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Carcinogenesis Advance Access originally published online on June 14, 2006
Carcinogenesis 2007 28(1):60-70; doi:10.1093/carcin/bgl092
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Discovery of novel epigenetic markers in non-Hodgkin's lymphoma

Huidong Shi, Juyuan Guo, Deiter J. Duff, Farahnaz Rahmatpanah, Rebecca Chitima-Matsiga, Mufadhal Al-Kuhlani, Kristen H. Taylor, Ozy Sjahputera, Melinda Andreski1, James E. Wooldridge1 and Charles W. Caldwell*

Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center University of Missouri School of Medicine, Columbia, MO 65203, USA
1 Department of Internal Medicine, Holden Comprehensive Cancer Center at University of Iowa Iowa City, IA 52242, USA

*To whom correspondence should be addressed at: Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri, 115 Business Loop I-70 West, Columbia, MO 65203, USA. Tel: +573 882 1234; Fax: +573 884 5206 Email: caldwellc{at}health.missouri.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-Hodgkin's lymphoma (NHL) is a group of malignancies with heterogeneous genetic and epigenetic alterations. Discovery of molecular markers that better define NHL should improve diagnosis, prognosis and understanding of the biology. We developed a CpG island DNA microarray for discovery of aberrant methylation targets in cancer, and now apply this method to examine NHL cell lines and primary tumors. This methylation profiling revealed differential patterns in six cell lines originating from different subtypes of NHL. We identified 30 hypermethylated genes in these cell lines and independently confirmed 10 of them. Methylation of 6 of these genes was then further examined in 75 primary NHL specimens composed of four subtypes representing different stages of maturation. Each gene (DLC-1, PCDHGB7, CYP27B1, EFNA5, CCND1 and RARß2) was frequently hypermethylated in these NHLs (87, 78, 61, 53, 40 and 38%, respectively), but not in benign follicular hyperplasia. Although some genes such as DLC-1 and PCDHGB7 were methylated in the vast majority of NHLs, others were differentially methylated in specific subtypes. The methylation of the candidate tumor suppressor gene DLC-1 was detected in a high proportion of primary tumor and plasma DNA samples by using quantitative methylation-specific PCR analysis. This promoter hypermethylation inversely correlated with DLC-1 gene expression in primary NHL samples. Thus, this CpG island microarray is a powerful discovery tool to identify novel methylated genes for further studies of their relevant molecular pathways in NHLs and identification of potential epigenetic biomarkers of disease.

Abbreviations: BFH, benign follicular hyperplasia; COBRA, combined bisulfite and restriction analysis; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; MSP, methylation-specific PCR; NHL, non-Hodgkin's lymphoma; PBL, peripheral blood lymphocyte; qMSP, quantitative MSP; TSA, Trichostatin A


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Non-Hodgkin's lymphoma (NHL) is the fifth most common malignancy in the United States, accounting for ~56 390 new cases in 2005 (1). Mature B-cell NHLs including B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), mantle cell lymphoma (MCL), follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) comprise the majority of all NHL cases (2) and each of these diseases is closely related to a normal counterpart in B-cell differentiation (3). Although advances in treatment over the past decade have improved outcomes for many patients with NHL, the survival time is quite variable, ranging from months to >20 years. Optimal clinical management relies on understanding the biological features and clinical behavior of each specific subtype. The currently used World Health Organization classification system is based on morphology, molecular abnormalities, immunophenotype and clinical presentation (2). Beyond genetic alterations, identification of novel epigenetic alterations may yield better diagnostic, prognostic and therapeutic information.

Epigenetics is a heritable change that modulates chromatin organization and gene expression without altering the DNA sequence. Methylation of cytosine residues at CpG dinucleotides in the promoter region of genes is a major epigenetic modification in mammalian genomes and can have profound effects on gene expression. Such aberrant epigenetic alterations have been shown to have causal effects in promoting tumor development (4). The profile of CpG island hypermethylation is unique for certain subtypes of leukemia or lymphoma (5). Although the most widely studied genes are the cell-cycle inhibitors p15INK4b and p16INK4a, the list of methylation-repressed genes in NHLs is expanding, including MGMT, RARß2, CRBP-1, SOCS-1, SHP-1, DAPK and others (6). Previous studies have mainly examined NHL as a single entity whereas only few studies (7–9) have examined methylation in specific NHL subtypes.

We developed a CpG island microarray-based technique for genome-wide methylation analysis in breast and ovarian cancer (10,11). In this study, we used the same approach to identify a group of genes silenced by DNA methylation in six NHL cell lines that are derived from different subtypes of NHL. A subpanel of the novel methylated genes was further examined in primary NHL samples from which we discovered stage-related gene methylation in NHLs. The CpG island microarray is an effective tool for discovering novel methylated genes and provides a solid foundation for further exploration of epigenetic alterations in the biology of NHL.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines and drug treatments
Human NHL lines RL, Daudi, DB, Raji, Granta 519 and Mec-1 were maintained in RPMI 1640 media. The germinal center-related cell line RL is derived from a male patient with FL and the t(14,18) translocation (12), and Daudi and Raji cells are also of germinal center derivation. The post-germinal center cell line DB is derived from a DLBCL patient and has undergone isotype switching (12). All four of these cell lines express surface CD10, thus suggesting a germinal center relationship (9). Granta 519 is an MCL cell line over-expressing cyclin D1 (13) and Mec-1 is a transformed CLL cell line (14). For gene reactivation experiments, cells were cultured in the presence of vehicle (PBS) or 1.0 µM 5-aza-2'-deoxycytidine (DAC) with medium changed every 24 h. After 4 days, cells were either harvested or further treated with TSA (1.0 µM) for 12 h and then harvested. Some cells were also treated with TSA alone for 12 h before harvest. Genomic DNA or total RNA was isolated using Qiagen kits (Qiagen, Valencia, CA) and used for methylation and gene expression analysis, respectively.

Tissue samples
Tissue and blood samples were obtained from patients after diagnostic evaluation for suspected lymphoma at the Ellis Fischel Cancer Center (Columbia, MO) and the Holden Comprehensive Cancer Center (Iowa City, IA) in compliance with local Institutional Review Boards. DNA was isolated from a total of 127 specimens; 7 from peripheral blood of healthy volunteers, 17 from patients with benign follicular hyperplasia (BFH), 13 MCL (mean age: 52.7 years; range: 39–87 years), 31 with B-CLL/SLL (mean age: 66.9 years; range: 56–84 years), 30 from FL (mean age: 62.0 years; range: 50–75 years) and 29 DLBCL (mean age: 57.0 years; range: 45–75 years). All cases of B-CLL/SLL had peripheral blood and bone marrow involvement, and thus were technically categorized as CLL. These are all referred to as CLL in this study. Retrospective analysis of flow cytometric data collected at the time of diagnosis for a subset of cases revealed that FL specimens comprised 75% neoplastic B-cells (n = 9, range 36–90%), MCL specimens comprise 88% neoplastic cells (n = 4, range 85–91%), CLL specimens comprise 80% neoplastic cells (n = 12, range 39–94%) and DLBCL specimens comprise 75% neoplastic cells (n = 7, range 38–99%). Total RNA was extracted from 2 samples of normal peripheral blood lymphocytes (PBL), 4 normal lymph nodes (LN), 9 DLBCL, 10 FL, 11 CLL and 9 MCL patient samples using the RNeasy kit (Qiagen). A 2-spin method of separating plasma from cellular elements (15) was used in our study. Plasma DNA was isolated from peripheral blood of 15 NHL patients using the QiaAmp Blood kit (Qiagen).

Preparation of CpG island microarray
The microarray panel containing 8640 CpG island clones was prepared as described previously (11). Amplified PCR products were spotted, in the presence of 20% DMSO, on UltraGap slides (Corning Life Science, Acton, MA). The slides were post-processed immediately before the hybridization using Pronto Universal Microarray Reagents (Corning Life Science). In addition, sequences from CpG islands of 42 known tumor suppressor genes were PCR amplified and printed on the same slides. The whole CGI library was sequenced recently by the Microarray Centre of University Health Network, Toronto, Canada and the sequences can be viewed at http://s-der10.med.utoronto.ca/CpGIslands.htm. Out of the 8640 CpG island fragments, 4564 unique genomic loci were identified (16).

Preparation of amplicons for methylation analysis
Amplicon preparation for methylation analysis was performed as described previously (17,18). Briefly, 2 µg genomic DNA was digested with MseI and then ligated to a PCR-linker. The ligated DNA was then directly digested with methylation-sensitive endonucleases, HpaII and BstUI, and amplified with a linker primer by PCR (11). The amplified products (or amplicons) were purified for fluorescence labeling. Incorporation of amino-allyl dUTP (aa-dUTP) into amplicons (5 µg) was conducted using the Bioprime DNA Labeling System (Invitrogen, Carlsbad, CA). Cy5 and Cy3 fluorescence dyes were coupled to aa-dUTP-labeled test and reference amplicons, respectively, and co-hybridized to the CpG island microarray panel. Hybridization and the post-hybridization washing were performed according to the manufacturer's procedures (Corning Life Sciences). Hybridized slides were scanned with the GenePix 4200A scanner (Axon, Union City, CA) and the acquired images were analyzed using the software GenePix Pro 5.1.

Microarray data analysis
The Cy3 and Cy5 fluorescence intensities were obtained for each hybridized spot. Array spots with fluorescence signals close to the background signal, reflecting PCR or printing failures, were excluded from the data analysis. Because Cy5 and Cy3 labeling efficiencies varied among samples, the Cy5/Cy3 ratios from each image were normalized according to a global mean method in Genepix Pro 5.1. This internal control panel included 20 MseI fragments that have no internal BstUI and HpaII restriction sites spotted at several concentrations on each array. The adjusted Cy5/Cy3 ratio for each CGI locus was then calculated and data were exported in a spreadsheet format for analysis. The hybridization experiments were repeated and only those reproducible spots were chosen for analysis. Cluster analysis was conducted using Cluster 3.0 and TreeView software (19).

Methylation-specific PCR and combined bisulfite and restriction analysis
Genomic DNA (2 µg) was treated with sodium bisulfite according to the manufacturer's recommendations (Ez DNA methylation kit; Zymo Research, Orange, CA). For the preparation of 100% methylated DNA, a PBL DNA sample was treated with M. SssI methyltransferase (New England Biolabs, Beverly, MA) that methylated all cytosine residues of CpG dinucleotides in the genomic DNA. Sodium bisulfite modification of the test and SssI-treated DNA samples was then performed as described above. Bisulfite-treated genomic DNA was used as a template for PCR with specific primers located in the CpG island regions of each selected gene. For methylation-specific PCR (MSP), allele-specific primers that cover 2–3 CpG dinucleotides were designed to differentiate methylated and unmethylated sequences. Amplification was performed using AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). For combined bisulfite and restriction analysis (COBRA), after amplification, PCR products were digested with the restriction enzyme BstUI (New England Biolabs), which recognizes sequences unique to the methylated and bisulfite-unconverted alleles. The digested DNA samples were separated in parallel on 3% agarose gels, stained with SYBR green and quantified using a Kodak gel documentation system. The COBRA and MSP primers are listed in Supplementary Table S1.

Bisulfite genomic sequencing analysis
Genomic DNA was treated with sodium bisulfite as described above. Primer sequences and PCR conditions are the same as for the COBRA assay. Amplified PCR products for CCND1, CYP27B1 and EFNA5 were subcloned using the TOPO-TA cloning system (Invitrogen). Plasmid DNA of 6–8 insert positive clones was isolated using the Montage Plasmid MiniprepTM kit (Millipore Corporation, Billerica, MA) and sequenced using ABI 3730 sequencing systems (Applied Biosystems).

DLC-1 real-time quantitative MSP assay
The real-time MSP uses two amplification primers specific for methylated sequences (listed in Supplementary Table) and an additional, amplicon-specific and fluorogenic hybridization probe (probe, FAM/AAG TTC GTG AGT CGG CGT TTT TGA/BHQ1) whose target sequence is located within the amplicon. The probe was labeled with two fluorescent dyes, with FAM at the 5' end and BHQ1 at the 3' end, and synthesized by IDT (Coralville, IA). The bisulfite-treated DNA was used for PCR amplification with the QPCR mixture (ABgene, Rochester, NY) as manufacturer's recommendations. The reaction was carried out in 40–45 cycles using a SmartCycler real-time PCR instrument (Cepheid, Kingwood TX). The quantitative MSP (qMSP) primers and probe for ß-Actin do not contain the CGs and therefore represent the quantitative estimate of input DNA in the PCR (20). Separate parallel reactions were run for ß-Actin using a series of diluted SssI-treated DNA samples as templates to generate standardization curves. The DLC-1 methylation levels were derived from the standardization curves and expressed as relative changes after normalization to those of ß-Actin.

Real-time RT–PCR
Total RNA (2 µg) was pre-treated with DNase I to remove potential DNA contaminants and reverse-transcribed in the presence of SuperScript III reverse transcriptase (Invitrogen). The generated cDNA was used for PCR amplification with the system described above. The Taqman probe and primer sets for real-time PCR were purchased from Applied Biosystems. Separate parallel reactions were run for GAPDH cDNA using a series of diluted cDNA samples as templates to generate standardization curves. The mRNA levels were derived from the standardization curves and expressed as relative changes after normalization to those of GAPDH.

Western blot analysis
NHL cells (1–2 x 106) were collected and lysed in RIPA buffer containing proteinase inhibitors. Protein concentrations of the supernatant were determined with the Bio-Rad Assay Kit, and 5 or 50 µg of protein were subjected to SDS–PAGE and transferred to immobilon-PSQ transfer membranes (Millipore Corporation). Membranes were incubated in Tris-buffered saline with 5% non-fat dry milk containing rabbit polyclonal antibodies against DLC-1 (1:200), cyclin D1 (1:200) or GAPDH (1:10000) (Santa Cruz Biotech, Santa Cruz, CA). Goat anti-rabbit secondary antibodies labeled with horseradish peroxidase were used to bind the primary antibodies, and detection was performed by a chemiluminescence system as per the manufacturer's instructions (Upstate, Charlottesville, VA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Methylation profiling in NHL cell lines
The microarray (17) was used to identify hypermethylated CpG island loci in the six NHL cell lines. Cy5- and Cy3-labeled amplicons were used as targets for microarray hybridization. These represented differential pools of methylated DNA in NHL cell lines relative to normal lymphocyte samples in a gender matched manner. Genomic DNA fragments containing methylated restriction sites were protected from the digestion and could be amplified by linker-PCR, whereas the equivalent allele fragments containing the unmethylated restriction sites were digested and thus could not be amplified in the normal lymphocyte DNA. Similar to cDNA microarray experiments, the significance of methylation changes is determined by the comparison of the ratio of two reporters, Cy5 and Cy3. These hypermethylated CpG island loci appeared as ‘red’ spots after microarray hybridization because greater signal intensities were obtained from the Cy5-labeled (red) NHL amplicons, than from those of the Cy3-labeled (green) control amplicons. When a cut-off value of the normalized Cy5/Cy3 ratio was set at >2 for the positive loci, a total of 86 methylated CpG loci (1.88% of 4564 CpG island fragments) were identified in Raji, 74 (1.62%) in Daudi, 68 (1.49%) in RL, 71 (1.55%) in DB, 51 (0.87%) in Mec-1 and 26 (0.56%) in Granta 519. Fifty-two loci (1.14%) were found commonly methylated in at least four of the six NHL cell lines. This same cut-off ratio was effective in identifying hypermethylated CpG islands in breast tumors in our previous study (11). Cluster analysis was conducted using the methylation microarray data of 83 named genes, all of which are methylated in at least two cell lines. Clustering of the pattern of methylation yielded a profile that discriminated between germinal center-derived lymphomas DB and RL, and non-germinal center lymphoma cell lines Granta 519 and Mec-1 (Figure 1A). Interestingly, the Burkitt's lymphoma cell lines possess different patterns of methylation in which Raji is grouped with DB and RL and Daudi is grouped with Granta and Mec-1. The cluster is somewhat related with the BCL6 and CD10 expression pattern as measured by real-time PCR and flow cytometry (data not shown). BCL6 and CD10 positive cell lines seemed to have acquired more methylation during transformation than BCL6 and CD10 negative cell lines.


Figure 1
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  		Fig. 1 DNA methylation analysis of six NHL cell lines. (A) Cluster analysis of the methylation microarray data derived from six NHL cell lines using Cluster 3.0 and Treeview software. BCL6 expression was measured by real-time PCR and CD10 expression by flow cytometry as described in Materials and methods. (B and C) Analysis of DNA methylation from 10 pertinent genes in a panel of 6 NHL cell lines. MSP (B) and COBRA (C) were used to determine methylation status of the indicated CpG island loci in cell lines. "U" and "M" indicate either PCR products from unmethylated, and methylated DNA in top two panels (MSP assay), respectively, and BstUI digested fragments resulting from unmethylated and methylated DNA in bottom eight panels (COBRA assay). (D) Bisulfite genomic sequencing of three selected genes in four NHL cell lines. From left to right panels represent CCND1 and CYP27B1 and EFNA5, respectively. Each row represents the sequence of individual clones, closed circles indicate methylated CpG sites, and open circles indicate unmethylated CpG sites. In all the gene-specific analysis described above, normal PBL and in vitro methylated DNA (SssI-treated DNA) serve as negative and positive controls, respectively.

 
Independent verification of methylation
Among the 30 most interesting genes based on review of literature (Table I), we chose to independently confirm the microarray findings of 10 known genes (PCDHGB7, EFNA5, CYP27B1, CCND1, DLC-1, NOPE, RPIB9, FLJ39155, PON3 and RARß2) whose function might relate to cancer by COBRA and MSP analyses. Since DLC-1 and RARß2 methylation has been reported previously (21,22), published MSP assays for these two genes were used. For all other genes COBRA assays were designed using the MethPrimer Software (http://www.urogene.org/methprimer/). We found hypermethylation of these genes in the six NHL cell lines (Figure 1B and C) and the results confirmed our microarray findings. The most frequently methylated DLC-1 was methylated in all six cell lines. The remaining nine genes were predominantly methylated in the germinal center-derived cell lines, and to a lesser extent in the Mec-1 and Granta 519 cell lines which correspond to the microarray findings in general. By semiquantitative COBRA assays, we found that NOPE and RPIB9 were partially methylated in Mec-1 and Granta 519 cell lines, but completely methylated in the other four germinal center-related NHL cell lines. Furthermore, the methylation status of CCND1 in the Granta 519 cell line is consistent with the findings of a recent report (23). The COBRA and MSP assays seem to be more sensitive than the microarray-based method. For instance, methylation of RARß2, CCND1, RPIB9 and PON3 can be detected in more cell lines by COBRA and MSP assays than methylation microarray hybridization. We also conducted bisulfite genomic sequencing of CCND1, CYP27B1 and EFNA5 (Figure 1D). Consistent with the COBRA results, all three genes are significantly methylated in RL and DB cells, but differentially methylated in Mec-1 and completely unmethylated in Granta 519 cells. Although CCND1 and EFNA5 were completely unmethylated in a PBL sample, we observed sparse methylation in the several CpG sites in the CYP27B1 fragment in the same sample. Since the bisulfite sequencing fragment covers both the first intron and the first exon of CYP27B1 gene, it is possible, though unproven, that we have just touched the boundary of the CpG island in the first intron of CYP27B1.


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  		Table 1 List of genes most frequently methylated in NHL cell lines

 
Epigenetic reactivation of methylated genes
We performed real-time RT–PCR on 4 of these 10 genes in cell lines treated with DAC and TSA (Figure 2A). We found that CYP27B1 and RARß2 were weakly to moderately up-regulated after DAC treatment, but there was a synergistic effect after combined DAC and TSA treatment in most of the cell lines. There was a synergistic effect for CCND1 in Raji, RL, Daudi and DB cell lines in which CCND1 was significantly methylated, but not in Mec-1 or Granta 519 cells in which CCND1 was not methylated. Interestingly, the treatment with DAC down regulated CCND1 expression in the Granta 519 cell line. DLC-1 was induced only under combination drug treatment indicating involvement of both methylation and histone deacetylation in its epigenetic control. However in Daudi cell lines, combined epigenetic drug treatments failed to reactivate DLC-1 expression and a similar result was obtained for RARß2 in the Granta 519 cell line. Although CYP27B1 was not methylated in the Granta 519 cells as shown by COBRA, DAC and TSA treatments did up-regulate its expression, suggesting the involvement of epigenetic control in regulating its expression. However, we are uncertain at this point, if methylation occurs in the CYP27B1 promoter other than the CpG sites surveyed by bisfulfite sequencing and COBRA assay; it is also possible that the silencing mechanism in Granta 519 cells is different from the other five NHL cell lines. Khorchide et al. (24) reported that treatments with DAC and TSA resulted in elevation of CYP27B1 in human normal prostate PNT-2 cells, which is very similar to what we reported here. To determine if silencing at the mRNA level leads to down regulation of protein expression, we conducted western blotting to analyze the protein expression levels of DLC-1 and CCND1. These two genes were chosen because of the availability of antibodies and the optimized experimental conditions. Western blot (Figure 2B) shows DLC-1 expression in a normal lymph node but not in any of the NHL cell lines examined, which is consistent with its mRNA expression and promoter methylation status. Similar to other reports (23), we did not detect cyclin D1 expression in normal lymph node or any of five NHL cell lines other than Granta 519.


Figure 2
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  		Fig. 2 (A) RT–PCR analysis of four selected genes in six NHL cell lines: total RNA (2 µg) isolated from treated (A, DAC; T, TSA; and AT, DAC+TSA) or untreated (C) cells was used to generate cDNA for real-time RT–PCR. cDNA generated from a normal lymph node served as a positive control (set at 100). Expression of GAPDH was used to normalize the gene expression under different conditions. (B) Western blot analysis of the CCND1 and DLC-1 protein expression in lysates (50 µg/lane) of six NHL cell lines and a normal lymph node sample. GAPDH serves as a loading control.

 
Hypermethylation in primary NHLs
The methylation profile of cancer cell lines does not always reflect the pattern of methylation in primary tumors. Therefore, we selected a subset of genes and confirmed the promoter methylation of six of these in a larger panel of NHLs (75 cases) including CLL, MCL, FL and DLBCL by COBRA and MSP analysis. Representative COBRA results of four genes are illustrated in Figure 3A. We also studied CCND1, CYP27B1 and EFNA5 methylation in three cases of primary NHL using bisulfite sequencing. The sequencing results were completely in agreement with COBRA results (Figure 3B). It is important to mention that the lower frequency of methylation in primary tumor compared to corresponding cell lines could be the presence of some normal cells in the primary tumor samples which may cause underestimation. All six of the methylation-silenced genes in the cell line models were also methylated in a significant proportion of NHL across the spectrum of subtypes (Figure 4A). CpG island promoter hypermethylation of DLC-1 was the most common, being present in 87% of primary NHL, whereas PCDHGB7 was the second most commonly methylated in 78% of NHL cases studied. Aberrant methylation was also detected in 61% of primary NHL for CYP27B1, 52% for EFNA5 and 40% for CCND1. Overall, RARß2 methylation was found in 38% which is consistent with previous reported findings (25). Interestingly, a lymphoma subtype-related profile was observed (Figure 4B). For example, CCND1 was methylated in FL and CLL, but not in MCL (P = 0.001). This corresponding reciprocal relationship is consistent with high levels of expression of cyclin D1 in MCL but not in FL or CLL (2). CYP27B1 and RARß2 were mainly methylated in FL and DLBCL as compared with MCL and CLL (P < 0.001). Except for CYP27B1, the remaining five genes were unmethylated in normal lymphocytes and BFH, confirming that the aberrant methylation is associated with malignancy. COBRA analysis of CYP27B1 genes in normal lymphocytes and BFH shows residual amount of methylation in these control samples, and the results were confirmed by bisulfite sequencing (Figure 1D). However, the methylation in normal samples mainly occurs in several CpG sites near the intronic region of CYP27B1. Increased methylation was observed in NHL cell lines and primary NHL samples and the methylation seems to spread from the intron region to the first exon region (Figures 1D and 3B).


Figure 3
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  		Fig. 3 (A) Confirmation by COBRA of promoter hypermethylation in representative NHL cases. COBRA was performed as described in the Materials and methods section. As shown, the digested fragments reflect BstUI methylation within a CpG island. P, positive control DNA methylated in vitro with the SssI methylase; N, negative control (PBL) DNA. For each gene analyzed, only selected gel electrophoresis pictures representing different samples from each class of NHLs were shown. The overall results were summarized in Figure 4A. (B) Bisulfite genomic sequencing of three selected genes in three primary tumors of NHLs (one sample each from CLL, FL and MCL). Each row represents the sequence of individual clones, closed circles indicate methylated CpG sites, and open circles indicate unmethylated CpG sites.

 


Figure 4
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  		Fig. 4 Comparative analysis of methylated genes across NHL subtypes. (A) Methylation distribution of 6 genes among 75 primary NHL samples. Closed box, methylated; open box, unmethylated; N/A, not determined. We quantified the COBRA gel pictures and calculated the percentage of methylation as the intensity of methylated fragments relative to the combined intensities of both unmethylated and methylated fragments. Since there was residual amount of CYP27B1 methylation in the BFH samples (average 15%, range from 9 to 22%), those samples with >30% methylation were considered hypermethylated in Figure 4A. (B) Comparison of frequencies of aberrant methylation in NHL samples. (C) Comparison of mean methylation indices in NHL subtypes. Frequencies of methylation of two groups were compared using Fisher's exact test. P-values are shown when there was a significant difference between two groups. The MI is defined as the total number of genes methylated divided by the total number of genes analyzed. To compare the extent of methylation for a panel of genes examined, we calculated the MIs for each case and then determined the median for the different groups. Mann–Whitney U-test was used to compare the median MIs between two variables.

 
Overall, simultaneous promoter methylation in ≥3 genes occurred in 9/14 (64%) of CLL, 2/10 (20%) of MCL, 15/15 (100%) of FL and 12/13 (92%) of DLBCL. As shown in Figure 4A, only two cases of MCL were completely unmethylated for all six genes studied. Therefore, using the six epigenetic markers it is possible to detect 96% of NHL cases suggesting that the gene methylation might be used as a diagnostic method. To determine whether different types of NHLs displayed evidence of coordinated methylation at multiple loci, the Mann–Whitney U-test was used to compare the mean methylation indices. This index is defined as the ratio of the number of methylated genes divided by the total number of genes analyzed between two variables. Significant differences were found between the subtypes of NHLs; MCL versus CLL, FL or DLBCL (P < 0.001), CLL versus FL or DLBCL (P < 0.01). There is no statistical difference between FL and DLBCL (P > 0.05). In general, germinal center-related lymphomas (FL, DLBCL) have more methylation than non-germinal center lymphoma (MCL, CLL) (P < 0.001, Figure 4C). Although MCL patients are relatively younger on average, there is no statistical difference in age between CLL, FL and DLBCL (P > 0.05).

Quantitative analysis of DLC-1 methylation in tumor and plasma samples of NHL patients
To test the feasibility of utilizing DLC-1 as a biomarker, we designed a real-time qMSP assay targeting the promoter region of the DLC-1 gene (Figure 5A) and expanded the methylation analysis from all the samples described above to now include additional samples from patients with BFH, MCL, CLL, FL and DLBCL. When a threshold ratio of DLC-1: ß-actin x 1000 was set as 15, the DLC-1 methylation frequencies were 71, 62, 83 and 83%, respectively (Figure 5B). When this qMSP method was compared to standard MSP, the agreement between the two methods was 93%. All of the discordant cases demonstrated methylation by standard MSP but not by qMSP. This may be because of the increased specificity by the additional probe in the qMSP assay. The relative methylation level of each sample, as measured by the ratio of DLC-1: ß-actin x 1000, varies among the four subclasses of NHL studied. The median methylation level was 135 (range from 0 to 1099) for MCL, 141 (range from 0 to 5378) for CLL, 348 (range from 0 to 5683) for FL and 295 (range from 0 to 5912) for DLBCL (Figure 5B). Interestingly, both the frequency and relative level of methylation of DLC-1 seem to correlate with the putative stages of differentiation. The methylation level is relatively higher in germinal center-related NHLs such as FL and DLBCL (some cases are post-germinal center), as compared with MCL and CLL which are usually derived from pre- or post-germinal center cells. The increased methylation level was not attributable to the variability in tumor cell percentage or age (P > 0.05).


Figure 5
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  		Fig. 5 Quantitative analysis of DLC-1 expression and methylation and in primary NHLs. (A) Schematic representation of the DLC-1 promoter region of interest. Relative positions of CG dinucleotides are illustrated as vertical bars, the forward and reverse primers are indicated as mF and mR, respectively, and the area covered by the fluorescent probe. (B) Methylation analysis by qMSP from controls and tumors of NHLs as indicated. Each circle represents a unique sample and the solid horizontal bar indicates the median ratio of methylated DLC-1/ß-Actin ratios x 1000 within a group of patients. (C) Expression analysis of DLC-1 by real-time RT–PCR from controls and tumors of NHLs as indicated. All values were normalized to GAPDH expression for each sample. DLC-1/GAPDH ratios x 1000 were plotted in log scale because of the large dynamic ranges of DLC-1 expression in normal lymph node and NHL samples. In both cases, cells from LN with BFH and PBL were used as controls.

 
For a subset of 15 patients with CLL, FL or DLBCL, paired tumor and plasma samples were available. Of these, 12/15 samples demonstrated concordant results, with 10/12 samples showing methylation in both the tumor and the plasma and 2/12 did not show methylation in either the tumor or in plasma (Figure 6A). The three discordant samples all demonstrated tumor methylation, but none was detected in the plasma samples. Two of these three were from patients with localized stage I FL and thus may suggest the levels are too low in early limited stage disease to be detected by our method. A larger study is under way to examine this issue. For all these samples, we examined DLC-1 methylation not only in the tumor and in the plasma, but also from buffy coat preparations of peripheral blood cells. In all cases of CLL and FL where methylation was present in the tumor, it was also present in buffy coat cells. However, in the case of DLBCL, methylation was present in the tumor and the plasma, but not in buffy coat cells, which is consistent with the fact that most patients with DLBCL (other than those with advanced disease) do not have detectable circulating tumor cells in blood (Figure 6B) but those with CLL frequently do. The result clearly demonstrates the value of detecting methylation in plasma of DLBCL patients. We also conducted a mall preliminary study of the potential use of DLC-1 methylation in plasma for monitoring the progress after chemotherapy. Plasma samples from one FL patient and one DLBCL patient, who were positive for DLC-1 methylation, were taken at the time of diagnosis and their revisit to the clinics during chemotherapy and were analyzed by qMSP. The shaded areas in Figure 6C indicate the time of treatment. Our results demonstrated that after chemotherapy, when the patients achieved complete or partial remission, the DLC-1 methylation in the patients’ plasma became undetectable. More experiments are needed to test this method for monitoring therapeutic responses and potentially for detection of relapse.


Figure 6
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  		Fig. 6 DLC-1 methylation in tumor, plasma and buffy coat leukocytes. (A) Detection of DLC-1 methylation in primary tumor and matched plasma samples. (B) Quantitative DLC-1 methylation analysis for matched primary tumor cells, buffy coat leukocytes and plasma samples from a grade II DLBCL. (C) DLC-1 methylation in plasma becomes undetectable after chemotherapy. Plasma samples from one FL patient and one DLBCL patient, each positive for DLC-1 methylation were taken at the time of diagnosis and upon their revisit to the clinics during chemotherapy; both sample sets were analyzed by qMSP. Shaded areas indicate the time of treatment. Each bar indicates the median ratio of methylated DLC-1/ß-Actin ratios x 1000 for each sample.

 
Down-regulation of DLC-1 gene expression in primary NHLs
The mRNA expression level of DLC-1 was quantified by real-time RT–PCR in 6 normal controls and 39 primary NHL samples. As shown in Figure 5C, DLC-1 mRNA could be detected in normal lymph node samples and weakly in PBL suggesting a tissue or developmental stage-specific expression or possibly that other silencing mechanism might exist in normal leukocytes other than methylation. DLC-1 mRNA was also weakly expressed in some cases of MCL, CLL and FL, and somewhat stronger in DLBCL cases. When overall DLC-1 mRNA expression was compared between tumor and normal LN, its expression was much lower in tumors. Among the 45 samples studied, we have matched methylation and expression data for 17 of them. Of 17 samples 13 show concordant results: higher methylation levels and low expression levels. Only four NHL cases have discordant results: one case with both higher expression and methylation and three other cases with no methylation but lower expression. Although most of our clinical samples contain >75% tumor samples we cannot rule out the interference of normal lymphoid cells on the quantification of DLC-1 expression. Nevertheless, the reciprocal relationship between DLC-1 promoter methylation and its expression suggests that promoter methylation is a major mechanism for DLC-1 silencing in germinal center related NHLs.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The molecular pathophysiology of NHL is characterized by selective association of different clinicopathological categories of the disease and reveals relationships to putative normal stages of B-cell differentiation. For example, we reported previously that the AR gene is preferentially methylated in FL compared with other subtypes (9). In the current study, we identified a substantial number of additional genes that are aberrantly methylated in NHL cell lines and in primary NHLs. A combination of methylated genes can potentially be used as a molecular marker panel for detection or diagnosis using highly sensitive qMSP technology. The advantage of this approach is that methylated genes are derived from patients’ tumor DNA, which is a more stable specimen than RNA. Although a growing number of genes have been identified as aberrantly methylated in lymphoma (5,6,25), to date few studies (7–9) have studied promoter hypermethylation in specific NHL subtypes in detail. We not only identified genes such as DLC-1 and PCDHGB7 which are methylated in the vast majority of NHLs, but also some subtype-specific markers such as CCND1, CYP27B1, RARß2 and EFNA5 which are preferentially methylated in one or two subtypes of NHLs. Using DLC-1 as an example, we show that we can detect aberrantly methylated DNA in 77% of tumor and 67% of plasma samples from NHL patients using quantitative real-time MSP. Although studies with a larger population are needed to further evaluate the predictive ability of these markers, they could potentially be used as biomarkers in diagnosis and classification of NHLs, especially for early detection and monitoring therapy.

We show that a candidate tumor suppressor gene, DLC-1, is a frequent target of aberrant methylation in NHLs. Although methylation of the gene has been reported previously in several types of non-lymphohematopoietic tumors (26–29), this is the first report of its involvement in NHL. The DLC-1 gene was mapped on to 8p21.3–22, a region suspected to harbor tumor suppressor genes and recurrently deleted in several solid tumors (29–31). The DLC-1 sequence shares high homology with rat p122RhoGAP, a GTPase-activating protein for Rho family proteins and DLC-1 protein was shown to be a RhoGAP specific for RhoA and Cdc42 (32). Recent evidence suggests that RhoA GTPase regulates B-cell receptor (BCR) signaling and may be an important regulator of many aspects of B-cell function downstream of BCR activation (33). Therefore, epigenetic silencing of DLC-1 might have a profound influence on lymphoid cell function and lymphomagenesis. Interestingly, DLC-1 is only weakly expressed in PBL but is expressed in the normal lymph node when examined by real-time RT–PCR for DLC-1 mRNA and suggests tissue-specific or developmental stage-dependent expression. However, no methylation was found in the normal B-cells regardless of their expression status. In addition, reactivation of methylated DLC-1 in NHL cells required both DAC and TSA (Figure 2) suggesting that DNA methylation is not the only process involved in DLC-1 gene silencing.

The chromosome translocation t(11;14)(q13;32) is seen in most MCLs (2,34), and as a result, CCND1 is overexpressed in >90% of MCL cases (2). However, cyclin D1 is known to be expressed at low levels in normal lymphoid tissues and several other types of lymphoma such as MCL and CLL (35,36). A recent finding of complete hypomethylation at the CCND1 promoter in normal B cells suggests that, although the CCND1 gene is inactive transcriptionally, the CCND1 promoter is still unmethylated in lymphoid cells that do not contain the translocation (23). It is possible that the mechanism of de novo methylation is dysregulated in NHLs, resulting in aberrant methylation of CCND1 despite its transcriptional status. This finding indicates that such DNA regions in the genome are prone to be methylated in cancer cells, which is consistent with an earlier report (37), although the factors that determine such susceptibility to methylation remain unresolved. Our results suggest that hypermethylation in the CCND1 promoter might not directly control the gene expression. However, this type of de novo methylation could be potentially developed as a biomarker that can assist in diagnosis or classification of NHLs.

CYP27B1 encodes 1{alpha}-hydroxtylase (1{alpha}-OHase), an important enzyme in the vitamin D metabolic pathway. The loss of 1{alpha}-OHase and/or VDR activity could contribute to the ability of cancer cells to escape growth control mechanisms of vitamin D (38). Several studies have shown that reduced 1{alpha}-OHase activities in cancer cells decreased the susceptibility to 25(OH)D3-induced growth inhibition (39). Ephrin-A5, a member of the ephrin gene family is encoded by EFNA5. The EPH and EPH-related receptors comprise the largest subfamily of receptor protein-tyrosine kinases and have been implicated in mediating developmental events, particularly in the nervous system. Himanen et al. (40) found that ephrin-A5 binds to the EphB2 receptor, a tumor suppressor gene (41), leading to receptor clustering, autophosphorylation and initiation of downstream signaling. PCDHGB7 is a member of the protocadherin gamma gene cluster, one of three related clusters tandemly linked on chromosome five. These gene clusters have an immunoglobulin-like organization (42), suggesting that a novel mechanism may be involved in their regulation and expression (43). The two cell surface molecules are known to play a role in the nervous system, but any role they may have in NHL is unclear.

Remarkably, we found that there were significant differences in DNA methylation between pre-germinal and germinal center-derived NHLs. The mean methylation index (MI) of non-germinal center NHLs was lower than germinal center-related NHLs. The results are consistent with our previous findings on 38 candidate tumor suppressor genes using a methylation-specific oliogonucleotide microarray (44). The mechanism and biological significance behind this experimental observation is not clear at this point, but provides strong evidence for further investigations that are now in progress. Although we cannot exclude the effect of age on the increase in methylation when we compare MCL with FL and DLBCL, age-related methylation cannot explain the difference in methylation between CLL, FL and DLBCL. One interesting question to ask is whether the increased methylation observed in germinal center-derived NHL is associated with overexpression of BCL6 (Figure 1). BCL6 is a Kruppel-associated box (KRAB) domain-containing zinc finger protein which is involved in the pathogenesis of NHL. A recent study showed that gene silencing induced by the KRAB-associated protein 1 complex was followed by regional DNA hypermethylation at the promoter of its target genes (45) and sheds light on the potential role of DNA methylation in BCL6-mediated gene silencing.

In summary, we have performed analysis of methylation alterations at the genome level in six cell lines derived from a spectrum of NHL subtypes and identified a group of aberrantly methylated genes that can potentially be used as epigenetic biomarkers for detection of NHL. We have further demonstrated that NHL exhibits non-random methylation patterns in which germinal center tumors seem to be prone to de novo methylation. The mechanism behind such experimental observations is unclear. However, it is unlikely that all of these methylation events were induced by global deregulation of methyltransferase activity. Instead, we suggest that dysregulation of a given transcriptional regulator or signaling pathway may selectively lead to the aberrant methylation of a portion of downstream genes and confer a growth advantage to the tumor cells. Further investigation is needed and will provide better insight into the pathogenesis of NHL.


   Acknowledgments
 
We thank Susan Souchek and Kathy Olson for their assistance in cell banking and flow cytometric analysis and Angel Surdin for her expert editorial assistance and contributions to writing this manuscript. This study was supported by National Cancer Institute grants CA100055 and CA097880 (C.W.C.) and grants from the CRC Missouri Chair in Cancer Research, P50 CA097274 (J.E.W.) and the Multiple Myeloma Research Foundation (H.S.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 6, 2006; revised May 18, 2006; accepted May 19, 2006.


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