A New MicroRNA Risk Model for Prediction of Clinical Outcome In Chronic Lymphocytic Leukemia

Increasing experimental evidence supports the idea of aberrant microRNA (miRNA) expression in cancer pathogenesis, especially in chronic lymphocytic leukemia (CLL). Recently, aberrant expression of miR-29c and miR-223 has been associated with CLL outcome. Moreover, low expression of miR-34a in CLL is associated with p53 inactivation and also chemotherapy-refractory disease.

Therefore, we investigated miR-29c, miR-223 and miR-34a expression in a large representative cohort of 110 CLL patients in order to assess the role of these miRNAs in risk prediction in B-CLL.

Mononuclear cells of B-CLL were isolated from whole blood by centrifugation on a Ficoll/Hypaque gradient and cryopreserved in liquid nitrogen. MicroRNA was extracted from native liquid-nitrogen frozen cells using the QIAGEN miRNeasy® mini kit (Qiagen, Hilden, Germany). The relative expression of mature miRNAs was quantified using the TaqMan MicroRNA RT kit and TaqMan® MicroRNA Assays (Applied Biosystems, Foster City, California) on the ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, California). RNU6B was used as internal control. The relative expression of each microRNA of interest to RNU6B was calculated using the formula miRNA of interest/RNU6B=2^{-deltaCt(miRNA-RNU6B)}.

The impact of the three miRNA expressions on treatment-free survival (TFS) and overall survival (OS) was assessed by performing “Receiver Operating Characteristics” (ROC) curve analysis. Patients with low miR-29c, miR-223 or miR-34a expression had a shorter TFS and OS than those patients with high expression:

miR-29c: TFS 49 vs 91 months (p=0.081); OS 164 months vs not reached (p=0.093)

miR-223: TFS 27 vs 84 months (p=0.035); OS 132 months vs not reached (p=0.001)

miR-34a: TFS 46 vs 83 months (p=0.084); OS 132 months vs not reached (p=0.001)

Furthermore, we investigated whether combining information on the expression of miRNAs could help refine the prognostic information provided by either of the three risk factors (RF) alone. Adding the number of risk factors this approach allowed for highly significant separation of our patient cohort into four subgroups, which differed significantly with regard to their clinical outcome.

TFS: 0 RF 99 months; 1 RF 75 months; 2 RF 26 months; 3 RF 22 months (p=0.008)

OS: 0 RF not reached; 1 RF not reached; 2 RF 164 months; 3 RF 132 months (p<0.0001)

Moreover, in multivariate analysis the miRNA risk model was a significant independent prognostic factor (HR 1.3; 95%CI 1.0–1.8; p=0.04) next to the stage of Binet (HR 2.1; 95%CI 1.4–3.4; p=0.001) and ZAP-70 status (HR 3.0; 95%CI 1.5–5.9; p=0.002).

Combined analysis of miR-29c, miR-223 and miR-34a expression in a risk model may help optimize currently available prognostic instruments. Future studies are warranted to elucidate the role of miRNAs as a prognostic factor for response and clinical outcome.

No relevant conflicts of interest to declare.