MicroRNA-29c and 223 Are Powerful Prognostic Factors for Chronic Lymphocytic Leukemia and Improve Risk Stratification When Combined with ZAP70 and LPL in a qPCR Score.
Authors: Stamatopoulos Basile , Meuleman Nathalie , Bron Dominique , Haibe-Kains Benjamin , Saussoy Pascale , Martiat Philippe , Lagneaux Laurence 
Published: April 2020

Abstract
Background: MicroRNAs (or miR) are a novel class of small noncoding RNA involved in gene regulation. Aberrant microRNA expression has been recently associated with chronic lymphocytic leukemia (CLL) outcome. Currently, the heterogeneous evolution of this disease can be predicted by several prognostic factors. Nevertheless, a better individualization of the outcome in a given patient is still of utmost interest.
Methods: In the current study, we investigated the expression of two microRNAs, miR-29c and miR-223, compared them to other biological or clinical markers and proposed a quantitative real-time PCR (qPCR) score to better assess CLL outcome. All cut-offs were calculated by ROC curve analysis maximising the correlation with the immunoglobulin variable heavy chain (IgVH) mutational status; statistical differences were evaluated by Mann Whitney test or Kruskal-Wallis test ; treatment-free (TFS) and overall (OS) survival differences were investigated by log-rank test or Cox proportional hazard ratio (HR).
Results: miR-29c and miR-223 expression decreased significantly with progression along Binet Stage A to C (P=0.0010 and P=0.0183, respectively), and were significantly lower in poor prognosis subgroups defined by cytogenetic abnormalities, IgVH mutational status, lymphocyte doubling time, solubleCD23, β2-microglobulin, ζ-associated protein 70 (ZAP70), lipoprotein lipase (LPL) and CD38 expression. Furthermore, miR-29c and miR-223 could predict TFS (n=110, P=0.0015 and P&lt;0.0001, respectively) and OS (n=110, P=0.0234 and P=0.0008, respectively). Regarding all these results, we developed a qPCR score (from 0 to 4 poor prognostic markers) combining miR-29c, miR-223, ZAP70 and LPL in order to stratify treatment and death risk in a 110 patient cohort with a median follow-up of 72 months (range, 2–312). Patients with a score of 0/4, 1/4, 2/4, 3/4, and 4/4 had a median TFS of &gt;312, 129, 80, 36 and 19 months, respectively (HR=17.00, P&lt;0.0001). Patient with a score of 0–1/4, 2–3/4 and 4/4 had a median OS of &gt;312, 183 and 106 months, respectively (HR=13.69, P=0.0001). Interestingly, during the first 50 months after diagnosis, only 10% of patients with a 0/4 score required a treatment, when compared to 100% of the 4/4. Furthermore, during the total follow-up (312 months), patients with a 4/4 score had a 27-fold higher risk to be treated and a 31-fold higher risk to die comparing to patients with a 0/4 score. This score was validated by a 10-fold cross-validation (prediction accuracy of 82%). Finally, in Binet stage A patients (n=77), this score remained relevant and significant for TFS and OS prediction (HR=18.56, P&lt;0.0001 and HR=12.5, P=0.0068, respectively).
Conclusions: we showed that (i) miR-29c and miR-223 levels were decreased in poor prognosis patients regarding several well-known prognostic factors; (ii) a low level of these two microRNAs is thus associated to disease aggressiveness, tumor burden and poor clinical evolution; (iii) we also showed that these two microRNAs could predict TFS and OS; (iv) we proposed a qPCR score to better individualize evolution of a particular CLL patient. This score will help to identify patients who will need early therapy and require thus a closer follow-up.