MicroRNA Expression Profiling of CLL B Cells From Relapsed/Refractory CLL Patients Reveals a Signature Group of Genes That Are Associated with Clinical Responses: Results of An ECOG Trial E2903.

MicroRNA (miRNA) profiling in CLL has revealed a leukemia specific gene signature in CLL patients. Our earlier work found a unique signature of 13 deregulated microRNAs from 190 previously untreated CLL patients. This signature included some of the most frequently deregulated miRNAs in different types of hematological malignancies (miRNA-15-a/16-1, miRNA-29 family, miRNA-155, miRNA-221) and correlation with CLL prognostic factors (ZAP-70 expression and immunoglobulin heavy chain variable region (IGVH) mutations). Since these miRNAs have both diagnostic and prognostic implications, we wished to extend our observations to a cohort of relapsed/refractory CLL patients entered on the ECOG trial E2903. This trial is a chemoimmunotherapy protocol that uses 6 cycles of pentostatin, cyclophosphamide and rituximab to induce responses followed by consolidation with Alemtuzumab.

To assess miRNA status, we used our previously described method which employs a bead-based profiling that interrogates 649 microRNA genes. We used the Trizol extracted RNA from the CLL B-cells of 33 patients entered onto the E2903 clinical trial. All patients were also characterized by determining their CD38, ZAP-70, fluorescence in situ hybridization (FISH) and IGVH status. All microRNA and prognostic parameters were done on baseline CLL B-cells obtained from the patients prior to going on study.

The demographics of this CLL patient population included 28 males with median age of 61 (range; 47-76), 1 Rai stage 3 and 24 Rai stage 4. The median number of prior treatments was 2 (range;1-5). To date with PCR treatment only assessed, there were 15 partial responses (PR), 15 stable disease, and 3 unevaluable patients. Because of a batch effect, we restricted our analysis in this report to only 24 patients. We decided to initially compare the microRNA profiles for CLL patients segregated on the following; responders (CR/PR) vs. non responders (SD or less), ZAP-70+ vs. ZAP-70- (20 % cutoff), CD38+ vs. CD38– (30% cutoff), Low-risk FISH (normal, 13q- or trisomy 12) vs. High-Risk FISH (17p- or 11q- or complex) and age '65 vs. ≥ 65. In addition, we did combine some of the prognostic parameters or responses together for comparison of microRNA status (ZAP-70 with either response, IGVH status, or CD38 status). This analysis revealed a set of 10 microRNA genes with a median false discovery rate of 20% that were aberrantly expressed when we compared the ZAP-70- responders to the ZAP-70+ non-responders. In addition, we found 15 differentially expressed miRNAs when we compared ZAP-70- to ZAP-70+ cohorts. We then evaluated the association of the 15 genes found in the ZAP-70 comparison with the overall survival and progression free survival (PFS). We found that miR-625* was significantly associated with PFS (p=0.01). The expression level of miR-625* was more than 2-fold increased in ZAP-70- compared to ZAP-70+. miR-625*, located at 14q23.3, has more than 60 putative target genes, including nuclear and cell cycle, and cell adhesion targets.

Interrogation of the genes that are impacted by the deregulated miRNAs uncovered in our analysis of this previously treated CLL cohort may provide valuable information regarding the mechanism of resistance commonly seen in this cohort of heavily pretreated CLL patients.

Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees.