BIOLOGICAL AND CLINICAL RELEVANCE OF MIRNAS INDUCED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS DURING ADMINISTRATION OF THERAPY IN VIVO AND IN VITRO

ABSSUB-5225

Background: MiRNAs are known to be involved in the pathogenesis of CLL and affect its clinical course. However, their function in the apoptotic pathways in CLL B-cells, and their roles in primary and/or acquired resistance to therapy are unclear.

Aims: The aim of this study was to reveal miRNAs changed with apoptotic response of CLL cells and to test their role in response to FCR treatment in vivo.

Results: To screen for DNA damage-regulated miRNAs we used RNAs isolated from patients (pts.) B-cells (purity>95%, CD5+19+) treated in vitro with fludarabine dose of ~LC50 (3.5 μg/ml; 48hrs). Five paired samples (n=10) were analyzed for miRNAs expression each using 2 next-generation sequencing (NGS) approaches (SOLiD; HiSeq). Obtained sequences were mapped to miRBase and miRNA changes were analyzed by a pair-wise comparison with edgeR and baySeq packages (Bioconductor). The overlap of 2 NGS sequencing platforms analyzed by the 2 mentioned statistical approaches identified 6 miRNAs significantly changed with DNA damage (FDR<0.1). HiSeq validation on 5 additional independent paired samples (n=10) confirmed changed expression of all 6 miRNAs (3 down-, 3 up-regulated). We further screened the expression of the two most constantly up-regulated miRNAs, namely miR-34a and miR-1246, in a cohort of pts. (n=40) treated with fludarabine, cyclophosphamide and rituximab (FCR) regimen in vivo. We observed significant induction of miR-34a and miR-1246 at 48 hrs post FCR administration (fold induction [FI]=1.8; p<0.001; FI=2.2; p=0.01, respectively). Interestingly, the basal miR-34a levels before the therapeutic intervention were able to distinguish pts. with more aggressive disease. The pts. with low miR-34a expression (lowest quartile) had a shorter time to treatment failure (1.04 yrs. vs. 2.16 years; p=0.002; HR=4.3; 95 % CI=1.68-10.98) and a remarkably shorter time to relapse after FCR-achieved remission (1.28 yrs. vs. not reached; p=0.05; HR=3.07; 95% CI=0.97-9.65).

We further screened the expression of miR-34a in 158 CLL pts. using an in-house designed assay for its absolute quantification. We defined a cut-point (copies of miR-34a) that segregates pts. with extremely unfavorable prognosis (overall survival [OS] 1.37 yrs. vs. not reached; p=0.0001; HR=3.89; CI=2.05-7.39) and this was independent of other prognostic markers (FISH, IgHV, age, sex) in a multivariate analysis. We have previously described that low miR-34a levels associate with p53 mutations, and low-miR-34a identified such CLL cases with a sensitivity and specificity of 0.75 and 0.91, respectively. In an OS multivariate analysis limited to wt-TP53 samples (n=116) miR-34a was the strongest predictor of OS (1.29 yrs. vs. not reached; p=0.002; HR=9.82; CI=2.30-42.05).

Summary/Conclusion: We have defined that FCR-induced miR-34a can be used as a strong predictive and prognostic marker. We have newly identified other 5 miRNAs involved in DNA damage response in CLL (including miR-1246). The investigation of the biological and prognostic role of miR-1246 and other miRNAs is ongoing. These miRNAs likely have convergent functions since the DNA damage induced miRNAs share >50 predicted targets with miR-34a, and thus could affect therapy response and aggressiveness of CLL. This is currently being investigated using integrated analysis of miRNA and mRNA profiling.

Supported by: EHA Research Fellowship award; MUNI/A/0830/2013; SoMoPro II Programme; GACR 14-22712P; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; CZ.1.07/2.3.00/30.0009; NGS-PTL/2012-2015/no.306242; MSMT (2013-2015, no.7E13008);  VaVPI CEITEC (CZ.1.05/1.1.00/02.0068); co-financed from EU, South-Moravian region and Czech Rep.

Keywords: B cell, Chronic lymphocytic leukemia, RNA interference (RNAi)

http://www.ehaweb.org/