Kategorie: Maligní lymfomy a leukémie
Téma: Chronic lymphocytic leukemia - Translational Research
Číslo abstraktu: P077
Autoři: MUDr. Mgr. Marek Mráz, Ph.D.; Kateřina Černá; Mgr. Kateřina Musilová; RNDr. Jitka Malčíková, Ph.D.; Mgr. Karla Plevová (roz. Malinová), Ph.D.; Mgr. Šárka Pavlová, PhD; MvDr. Boris Tichý, Ph.D.; MUDr. Yvona Brychtová, Ph.D.; prof. MUDr. Michael Doubek, Ph.D.; prof. MUDr. Jiří Mayer, CSc.; MUDr. Martin Trbušek, PhD; prof. RNDr. Šárka Pospíšilová, Ph.D.
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.
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.
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.
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Datum přednesení příspěvku: 14. 6. 2013