Abstract
Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.
Highlights
The transformation of normal cells into cancer cells is a multistep process, involving irreversible changes of the DNA sequence [1]
2.1 Patients and Cell Lines In the present study we included DNA from 62 fresh-frozen primary diagnostic biopsies of patients diagnosed with B-cell lymphoma (activated B cell like diffuse large B-cell lymphoma (DLBCL activated B-cell type (ABC)); n = 18, germinal centre cell like diffuse large B-cell lymphoma (DLBCL germinal center B-cell type (GCB)); n = 17, primary mediastinal B-cell lymphoma (PMBL); n = 6, follicular lymphoma (FL); n = 14 and Burkitts lymphoma (BL), n = 7) and 52 various healthy donors (CD19+-B-cells isolated from buffy coat with CD19+ Dynabeads (Invitrogen) as previously described [20]; n = 20, follicular hyperplasia samples; n = 9, peripheral blood mononuclear cells; n = 10, bone marrow, n = 3 and tonsils; n = 10)
The epigenetic drug treatment of Bcell lymphoma cell lines (BL: Raji, BL41, Ramos; DLBCL ABC: HLY-1, OciLy3, OciLy10; DLBCL GC: SUDHL4, SUDHL6; FL: K422, SC-1, ROS50) with 5-aza-29deoxycytidine and Trichostatin A (TSA) and a subsequent genome-wide expression analysis revealed that 2027 array elements were upregulated a minimum of two fold in at least 6 out of the 11 analyzed cell lines
Summary
The transformation of normal cells into cancer cells is a multistep process, involving irreversible changes of the DNA sequence [1]. The number of methylated genes found in NHL is constantly increasing [10,11], most studies are focusing on only one NHL type [12,13,14,15] and so far only a handful studies have examined the putative use of methylation markers as diagnostic or prognostic tools [16,17,18]. This is of importance since, due to new treatment regimes, established biomarkers for NHL are losing the power of predicting patients outcome. We further analyze the ability of the candidate genes to differentiate lymphoma patients from healthy controls
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