Changes Identified by Flow Cytometry and WT1 Expression in Consecutive Bone Marrow Samples in Refractory Cytopenia of Childhood and Aplastic Anemia Before Start of the Therapy
Authors: Reiterova Michaela , Kramarzova Karolina , Sukova Martina , Campr Vit , Vodickova Elena , Zapletal Ondrej , Blatny Jan , Zemanova Zuzana , Lizcova Libuse , Pelkova Vendula , Trka Jan , Stary Jan , Hrusak Ondrej , Mejstrikova Ester 
Published: October 2019

Abstract
Abstract 1342

Introduction.
Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. The most common subtype of MDS is refractory cytopenia (RC). Both diseases typically exhibit with overlapping features and in both disorders dysregulation of immune system variably contributes to the degree of bone marrow (BM) failure. In the diagnostic algorithm plays role also analysis of consecutive BM samples by morphology.

Patients and methods.
Patients diagnosed between 2005 – 2011 with at least two BM samples analyzed by flow cytometry (FC) before treatment has started and with centrally evaluated BM biopsy according to EWOG MDS criteria were included into the study. We compared first and the last available sample before treatment (immunosupression or stem cell transplantation). By FC we analyzed following parameters: cell subsets (granulocytes, monocytes, lymphoid cells, erythroid precursors), BM precursors (CD34pos, CD117pos), T cells (CD3pos, CD3pos4pos, CD3pos8pos, CD3posHLA DRpos out of all cells, HLA DRpos out of CD3pos/CD3pos4pos/CD3pos8pos cells); B cells (CD19pos, CD19pos10pos, CD19pos45dim to neg, CD19pos34posout of all cells, CD10pos and CD20pos10neg out of CD19pos). In total 22 patients with AA (12 girls, 10 boys, mean age 11 years; 1.1–18 years) and 20 patients with RC (11 girls, 9 boys, mean age 11 years; 3.7–18) were included into the study. Median of time interval between both samples was 139 (1–1343) days in RC and 15 (1–56) days in AA. WT1 expression on mRNA level was analyzed in the sample before treatment with the highest number of CD34pos precursors to avoid blood contamination. All patients were screened by FISH for changes on chromosome 7 and 8. We asked following questions: Are there differences in the parameters in both bone marrow samples between SAA and RC? Is there any different pattern between d0 and before therapy sample between AA and RC? Are there any differences in WT1 expression between AA and RC group?

Results.
RC and AA significantly differ in both time points. AA patients have significantly decreased precursors (CD34, CD117); the difference is more pronounced at the later time point. More lymphocytes (both B and T) and less granulocytes are present at later time point in AA patients (p<0.05, Mann-Whitney test). Activation of CD8 cytotoxic T cells according to HLA DR expression is more distinct in AA patients at later time point. The most significant different parameter between RC and AA is a ratio CD19/CD34 also with the significant trend between two time points (Two way ANOVA, p<0.05). WT1 expression is statistically higher in RC patients; the higher expression is associated with presence of monosomy 7.

Conclusion.
By FC statistical differences can be identified in both samples (d0 and before treatment) between RC and AA. More pronounced differences are at later time point, which can be explained by further destruction of precursor and myeloid compartment more pronounced in AA patients compared to RC. WT1 expression is typically high in patients with RC and monosomy 7.

Disclosures:
No relevant conflicts of interest to declare.