Monitoring of Wilms’ Tumor 1 (WT1) Expression in Peripheral Blood (PB) of Patients with Myelodysplastic Syndromes (MDS) By a Standardized European Leukemia Net (ELN)-Certified Assay to Detect Minimal Residual Disease (MRD) and Guide Therapeutic Interventions Following Allogeneic Transplantation
Authors: Schroeder Thomas , Pechtel Sabrina , Lamers Marius , Dienst Ariane , Schuler Esther , Rachlis Elena , Wegener Nadija , Kündgen Andrea , Kondakci Mustafa , Fenk Roland , Gattermann Norbert , Haas Rainer , Germing Ulrich , Kobbe Guido 
Published: October 2019

Background:

Since the success of therapeutic interventions to prevent or treat relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with MDS and secondary acute myeloid leukemia (sAML) especially depends on disease burden, early detection of MRD is essential. Despite the discovery of various mutations during the last years, no reliable MRD marker has been defined, which can be quantitatively measured by a standardized assay in a large proportion of MDS patients. Measurement of PB WT1 mRNA expression facilitates quantitative MRD monitoring in >90% of de novo AML patients in the pre- and posttransplant period. WT1 overexpression has also been reported in a proportion of MDS patients suggesting its potential use as MRD marker. Still, because of limited patients numbers, differing sample sources (PB or BM) and the use of in-house methods without reproducible cut-off levels, WT1 has been not established as MRD marker in MDS so far.

In the current study we used a standardized ELN-certified assay (1) to measure WT1 expression in PB of a large group of all MDS subtypes enabling correlation with disease characteristics, (2) to determine its’ value as MRD marker in patients with MDS undergoing allo-HSCT, and (3) to monitor WT1 levels during salvage therapy.

Patients and Methods:

WT1 levels were measured in PB using the Ipsogen® WT1 ProfileQuant® Kit (Qiagen, Hilden, Germany) in accordance with the manifacturers’ instructions. This standardized assay has a validated PB cut-off level of 50 WT1 copies/104 ABL copies to distinguish between normal and overexpression of WT1.

Our study included a total of 92 untreated MDS patients (7 RCUD, 30 RCMD, 7 MDS del5q, 7 CMML, 11 RAEB-1, 15 RAEB-2, 15 sAML). Samples of 12 healthy volunteers and 18 patients with non-MDS related cytopenias served as controls.

In MDS patients, who had WT1 overexpression prior transplant, WT1 levels were serially monitored after allo-HSCT. Finally, in an independent group of patients with MDS or AML relapsing after allo-HSCT WT1 expression was measured during salvage therapy with Azacitidine (Aza) and donor lymphocyte infusions (DLI).

Results:

Overall, PB WT1 overexpression was found in 48 of 92 MDS patients (52%) and enabled not only to significantly discriminate between MDS and healthy controls (0/12 samples, 0%, p=0.0004), but also between MDS and cytopenias related to other causes (0/18 samples, 0%, p<0.0001)

WT1 expression significantly correlated with disease category according to WHO classification as indicated by the frequency of WT1-overexpressing patients (RCUD 14%, MDS del5q 28%, RCMD 40%, CMML 57%, RAEB-1 55%, RAEB-2 87%, sAML 67%, p=0.0011) as well as by absolute WT1 levels of the respective subentities (p=0.0192). In addition, there was a clear association between WT1 expression and risk categories according to IPSS as well as IPSS-revised. While no differences were found with regard to WT1 expression between patients with a normal or abnormal karyotype, WT1 overexpression was significantly associated with increased BM blasts.

In 16 patients with WT1 overexpression prior transplant, we monitored WT1 expression in the posttransplant period. All patients had a rapid decline to normal WT1 levels below the normal threshold after transplant. Nine patients exhibited low and stable WT1 levels and remained in remission throughout follow-up. In contrast, 7 patients relapsed after allo-HSCT and all of them had an increase of WT1 expression above the cut-off level at/or before relapse suggesting stable correlation between WT1 and disease burden after allo-HSCT.

In line with this we found a 3,32-log-fold (range: 0,61 to 4,14-log) decrease of WT1 expression to normal levels in 8 patients achieving CR after salvage therapy with Aza and DLI. All 20 patients, who did not respond, remained above the normal WT1- threshold.

Conclusion:

Using a standardized assay with reproducible cut-off levels WT1 overexpression can be detected in PB of nearly half of MDS patients. WT1 overexpression was in particular found in those MDS patients with an increased BM blast count and expression levels correlated with disease burden in the posttransplant period. These findings suggest that WT1 expression might be used as MRD marker in a broader fraction of MDS patients for detection of relapse and for guidance of therapeutic interventions after allo-SCT.

Disclosures

Gattermann: Novartis: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.