count of CD34+ cells infused: 5×106/kg (range 3.3-8.5×106/kg). RT-PCR quantification of WT1 transcript was performed using TaqMan technology starting from 1 μg of RNA extracted from mononucleated cells of fresh (4) or cryopreserved (5) PBSC samples. The housekeeping gene ABL was used as the control gene for these quantifications with WT1 level being normalised to 104 copies of ABL per sample. We used the Mann-Whitney-U-test to determine if median WT1 levels in the PBSC products of relapsed and not-relapsed pts was statistically different. Than we used the log-rank test to compare RFS and median WT1 value in the PBSC products.

Results
at last follow-up 4 pts relapsed and 5 were still in CR. The WT1 levels in the autologous PBSC of the 4 relapsed pts were 89.96, 193.87, 779.43 and 839.63, of the 5 CR pts were 8.40, 16.96, 36.45, 74.89 and 82.49. Overall median WT1 in the PBSC products was 82,49 copies. The median WT1 levels in the PBSC products of relapsed and not-relapsed pts were 486.65 (89.96–839.63) and 36.35 (8.40–82.49) copies, respectively; this difference was statistically significant (p<0.05). Overall, median relapse free survival (RFS) from ASCT was 534 (93-1096) days. Median RFS was 360 days for pts with a WT1 level > 82,49 copies (n=4), and has not been reached for pts with a WT1 level ' 82,49 (n=5) (p=ns).

Conclusions
these results suggest that RT-PCR quantification of the WT1 transcript in autologous PBSC could predict AML relapse in pts who receive ASCT in CR. Higher WT1 levels should reflect higher PBSC product contamination with leukemic blasts, indicating an increased risk of relapse after ASCT. These last pts could probably benefit of other strategies than ASCT. We conclude that, if our preliminary data will be confirmed in a larger number of pts, RT-PCR quantification of WT1 transcripts should be used for MRD detection and quantification in autologous PBSC, before proceeding to ASCT; according to our preliminary data the cut-off level could be about 80 WT1 copies to discriminate which pts should receive the ASCT and which should not.

Disclosures:
No relevant conflicts of interest to declare.