THE EXPRESSION OF A SINGLE MICRORNA - MIR-34A – IS A RELIABLE MARKER FOR IMPAIRMENT OF P53-PATHWAY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:

We and others have shown that miR-34a is down-regulated in the aggressive CLL subtype harbouring p53 aberration (Mraz, 2009; Zenz, 2009; Asslaber, 2010). However, these studies only specifically studied the expression of miR-34a or few other selected miRNAs that were previously shown to be associated with cancer biology.

Aims:

Here we performed an unbiased search for miRNAs that are involved in fludarabine-induced cell death in CLL cells in vitro and in vivo to investigate the relevance of miR-34a as a marker for impairment of p53 pathway and resistance of CLL to therapy.

Results:

To test for miRNAs that are involved in apoptotic pathways in CLL cells we treated in vitro ten purified primary CLL B-cell samples with fludarabine (IC50 dose of 3.5 ug/mL, 48hrs), and profiled the expression of 750 miRNAs (TaqMan miRNA Cards, ABI). This identified induction of 15 miRNAs (fold induction [FI] >1.5) with miR-34a being the most prominently up-regulated miRNA (FI 3.7). To verify this observation in vivo we obtained peripheral blood from CLL patients treated with a FCR regimen that includes administration of rituximab (R) on day 1, followed by fludarabine (F) and cyclophosphamide (C) on day 2, 3 and 4. The samples were obtained from patients before administration of therapy, 24hrs after administration of R and 48hrs after FC. The administration of R did not induce miR-34a expression, which was however clearly induced in most samples (96%) after FC administration (FI 2.2, P<0.0001). Additionally, the levels of miR-34a before or after FCR administration were strongly correlated with P53-aberration present in 5 out of 52 FCR-treated patients; with patients harbouring P53-aberration having the lowest miR-34a levels from the whole cohort. Interestingly, CLL cases with deletion of ATM (del11q23, N=19), an up-stream regulator of p53, had similar basal and induced levels of miR-34a compared to cases without del11q23 (N=28). Importantly, when the FCR-treated patients were divided into terciles based on the level of miR-34a after FC, those with the lowest levels of miR-34a experienced significantly shorter time to treatment failure (1.1 years vs. 2.2 years; P=0.037; HR 3.01). These data and the broad screening of fludarabine-induced miRNAs confirmed that miR-34a is the preferential miRNA activated with apoptosis and DNA damage pathway in CLL. This prompted us to develop an assay for absolute quantification of miR-34a that allowed us to  determine  the copy numbers of miR-34a and to define the  precise cut-offs (similarly to assays for bcr-abl quantification in CML). Using this assay, we profiled the expression of miR-34a in 200 CLL patients (miR-34a expression ranged from 1 to 81820 copies/10e6 copies of internal standard). Significantly, miR-34a levels below 2500 copies (N=47) were correlated with shorter overall survival (9.6 years vs. not-reached, HR 2.2 [CI 1.1-4.5], P=0.03). The low miR-34a levels were also apparently associated with the deletion & p53 mutation of 17p13 (N=18) or sole p53 mutation (N=13) in this second CLL cohort (P<0.005).

Summary / Conclusion:

Our data provide evidence and novel tool for the use of miR-34a as a marker of p53-aberration, which can be especially useful in cases with sole p53 mutation not accompanied by 17p13 deletion that cannot be discriminated by routine FISH. Patients with sole p53 mutation represents approx. 1/3 of p53 aberrant cases with inferior prognosis similar to cases with del17p13.

IGA MZCR NT11218-6/2010, FR-TI2/254.

Email address: hematoonkologie@gmail.com

Abstrakta v časopise Haematologica 2013, Suppl1

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