Molecular Characterization of High-Risk MDS Patients Treated with Azacitidine and Lenalidomide: Role of Inositide-Dependent Signalling, Mutations and Microrna

Background: Several clinical studies demonstrated the efficacy and safety of combining azacitidine with lenalidomide in Myelodysplastic Syndromes (MDS), but the molecular implications of this therapy (i.e. gene mutations and microRNA profiling) are still under investigation. Moreover, phosphoinositide-specific phospholipase C (PI-PLC) beta1 is modulated in MDS cells treated with azacitidine, whereas lenalidomide, restoring a normal erythropoiesis, can affect PI-PLCgamma1 pathways. Purpose: This study firstly aimed to assess the effect of azacitidine and lenalidomide combination therapy on the expression of inositide-dependent signalling, focusing particularly on PI-PLC isoenzymes. Moreover, an extensive analysis on gene mutations and microRNA expression profiling was also performed. Patients and Methods: This study included 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Paired samples, before and during treatment, were analyzed. Firstly, we quantified the expression of PI-PLC isoenzymes by Real-Time PCR and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In addition, the frequency of recurrent gene mutations associated with myeloid malignancies was evaluated, using an Illumina Cancer Myeloid Panel, and the expression of microRNAs was assessed using an Affimetrix array. Results: In our case series, 34/44 patients completed at least 6 cycles of treatment and were clinically evaluable, with an overall response rate of 76.5% (26/34 cases). Firstly, the therapy affectedthe MDS cell cycle. Moreover, the expression of PI-PLCs and Beta-Globin could be associated with a favourable clinical response to the combination therapy, hinting at a specific contribution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. At baseline, the most frequent gene mutations were ASXL1 (59%), TET2 (41%), RUNX1 (35%) and SRSF2 (29%). Interestingly, all patients showing SRSF2 mutations evolved into AML. Moreover, all patients maintaining a stable disease during the therapy had increasing variant allele frequencies, showing a genetic instability. Finally, microRNA analysis revealed specific clusters associated with the treatment: 14 microRNAs were overexpressed and 28 were under-expressed as compared to baseline levels. Interestingly, down-regulation of 5 microRNAs was significantly associated with the lack of response. Conclusions: Our results show that the combination of azacitidine and lenalidomide can affect PI-PLC signalling, possibly regulating MDS cell cycle, myeloid and erythroid differentiation. Moreover, the therapy can induce a change in the specific gene mutation and microRNA profile that, if confirmed by larger studies, could be important to better evaluate the response to this therapy.