High Fractions of CD34+CD38- Cells with Aberrant hMICL Expression Predicts Shorter Overall- and Progression Free Survival in Myelodysplastic Syndrome

Introduction

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal diseases marked by dysplasia in the bone marrow (BM) causing cytopenias in the peripheral blood and with an inherent tendency to progress to acute myeloid leukemia (AML). Myelodysplastic syndrome has generally been regarded a stem cell disease, which very recent research has emphasized to hold true also for low-risk MDS (Woll, P. S. et al Cancer Cell. 2014). In AML, we have identified the human Myeloid Inhibitory C-type lectin-like receptor (hMICL) (also named CLL-1 or CLEC12A) as a stable and reliable marker at diagnosis and as a tool for assessing minimal residual disease (Roug, A. S. et al Br. J. Haematol. 2014). Furthermore, hMICL has been proposed as a marker of the leukemic stem cell (LSC) since it is present on CD34+CD38- cells in the majority of CD34-positive AMLs, while it is absent on the CD34+CD38- cell compartment in normal controls (van Rhenen, A. et al Leukemia. 2007). Previous studies have shown aberrant marker-expression on CD34+CD38- cells in MDS, e.g. CD7 or CD2, but only in minor fractions of patients (Xie, W. et al. Cytometry A. 2010).

Using multicolor flowcytometry, we have examined the aberrant expression of hMICL on the CD34+CD38- stem cell compartment from 19 untreated MDS patients. We correlated the aberrant hMICL positive cells as a percentage of the total CD34+CD38- compartment (CD34+CD38-hMICL+/CD34+CD38-) to follow-up data.

Materials and methods

Bone marrow (BM) samples were obtained from 19 MDS patients diagnosed and followed at the Department of Hematology, Aarhus University Hospital from January 2010 to January 2011. Diagnosis was determined by morphology according to the 2008 WHO classification. Risk stratification of patients was done using the International Prognostic Scoring System (IPSS). All samples were collected before treatment with a cytoreductive agent or 5-azacytidine. Normal bone marrow (NBM) from 11 healthy volunteers served as controls.

Following lysis of red blood cells, BM was stained with the following monoclonal antibodies: anti-CD34 APC, anti-CD38 FITC, anti-CD45 PerCP-Cy5.5 and anti-hMICL PE. Data acquisition was performed on a FACSCanto II and subsequently analyzed using FACSDiva Software. Using the back-gating strategy, the CD34+CD38-hMICL+ cell subset was shown to cluster together in an FSC-SCC plot. A total number of 5 clustered events were considered significant for further analyses.

Results and perspectives

The median age at sampling was 69 years (range 44-83 years). According to IPSS, 1 patient was classified as low-risk, 11 patients as intermediate-1 and 3 patients as intermediate-2. Four patients could not be classified. Consistent with previous reports, hMICL was not found on CD34+CD38- cells in any of the 11 NBM examined (0.0%) whereas aberrant hMICL expression was present on the CD34+CD38- stem cells in 16/19 (84%) of MDS cases. In MDS, the CD34+CD38-hMICL+ cells amounted a median of 5.68% (range 0,0-56.94%) of the CD34+CD38- subset, which was significantly different from NMB (p<0.0001) (Figure 1A).

The median follow-up period for the cohort was 3.7 years. Patients with fractions of hMICL positive CD34+CD38- cells above the median (5.68%) showed a significantly shorter progression free survival (PFS) compared to patients with hMICL+ fractions below the median (p=0.01) (Figure 1B). The same trend was evident with regards to overall survival (OS), although borderline significant (p=0.06) (Figure 1C).

The aberrant expression of hMICL on CD34+CD38- stem cells from MDS patients belonging to both low- and intermediate risk groups identifies hMICL as a potential LSC-marker in this heterogeneous group of diseases. A considerable advantage of hMICL is its complete absence on normal hematopoietic stem cells and the fact that it was present in the majority of MDS cases studied. Despite the small patient-number in our cohort, the impact of a high hMICL+ stem cell fraction on PFS and OS is evident. Thus, hMICL marking of the CD34+CD38- stem cell compartment might serve as a prognostic indicator in the future.

Figure 1

View largeDownload slide

Figure 1

View largeDownload slide

Close modal