TGFβR2 Methylation Assessed by Quantitative-MSP in Multiple Myeloma Patients: An Independent Prognostic Marker

Introduction: Multiple myeloma (MM) is a B-cell neoplasm characterized by multiorgan dysfunction as a result of bone marrow infiltration by malignant cells and systemic damage of monoclonal circulating protein. Molecular studies have largely focused on acquired genetic aberrations in MM. The accumulation of genetic events is thought to be crucial for the malignant transformation of plasma cells. DNA methylation is associated with several changes in chromatin structure, including the regulation of histone methylation and acetylation and the recruitment of proteins to the methylated sites. This leads to the obstruction of the promoter, and subsequent gene silencing. Aberrant promoter methylation of genes has been described for several genes in MM. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function.

Objectives: We determined the aberrant DNA methylation status of 20 genes (AIM1, CCNA1, CCND2, CDH1, CDKN2A, CDKN2B, DCC, ESR1, GSTP1, HIC1, MGMT, MINT31, p14ARF, PTGS2, RARβ, RB1, SFN, SOCS1, TGFβR2, and THBS1) in 51 samples of MM and compared the methylation profile with clinicopathological characteristics of the patients.

Methods: DNA was isolated using the TRIzol reagent (Invitrogen), from bone marrow aspirates of MM patients. The promoter methylation pattern was determined by quantitative methylation specific PCR (QMSP).

Results: The QMSP analysis showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%), and TGFβR2 (39.2%) were frequently methylated in MM at diagnosis while hypermethylation of RARβ (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%), and THBS1 (4.1%) were rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. The median age of the 51 MM patients was 65 years (range, 27–80 years) and 56.8% were male. According the monoclonal component isotype, the patients were classified as IgG isotype (56.6%), IgA isotype (24.5%) and others (18.8%). The kappa light chain monoclonal protein was present in 64.7% of the patients, while the lambda protein was detected in 27.4% of the cases. Based on Durie Salmon staging system, 5.9% were IA, 3.9% were IIA, 52.9% were IIIA and 33.4% were IIIB, confirming that most of our patients were diagnosed at advanced stage disease. According to ISS system, 13.7% of cases were ISS 1, 31.4% ISS 2, 49% ISS 3. More than 86% of the cases have > 50% of monoclonal plasma cells in their bone marrow assessed by biopsies. Methylation of ESR1 was correlated positively with isotype IgA (p = 0.016), while methylation of THBS1 correlated negatively with isotype IgG (p = 0.031). The 3-year overall survival was 31.5%. The clinicopathological parameters such as Durie Salmon Stage III (p = 0.015), ISS 3 (p = 0.007) and non-transplanted cases (p = 0.019) were significantly associated with reduced overall survival. The aberrant DNA methylation analyses showed that hypermethylation of DCC and TGFβR2 were also correlated with poor survival (p = 0.0034 and p = 0.0016, respectively). The multivariate analysis showed ISS (95% CI, 1.24 – 5.86, p = 0.012) and methylated TGFβR2 (95% CI, 1.02 – 4.62, p = 0.044) strongly correlated with poor outcome.

Conclusion: The current study represents the first reported quantitative evaluation of MM methylation profile and our results demonstrated that aberrant promoter methylation is a frequent event in this disease. Furthermore, our data provide evidence that TGFβR2 methylation may be useful as prognostic indicator in MM.