New Targets in Cytometric Investigation of Acute Leukemia Selected From Gene Profiling Studies
Authors: Vaskova Martina , Mejstrikova Ester , Brdicka Tomas , Angelisova Pavla , Kalina Tomas , Kanderova Veronika , Cerna Daniela , Kovac Martin , Trka Jan , Lund-Johansen Fridtjof , Stary Jan , Hrusak Ondrej 
Published: October 2019

Abstract
Abstract 2536
BACKGROUND. Gene expression profiling (GEP) studies have identified previously unknown molecules that correlate with genotypes of acute leukemia (AL), prognosis and/or the malignant status itself. Although some of these molecules ignited enthusiasm upon their publication, confirmation on independent cohorts and using other reliable methods is needed. Using specific tools on gene or protein level will bring us closer to a conclusion as to what is the biologic importance of the molecules and whether each molecule is worth testing as potential new diagnostic or immunotherapeutic target. METHODS. We have selected genes with probable relevant correlation(s) with conditions listed above. Correlations were then tested using qPCR. Successfully tested genes were selected for the production of new monoclonal antibodies (mAbs), unless satisfactory mAbs were already available. The newly produced mAbs were tested together with those, which were commercially available, using cell lines and diagnostic bone marrow or peripheral blood specimens of children with AL; data were compared also to non-malignant tissues. Flow cytometry was applied both to entire cells and to a novel method of Size Exclusion Chromatography-Microsphere-based Affinity Proteomics (Size-MAP). RESULTS. Eighteen genes (CMTM2, AGPS, EFNB1, DBN1, Clic5a, MYO, LARGE, DDIT4L, TCEAL4, PCLO, ARHGEF4, HYA22, RAG1, Opal 1, Siva, EVI2b, CDC42EP3, and SH3BP5) were selected for testing on qPCR and/or cytometric level. Among them, correlations were confirmed in 9 molecules, excluded in 3 molecules, failed to conclude due to insufficient testing system (cell lines) in 2 molecules and pending in 1 molecule. In 3 molecules, we started mAb production without qPCR testing. The results were combined with those related to previously known mAbs. We have produced 56 novel mAb clones to 9 target molecules. Seventeen mAbs to 9 molecules were further tested using flow cytometry both on cells and by Size-MAP. Of these, mAb to drebrin (DBN1, Figure) was proven to correlate with TEL/AML1 positivity, while its correlation to better prognosis is waiting for a longer follow up.Figure:Two diagnostic specimens of leukemic cells that had negative (left) or high (right) expression of drebrin.Figure:. Two diagnostic specimens of leukemic cells that had negative (left) or high (right) expression of drebrin.
Anti-drebrin and anti-OPAL-1 mAbs both showed reproducible signals using Size-MAP. The data were compared with the strong predictors of TEL/AML1 status (CD27 and CD44) that we showed previously. CONCLUSIONS. Some but not all of the molecules that were presented by GEP as promising targets can be detected by flow cytometry and their correlations with molecular genetics was confirmed.
Grant support: National Program of Research II (NPVii, project 2B06064), GACR 301/10/1877, MZ CR NS10004-4, NS10480-3 and NS10473-3.

Disclosures:
No relevant conflicts of interest to declare.