Proof of Y-Loss As Clonal Abnormality in MDS by Comparative Analysis of CD34+ and CD3+ Peripheral Blood Cells,
Authors: Ganster Christina , Braulke Friederike , Shirneshan Katayoon , Kämpfe Dietrich , Platzbecker Uwe , Söling Ulrike , Bramlage Carsten P , Legler Tobias J , Haase Detlef , Schanz Julie 
Published: October 2019

Abstract
Abstract 3800

Introduction:
In an ongoing diagnostic study we are currently following chromosomal anomalies in immunomagnetically enriched CD34+ peripheral blood cells in patients with suspected or proven myelodysplastic syndromes (MDS) at short intervals using fluorescence in situ hybridization (FISH) analysis every two to three months over three years. A loss of the Y chromosome was detected in 4% of these patients, as a single anomaly in 2%. Since it is controversially discussed whether loss of the Y chromosome is an age-related or a clonal event in patients with MDS, we aimed to examine whether a Y-loss is clonal or an age-related event in our patients.

Methods:
For patients with known Y-loss, we used peripheral blood not only to immunomagnetically enrich clonal CD34+ cells, but also CD3+ T-cells not belonging to the MDS clone. Subsequently, we performed FISH analysis to compare the clone sizes of cells with Y-loss in CD34+ and CD3+ cells. As our laboratory threshold for the FISH probe in CD34+ peripheral blood cells is 5%, we included 18 patients with clone sizes exceeding this threshold in CD34+ cells. The median age of the patients was 76 years (range 62–89). To establish a laboratory threshold for the FISH probe in CD3+ peripheral blood cells, we analyzed T-cells of 25 healthy men with a median age of 27 years (range 19–35). Furthermore, we just initialized an investigation of the laboratory threshold for the FISH probe in CD3+ peripheral blood cells of elder men by measuring the frequency of loss of the Y chromosome in T-cells of this control cohort not suffering from hematopoietic diseases. Until now we could recruit 15 men with a median age of 75 years (range 66–84) for this purpose, further will follow soon.

Results:
In patients with suspected or proven MDS, the number of cells with -Y was significantly increased in CD34+ cells compared to CD3+ cells (p<0.0001). The median clone size was 64% (range 12–97) in CD34+ cells and 5% (range 1–14) in CD3+ T-cells. The clone size in CD34+ cells was at least four times higher than in CD3+ cells in all patients. We could not detect further chromosomal abnormalities in 16 patients. Chromosomal banding analysis revealed that cells with -Y and cells with -Y and +8 occurred in parallel in two patients. In men below the age of 35 Y-loss could not be detected. The median clone size of 0.5% (range 0–2) resulted in a laboratory threshold of 2%. Interim analysis of men over the age of 65 resulted in a median clone size of 2.5% (range 1–14) and a laboratory threshold of 13%. So far the FISH-signal corresponding to the Y chromosome was significantly more frequent missing in T-cells of elder than in T-cells of younger men (p=0.005).

Conclusion:
Regarding the absence of Y-loss in CD3+ peripheral blood cells of young healthy men compared to up to 14% -Y in CD3+ peripheral blood cells of elder men, the low proportion of -Y in CD3+ cells of our patients suggests an age related Y-loss in normal T-cells. As the number of CD34+ peripheral blood cells with -Y exceeds the number of CD3+ peripheral blood cells with -Y in all patients, we assume that Y-loss is clonal to some extent in all of them. We established a reliable method to test if loss of the Y chromosome is disease- or age-related in individual MDS patients. It can be used to determine a clonal disease in patients with suspected MDS and Y-loss as sole abnormality.

Disclosures:
No relevant conflicts of interest to declare.