Impact of Genemutations and Dynamics Methylation of CDKN2B and DLC-1 Promoters on Treatment Response with Azacitidine

Introduction: Azacitidine (AZA) is a DNA hypomethylating agent used to treat high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in patients who are not eligible for intensive strategies. In view of the variability of drug response, last years it has been studied the genes mutations as possible response biomarkers. In addition, although there seems to be no association between promoter genes methylation and response, dynamics methylation before and after AZA treatment could be a predictable tool of clinical response. CDKN2B gene is a regulator of cell cycle and its aberrant hypermethylation is recurrent in myeloid diseases. DLC-1 gene plays a critical role in cell migration and proliferation and although its abnormal methylation is common in different kinds of cancers, its role in hematological malignancies is slightly studied.

Aim: To assess the predictive value of gene mutations and dynamics methylation of CDKN2B and DLC-1 promoters on AZA response in a series of patients with myeloid neoplasms.

Methods: We studied 61 patients with MDS and 16 patients with AML who received AZA treatment. According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Ten genes with prior implication in the pathogenesis of myeloid diseases were analyzed: DNMT3A , IDH1 , IDH2 , ASXL1 , SETBP1 , TP53 , SF3B1 , SRSF2 , U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. The methylation levels of CDKN2B and DLC-1 were studied with unmethylated and methylated primers, positive and negative controls and analysis of the PCR products in a QIAxcel System (Qiagen). In all patients CDKN2B and DLC-1 methylation were analyzed pre-AZA treatment and in 57 patients with post-AZA DNA sample the methylation assay was also possible at that point. P values <0.05 were considered as statistically significant .

Results: Of the 77 patients analyzed, 30 patients (39%) showed AZA response (median cycles=6): CR=13, mCR=2, PR=8, and SDHI=7. Stable disease and treatment failure were observed in 19 (25%) and 28 patients (36%), respectively. Pre-treatment, genetic mutations were presented in 63 patients (82%) and methylationof CDKN2B and DLC-1 promoters were detected in 32 (42%) and 37 (48%) patients respectively (Figure 1). Patients with mutations in DNA methylators ( DNMT3A , IDH1/2 ) showed a significant worse AZA response than the remaining patients (9% vs. 44%, P =0.026) and furthermore DNMT3A patients presented a lower overall survival (OS) compared with DNMT3A patients (8 vs . 19 months, P =0.042). All SETBP1 patients also did not show AZA response and moreover transformed to AML in a few months. On the other hand, TP53 mutations did not influence the drug response although they were associated with complex karyotypes (92% vs. 8%, P <0.001) and with a lower OS (10 vs. 21 months, P =0.043). Conversely, splicing genes mutations ( SF3B1 , SRSF2 and U2AF1 ) were related to favourable karyotypes (78% vs. 54%, P =0.007), higher OS (28 vs. 13 months, P =0.038) and lower DLC-1 methylation (33% vs . 64%, P =0.010). Interestingly, patients with DLC-1 methylation showed higher levels (>5%) of bone marrow blasts (74% vs . 42%, P =0.005), higher frequency of complex karyotypes (29% vs. 9%, P =0.002) and lower OS (15 vs . 30 months, P =0.044). In addition,dynamics methylation of DLC-1 was significantly associated with the AZA response: 59% patients who reduced DLC-1 methylation levels post-treatment, responded favourably to the drug and 95% patients who maintained or increased them, did not respond ( P =0.002; Figure 2). Finally, CDKN2B methylated patients had a significant co-occurrence with favourable karyotypes (80% vs. 45%, P =0.014), presented a higher OS (28 vs. 13 months, P =0.018) but its dynamics methylation was not associated with the AZA response ( P =0.07).

Conclusion: In our seriesof patients treated with AZA, DNMT3A and IDH1/2 mutations have a negative impact on drug response. DLC-1 dynamics methylation is associated with the AZA response and clinical outcome.The combination of point mutations and monitoring methylation could contribute to give light to the heterogeneity of AZA response in myeloid neoplasms.

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