Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (<20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (>80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

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