PF363 UNDETECTABLE EXPRESSION OF WNT5A, A GENE ENCODING A ROR1 LIGAND, DEFINES MEMORY B-CELL LIKE CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: Chronic lymphocytic leukemia (CLL) is a malignancy with highly variable disease course among patients. Although many approaches distinguishing patients with worse and better prognosis have been published, IGHV somatic hypermutation status remains one of the strongest prognostic predictors. However, even within prognostically favorable IGHV-mutated (M-CLL) group, there is a subset of “grey-zone” patients with worse prognosis. We have shown recently that expression of WNT5A, a gene encoding a ligand activating WNT/PCP signaling pathway via ROR1 receptor, distinguishes the cases with worse prognosis within M-CLL (Janovska, Clin Cancer Res 2016). Still, over 80% of M-CLL patients had undetectable WNT5A expression, suggesting possible silencing of the gene expression through its methylation. Aims: To analyze methylation status of WNT5A promoter in the context of WNT5A mRNA expression, IGHV somatic hypermutations and methylation-based CLL classification (Querios, Leukemia 2015). Methods: Using bisulphite sequencing we analyzed methylation status of 3 CpG islands (CGI) located in WNT5A promoter and 5 marker CpG sites distinguishing methylation-based prognostic subgroups of CLL (located in B3GNTL1, CTBP2, TNF, SCARF1 and intergenic region on chromosome 14). mRNA expression was analyzed with quantitative real-time PCR using TaqMan probes (ThermoFisher Scientific): WNT5A, and HPRT1, TBP were used as controls. Results: Thirty-nine previously untreated CLL patients were chosen for the analysis. They were equally distributed among 4 groups based on IGHV status and WNT5A expression [WNT5A- M-CLL (10), WNT5A+ M-CLL (9), WNT5A- IGHV-unmutated CLL (U-CLL; 10), and WNT5A+ U-CLL (10), respectively]. Within the first of the three CpG islands of the WNT5A promoter (CGI1), we identified three CpG sites whose methylation status significantly correlated with the WNT5A expression in M-CLL (P = 0,004; P = 0,004; P = 0,0268; Mann-Whitney test). Furthermore, complete demethylation of the three CpG sites was associated with high WNT5A expression in M-CLL. In fact, complete demethylation of the whole tested CGI1 region was noted in these patients. Methylation analysis of 5 marker CpGs (Querios, Leukemia 2015) enabled us to classify patients into three methylation-based prognostic groups: 9/10 WNT5A- M-CLL patients belonged to the memory B-cell like CLL (m-CLL) group, and 7/9 WNT5A+ M-CLL patients to the intermediate CLL (i-CLL). U-CLL patients irrespective of WNT5A expression were assigned as naive B-cell like CLL (n-CLL) in 19/20 cases.Summary/Conclusion: Within M-CLL, methylation of the 3 CpGs located in the CGI1 of WNT5A promoter correlated significantly with WNT5A expression. Higher WNT5A expression was associated with complete demethylation of CGI1 locus. Our classification based on WNT5A expression and IGHV status overlapped with the methylation-profile-based CLL classification (m-CLL, i-CLL, n-CLL). Furthermore, memory B-cell like CLL group was associated with undetectable WNT5A level in our cohort. Supported by grants: MUNI/A/1105/2018, CEITEC2020 LQ1601, MZCR-RVO 65269705. Spanish Ministry of Health, Plan Estatal de I + D + I, ISCIII, FEDER, FIS PI15/01763ISIII PI15/01763 and PI18/01484.