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Originally published as JCO Early Release 10.1200/JCO.2005.11.353 on May 16 2005

Journal of Clinical Oncology, Vol 23, No 17 (June 10), 2005: pp. 3886-3896
© 2005 American Society of Clinical Oncology.
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Epigenetic Profiling of Cutaneous T-Cell Lymphoma: Promoter Hypermethylation of Multiple Tumor Suppressor Genes Including BCL7a, PTPRG, and p73

Remco van Doorn, Willem H. Zoutman, Remco Dijkman, Renee X. de Menezes, Suzan Commandeur, Aat A. Mulder, Pieter A. van der Velden, Maarten H. Vermeer, Rein Willemze, Pearlly S. Yan, Tim H. Huang, Cornelis P. Tensen

From the Departments of Dermatology and Medical Statistics, Leiden University Medical Center, Leiden, the Netherlands; and Division of Human Cancer Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH

Address reprint requests to Cornelius P. Tensen, MD, Leiden University Medical Center, Dermatology, Wassenaarseweg 72, Leiden ZH 2333AL, the Netherlands; e-mail: c.p.tensen{at}lumc.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
PURPOSE: To analyze the occurrence of promoter hypermethylation in primary cutaneous T-cell lymphoma (CTCL) on a genome-wide scale, focusing on epigenetic alterations with pathogenetic significance.

MATERIALS AND METHODS: DNA isolated from biopsy specimens of 28 patients with CTCL, including aggressive CTCL entities (transformed mycosis fungoides and CD30-negative large T-cell lymphoma) and an indolent entity (CD30-positive large T-cell lymphoma), were investigated. For genome-wide DNA methylation screening, differential methylation hybridization using CpG island microarrays was applied, which allows simultaneous detection of the methylation status of 8640 CpG islands. Bisulfite sequence analysis was applied for confirmation and detection of hypermethylation of eight selected tumor suppressor genes.

RESULTS: The DNA methylation patterns of CTCLs emerging from differential methylation hybridization analysis included 35 CpG islands hypermethylated in at least four of the 28 studied CTCL samples when compared with benign T-cell samples. Hypermethylation of the putative tumor suppressor genes BCL7a (in 48% of CTCL samples), PTPRG (27%), and thrombospondin 4 (52%) was confirmed and demonstrated to be associated with transcriptional downregulation. BCL7a was hypermethylated at a higher frequency in aggressive (64%) than in indolent (14%) CTCL entities. In addition, the promoters of the selected tumor suppressor genes p73 (48%), p16 (33%), CHFR (19%), p15 (10%), and TMS1 (10%) were hypermethylated in CTCL.

CONCLUSION: Malignant T cells of patients with CTCL display widespread promoter hypermethylation associated with inactivation of several tumor suppressor genes involved in DNA repair, cell cycle, and apoptosis signaling pathways. In view of this, CTCL may be amenable to treatment with demethylating agents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Primary cutaneous T-cell lymphomas (CTCLs) are extranodal non-Hodgkin's lymphomas derived from mature T cells presenting in the skin. CTCLs encompass a group of distinct entities with different clinical behavior.1 Management of patients with CTCL may be very difficult as few therapies are able to influence the generally progressive disease course of these malignancies.

In malignant T cells of patients with CTCL, many oncogenic alterations have been demonstrated, such as functional inactivation of the Fas receptor, the transforming growth factor beta receptors, p16, constitutive activity of STAT3, and chromosomal instability.2-6 Although only few causative genetic lesions have been identified, the epigenetic mechanism of promoter hypermethylation has been found to be associated with inactivation of several tumor suppressor genes in CTCL.6-9

Methylation of CpG islands located in the promoter or first exon of genes is associated with transcriptional downregulation through alteration of the chromatin conformation and interference with binding of transcription factors.10 Aberrant promoter methylation is increasingly recognized as an important factor in the pathogenesis of cancer as many tumor suppressor genes can be inactivated through this epigenetic mechanism.11 Both the frequency and the patterns of promoter hypermethylation vary markedly between different tumor types.12,13 Promoter hypermethylation in cancer cells is interesting from a clinical point of view as its detection can be exploited in the diagnosis and prognosis of cancer patients.14 The potential reversibility of gene silencing associated with promoter hypermethylation through pharmacologic manipulation with demethylating agents, such as 5-aza-2'-deoxycytidine, zebularine, and MG98, promises epigenetic approaches to cancer therapy.15

The notion that epigenetic dysregulation has an important role in the development and progression of CTCL is supported by the observation that these malignancies respond favorably to treatment with histone deacetylase inhibitors.16 Moreover, promoter hypermethylation, that often coincides with repressive histone modifications, is displayed by hematopoietic malignancies at a higher frequency than by other tumor types.12,13,17,18 Consistently, in the few studies performed thus far, promoter hypermethylation of the p15, p16, MLH1, and SHP1 genes has been demonstrated in CTCL.6-9

The purpose of this study was to analyze the occurrence of promoter hypermethylation in CTCL on a genome-wide scale. We analyzed DNA isolated from skin tumor biopsy specimens of 28 patients with CTCL, including CTCL entities with aggressive clinical behavior (transformed mycosis fungoides and CD30-negative primary cutaneous large T-cell lymphoma) and indolent CTCL (CD30-positive primary cutaneous large T-cell lymphoma). To identify novel methylation targets and to screen for global DNA methylation patterns, differential methylation hybridization (DMH) using CpG island microarrays was applied.19,20 Additionally, the methylation status of eight tumor suppressor genes, selected for their known involvement in lymphomagenesis, was examined using bisulfite sequence analysis (BSA).

In this study, we show that malignant T cells in CTCL exhibit widespread promoter hypermethylation suggestive of epigenetic instability. The novel methylation targets we identified in CTCL include the putative tumor suppressor genes BCL7a (B-cell CLL/Lymphoma 7a), PTPRG (protein tyrosine phosphatase receptor gamma), and THBS4 (thrombospondin 4). We found six of eight selected tumor suppressor gene promoters hypermethylated in CTCL including MGMT, p73, p16, p15, CHFR, and TMS1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Patient Samples
Snap-frozen biopsy specimens were obtained from skin tumors of 28 patients with CTCL, including 12 patients with tumor-stage mycosis fungoides with blast cell transformation (MF-TR) characterized by the presence of > 25% blast cells, five patients with CD30-negative primary cutaneous large T-cell lymphoma (CD30– LTCL) and 11 patients with CD30-positive primary cutaneous large T-cell lymphoma (CD30+ LTCL), defined by criteria of the European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas.1 MF-TR and CD30– LTCL have an estimated 5-year survival of approximately 25%; CD30+ LTCL has a 5-year survival of 96%.1,21,22 Skin biopsy specimens were required to contain at least 70% malignant T cells for inclusion in the study. As controls, seven CD4+ T-cell samples isolated from peripheral blood of healthy volunteers and three skin biopsy specimens from patients with inflammatory dermatoses (atopic dermatitis, discoid lupus erythematosus) were used. DNA was extracted using the Genomic Tip kit (Qiagen, Hilden, Germany). All 28 CTCL samples were analyzed using DMH; 21 CTCL samples were analyzed using BSA.

DMH Using CpG Island Microarrays
DMH was performed according to the detailed protocol published by Yan et al,20 with the only modification that tumor and control amplicons were hybridized to microarrays separately and fluorescence signal intensities instead of ratios were used as a measure of CpG island methylation. Briefly, DNA isolated from tumor tissue was digested by MseI, ligated to linkers, and sequentially digested with methylation-sensitive restriction enzymes (HpaII and BstUI). The digested linker-ligated DNA was used as template for polymerase chain reaction (PCR) amplification (20 cycles) and fluorescence labeling using Cy5 before hybridization to the CpG island microarray. As probes, a panel containing 8640 CpG island tags prepared from a genomic library arrayed on glass slides were used. The identity of selected CpG islands (CpGIs) was determined by sequence analysis of plasmids contained in the CGI library.

Statistical Analysis
Data from DMH experiments were normalized using variance stabilizing normalization implemented in the R statistical software package.23 A Wilcoxon rank-sums test was applied separately to each CpG island in order to identify those with consistently different intensity patterns in the 28 tumors when compared with the 10 controls. The resulting list of P values was corrected for multiple testing via the false discovery rate step-up procedure of Benjamini & Hochberg.24 To identify CpG islands methylated sporadically in only a subset of tumors, for which the Wilcoxon test is not suited, we first estimated the CpG island–specific intensity distribution based upon the 10 control samples and a gamma-distribution model. These estimates were used to compute upper-tail probabilities corresponding to the intensity of each tumor. A threshold {Delta} = 0.01 for these probabilities was applied, below which tumor intensities were classified as potentially methylated. Under the hypothesis that tumor intensities behave in the same way as control intensities, the number of potentially methylated intensities per CpG island follows a binomial distribution, with sample size 28 and probability {Delta} = 0.01. This yields a list of P values, which were then corrected for multiple testing as those from the Wilcoxon test. Considered as frequently hypermethylated were CpG islands that exhibited values exceeding this threshold in at least four tumor samples. The false discovery rate for CpG islands selected using the Wilcoxon test was 30% and for the sporadic test, 2.1%. Subsequently excluded were CpG islands that had fluorescence intensity values that were below a predetermined background level in all tumor samples or above this level in any of the control samples.

Bisulfite Sequence Analysis
Two µg of patient sample–derived DNA was bisulfite-converted using the EZ DNA methylation kit (Zymo Research, Orange, CA). Primers were designed to anneal to bisulfite-converted DNA as template. Primer sequences (Invitrogen, Breda, the Netherlands) are given in Table 1. PCR reactions were carried out in 25-µL volume using 50-ng bisulfite-converted DNA as template. Cycle parameters for all analyzed CpG islands were as follows: denaturing at 94°C for 5 minutes; annealing at temperatures varying from 63°C to 56°C according to primer set for 1 minute and extension at 72°C for 1 minute for five cycles; followed by denaturing for 30 seconds, annealing for 1 minute and extension for 1 minute at similar temperatures for 30 cycles. Following electrophoresis, PCR products were excised from the agarose gel and purified using the Qiaquick Gel Extraction Kit (Qiagen). Sequence analysis was performed on an ABI Prism 3700 DNA analyzer (Applied Biosystems, Nieuwerkerk aan den IJssel, the Netherlands) under standard conditions.


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  		Table 1. Primers for Bisulfite Sequence Analysis

 
Validation experiments using mixtures (1:1 and 1:3) of unmethylated semen and completely methylated CpGnome universal methylated DNA (Chemicon, Hampshire, United Kingdom) for each primer combination demonstrated absence of PCR bias and showed that methylation could accurately be detected if a minimum of 25% of total analyzed DNA was methylated. Cytosine peaks in the chromatogram with a height of 0.4 or more relative to the thymine peak height were considered as indicative of methylation. A CpG island was defined as aberrantly methylated if the density of methylated CpG dinucleotides contained in the amplified sequence exceeded 15%, consistent with the threshold value implemented in other studies.25,26

Cell Culture, 5-Aza-2'-Deoxycytidine Treatment
The MF cell line MyLa (courtesty of Dr K. Kaltoft) was cultured in RPMI 1640 (Invitrogen) supplemented with 200 U/mL penicillin, 200 µg/mL streptomycin, 2 µmol/mL L-glutamine, 20% fetal calf serum, and 12.5% crude T-cell extract at 37°C, 5% CO2.27 For demethylation studies, cells were cultured in this medium to which 5-aza-2'-deoxycytidine (Sigma, St. Louis, MO) was added to a concentration of 2 µmol/L for 4 days before extraction of RNA using the RNeasy kit (Qiagen).

Real-Time Quantitative PCR
cDNA synthesis was performed on 1-µg total RNA after treatment with RQ1 DNase I (Promega, Madison, WI) using Superscript III reverse transcriptase (Invitrogen) and an oligo(dT)12-18 primer. Quantitative PCR (qPCR) was performed using DyNAmo HS SYBR Green qPCR kit (Finnzymes, Espoo, Finland) in an ABI-Prism 7700 sequence detection system (Applied Biosystems). For normalization, cleavage and polyadenylation specific factor 6 (CPSF6) and TATA-box-binding protein (TBP) were used, genes stably expressed also after treatment with 5-aza-2'-deoxycytidine (T. van Wezel, personal communication, September 2004). Primer sequences are given in Table 2. Cycle parameters were as follows: denaturing for 15 seconds at 95°C, and annealing and extension for 60 seconds at 60°C for 40 cycles. Specificity of PCR products was confirmed by agarose gel electrophoresis, melting curve, and sequence analysis of test samples. Experiments were performed in duplicate. Cross-over point values were used to calculate the gene-specific input cDNA amount. Data were evaluated using Sequence Detection System software version 1.9.1 (Applied Biosystems) and the second derivative maximum algorithm.


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  		Table 2. Primers for Real-Time Quantitative Polymerase Chain Reaction

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Examination of DNA Methylation Patterns Using CpG Island Microarrays
To screen for hypermethylated CpG islands in the 28 CTCL tumor specimens, we applied DMH using CpG island microarrays. DMH allows screening of methylation of 8640 CpG islands, most of which are located in gene promoters, and has been established in previous studies to accurately identify DNA methylation patterns.20,28,29 This method is based on the use of methylation-sensitive endonucleases that digest unmethylated DNA sequences, whereas methylated sequences are protected from digestion. Following methylation-sensitive digestion of DNA extracted from tumor and control samples, methylated DNA sequences can therefore be selectively amplified by PCR, labeled, and hybridized to a high-density microarray of CpG islands.19 Differences in methylation of a particular CpG island between tumor and control samples are reflected in differences in fluorescence intensities of CpG island tags on the microarray corresponding to the amplified DNA sequences. Since T cells have been found to display heterogeneous DNA methylation patterns, we compared methylation characteristics of 28 CTCL samples with those of multiple controls including seven T-cell samples and three inflammatory skin disease biopsy specimens.30 A statistical algorithm suited for the analysis of methylation profiling data was used, which detects CpG islands methylated consistently as well as CpG islands methylated sporadically in four or more of the tumor samples. Thus 35 CpG islands were identified as differentially methylated in CTCL. The methylation patterns, identity, and location of these 35 CpG islands are presented in Figure 1. Fourteen of these CpG islands were identified as consistently methylated using the Wilcoxon test, 15 as sporadically methylated, and six CpG islands were identified by both methods. The CpG island associated with the BCL7a gene was among the most discriminating between CTCL and control samples. Methylation in CTCL was evenly distributed over the various chromosomes, in contrast to acute myeloid leukemia, where preferential methylation of CpG islands located on one particular chromosome was observed.31 Among the CpG islands hypermethylated in CTCL were the promoters of collagen alpha II type I and THBS4, genes previously shown to be methylated in colon cancer cells using different methylation detection methods.18,32



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  		Fig 1. Methylation patterns of 35 CpG islands identified as hypermethylated when comparing 28 cutaneous T-cell lymphoma (CTCL) samples (mycosis fungoides with blast cell transformation [MF-TR], CD30-negative large T-cell lymphoma [LTCL], and CD30-positive LTCL) with 10 control samples using differential methylation hybridization (DMH) on CpG microarrays. The colors reflect normalized fluorescence values that correspond to likelihood of methylation of the particular CpG island (red = high, white = low). The gene name, location of the CpG island, and locus and accession number (Acc. no.) of the gene are indicated. Patient samples were ordered according to CTCL entity; the 35 CpG islands were ordered using a hierarchical clustering algorithm based on the Euclidean distance and average-linkage method.

 
Confirmation of BCL7a, PTPRG, and THBS4 as Novel Methylation Targets in CTCL
Among the 35 CpG islands identified as hypermethylated in CTCL by DMH, we selected for confirmation studies CpG islands of BCL7a, PTPRG, and THBS4, as their epigenetic inactivation may be relevant in the pathogenesis of CTCL. These putative tumor suppressor genes were detected as consistently hypermethylated (THBS4), sporadically hypermethylated (PTPRG), or identified by both methods (BCL7a). Direct BSA, applied to confirm the results of DMH experiments, provides a map of average methylation densities for each of the cytosines contained in the DNA sequences amplified by PCR following bisulfite conversion.25,26,33 This method makes use of the fact that sodium bisulfite deaminates cytosine to uracil, which is replaced in subsequent PCR amplification by thymine, while leaving methylated cytosine unaltered. Each cytosine base present in the DNA sequence following bisulfite conversion and PCR amplification is indicative of methylated cytosine in the original DNA sequence. Figure 2
Fig 2. (continued) (C) Methylation density map of the BCL7a CpG island amplicon, containing 26 CpG dinucleotides, in 21 CTCL samples and nine controls. Unmethylated cytosine bases are indicated as ({square}), methylated cytosines as ({blacksquare}). Marked in gray are the patient samples considered as having a hypermethylated BCL7a CpG island. MF-TR, mycosis fungoides with blast cell transformation; LTCL, large T-cell lymphoma.

illustrates the results of BSA of the CpG island located in the first exon of BCL7a. As presented in Table 3, the CpG islands of BCL7a, PTPRG, and THBS4 were hypermethylated in 48%, 24%, and 52% of CTCL samples respectively, confirming DMH results. These three novel methylation targets were methylated among all three CTCL entities, as well as in the CTCL cell line MyLa, but not in any of the six control samples analyzed.



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  		Fig 2. Hypermethylation of the CpG island located in the first exon of the BCL7a gene. (A) Schematic representation of the first exon of BCL7a (accession number AC069503) on 12q24.31, the location of the CpG island and of the region amplified for bisulfite sequence analysis. (B) Exemplary results of bisulfite sequence analysis of the BCL7a CpG island in a tumor sample of a cutaneous T-cell lymphoma (CTCL) patient with an unmethylated (upper) and a CTCL patient with a hypermethylated (lower) BCL7a CpG island. Unmethylated and methylated bisulfite-converted sequences are indicated above the chromatograms.

 

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  		Table 3. Hypermethylation of Methylation Targets Identified by Differential Methylation Hybridization Analysis in Different Cutaneous T-Cell Lymphoma Entities, As Analyzed Using Bisulfite Sequence Analysis

 
Demethylation and Reactivation of BCL7a and PTPRG Expression
Next we analyzed whether methylation of the CpG island located in the first exon of BCL7a and in the promoter of PTPRG is associated with gene silencing. Promoter methylation of the THBS4 gene had previously been shown to be associated with gene silencing in cancer cells.18 The MyLa CTCL cell line with demonstrated methylation of BCL7a and PTPRG was exposed to 5-aza-2'-deoxycytidine and effects on expression of these genes before and after treatment with this demethylating agent was assessed by real-time quantitative PCR. As illustrated in Figure 3, expression of these genes, nearly undetectable before exposure to 5-aza-2'-deoxycytidine, was dramatically increased upon demethylation. Expression of BCL7a increased with a fold change, denoting the average expression of the gene of interest relative to two stably expressed genes, of approximately 489. Expression of PTPRG increased more than seven-fold. These results suggest that expression of BCL7a and PTPRG is epigenetically regulated and that promoter methylation as occurs in CTCL is associated with transcriptional downregulation.



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  		Fig 3. Results of demethylation using 5-aza-2'-deoxycytidine on expression of the BCL7a and PTPRG genes as measured using real-time quantitative polymerase chain reaction analysis. Expression levels of the genes of interest are represented as the average of 2–{Delta}Ct(gene of interest-CPSF6) and 2–{Delta}Ct(gene of interest-TBP). Error bars indicate standard deviation from duplicate experiments. (A) Methylation density of the CpG island located in the first exon of BCL7a and the CpG island in the promoter of PTPRG in the MyLa cell line before demethylation. (B) Histogram showing BCL7a gene expression in the MyLa cell line before and after exposure to 5-aza-2'deoxycytidine. (C) Histogram showing PTPRG gene expression in the MyLa cell line before and after exposure to 5-aza-2'-deoxycytidine.

 
Promoter Hypermethylation of Eight Selected Tumor Suppressor Genes in CTCL
Subsequently the methylation characteristics of promoters of eight selected tumor suppressor genes were examined in 21 CTCL patient samples using BSA. Hypermethylation of these genes was recently shown in MF (p15, p16, and MLH1), in nodal lymphomas (p73, MGMT, and SOCS1), or in solid tumors (TMS1 and CHFR).34-38 In addition, promoter hypermethylation of these genes is known to be associated with gene silencing in cancer cells. As presented in Table 4, aberrant promoter methylation of p73 (48%), p16 (33%), CHFR (19%), p15 (10%), and TMS1 (10%) could be detected in CTCL, but not in control samples. The CpG island located in the promoter of the MGMT gene was found to be methylated in all tested CTCL samples, but unexpectedly was also methylated in three of the six CD4+ T-cell samples included as controls. An overview of clinical features, disease course data of the CTCL patients, and the methylation status of the 11 genes investigated using BSA is presented in Table 5.


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  		Table 4. Hypermethylation of Selected Tumor Suppressor Genes in Different CTCL Entities, As Analyzed Using Bisulfite Sequence Analysis

 

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  		Table 5. Clinical Features, Disease Course Data, and Methylation Status of Tumor Suppressor Genes in 21 CTCL Patients

 
Promoter Hypermethylation in CTCL Entities With Different Clinical Behavior
To evaluate possible correlations between DNA methylation and CTCL entities with different clinical behavior, we compared the methylation characteristics of CTCL entities with known aggressive behavior (MF-TR, CD30– LTCL) with those of an indolent CTCL entity (CD30+ LTCL). As noticeable in Figure 1, the CpG island methylation patterns of aggressive and indolent CTCLs as identified using DMH show great similarity. Hierarchical clustering of patient samples using the entire set of 8640 CpG islands or the selection of 35 CpG islands hypermethylated in CTCL revealed that the two groups of CTCL entities with different clinical behavior could not be separated on the basis of their methylation pattern.

In the included CTCL patient samples, the frequency of promoter methylation did not differ significantly between patients with an aggressive CTCL entity (3.2 of 11 promoters) and an indolent CTCL entity (3.1 of 11 promoters). This contrasts with acute lymphoblastic leukemia, where prognostic significance of promoter hypermethylation frequency was recently reported.39 Of the 11 investigated tumor suppressor genes, only BCL7a was methylated significantly more frequently in the aggressive CTCLs MF-TR and CD30– LTCL than in CD30+ LTCL (64% v 14%; {chi}2-test, P < .05). Comparison of survival rates using the log-rank test showed no significant association between hypermethylation of any of these 11 genes and survival, possibly related to the limited number of 21 patients and short follow-up duration in this study group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Authors' Disclosures of...
 REFERENCES
 
In this study we evaluated the occurrence of aberrant DNA methylation in skin biopsy specimens obtained from patients with CTCL using a combination of a screening genomic approach, DMH on CpG island microarrays, and a candidate-gene approach, BSA. Using DMH analysis, 35 CpG islands were identified as hypermethylated in at least four of 28 studied CTCL patient samples. Hypermethylation of the putative tumor suppressor genes BCL7a, PTPRG, and THBS4 was confirmed. Subsequently, we showed that promoter hypermethylation of BCL7a and PTPRG is associated with gene silencing, as expression of these genes was restored by chemical demethylation in the MyLa CTCL cell line that exhibits hypermethylation of these genes. The THBS4 gene was previously shown to be silenced through promoter methylation in cancer cells.18

Additionally, we demonstrated hypermethylation of six of eight selected tumor suppressor gene promoters in CTCL using BSA. Promoters of the MGMT, p73, p16, p15, CHFR, and TMS1 genes were found to be hypermethylated in CTCL at varying frequencies.

Concerning the results of DMH analysis, the DNA methylation pattern exhibited by CTCL is tumor type–specific, as it was different from that of colon, ovarian, and breast cancer analyzed previously with similar methods.20,28,29 Hierarchical clustering revealed that MF-TR and CD30– LTCL could not be distinguished from CD30+ LTCL, which has a more favorable prognosis, on the basis of their methylation patterns. This may not be considered surprising as the three entities are very closely related, all originating from CD4+, CD45RO+, CLA+ skin-homing memory T cells. Akin to loss of heterozygosity, recurrent promoter methylation of genes in cancer cells may indicate that these genes encode proteins with tumor suppressive functions, loss of which confers growth advantage. In line with this assumption, the CTCL-specific methylation pattern included several putative tumor suppressor genes.

The CpG island of BCL7a, located in its first exon, was methylated in 48% of CTCL samples. Epigenetic inactivation of BCL7a may be related to clinical behavior of CTCL, as its CpG island was hypermethylated at a higher frequency in aggressive than in indolent CTCL entities. The BCL7a locus is on chromosome 12q24.31 at the site of a recurrent breaking point in B-cell lymphomas. The BCL7a gene was cloned as part of a complex chromosomal translocation in a Burkitt's lymphoma cell line and was subsequently found to be rearranged in another cell line derived from mediastinal large B-cell lymphoma.40 The resultant disruption of this gene is considered to be implicated in the pathogenesis of these lymphomas. However, the exact cellular function of BCL7a, which is expressed at low levels in a wide variety of normal tissues, and the consequences of its functional inactivation are currently unknown. Notably, gene expression profiling has shown that expression of BCL7a is diminished in MF skin lesions: BCL7a was among the seven downregulated genes, significant in separating MF from benign inflammatory skin diseases.41 In addition, BCL7a was recently found to be expressed at significantly lower levels in peripheral T-cell lymphomas when compared with lymphoblastic lymphomas.42 These observations combined with the finding that diminished expression of BCL7a is an unfavorable prognostic sign in patients with B-cell lymphoma strongly suggests that this gene functions as a tumor suppressor in lymphoid cells.43 Promoter hypermethylation is an important mechanism of its inactivation in T-cell lymphomas.

PTPRG, another novel target of epigenetic inactivation in CTCL, was hypermethylated in five (24%) of 21 CTCL patients as confirmed by BSA. Because we additionally observed frequent methylation of PTPRN2 using DMH in CTCL, and because methylation of PTPRO has been described in hepatocellular carcinomas, members of the tyrosine phosphatase gene superfamily may be a common target for epigenetic inactivation.44 Protein tyrosine phosphatases play a role in setting the levels of tyrosine phosphorylation in cells by balancing the activity of tyrosine kinases, thereby regulating signaling pathways that control cellular growth. Whereas constitutive activity of tyrosine kinases has been long recognized as an important oncogenic alteration, inactivation of tyrosine phosphatases is increasingly considered as an important event in tumorigenesis. The PTPRG tumor suppressor gene frequently contains missense mutations in colon carcinomas and is often deleted in lung and renal carcinoma.45,46 Therefore, it has been concluded that PTPRG, which is normally expressed in lymphocytes, is a tumor suppressor gene. However, the functional significance of epigenetic silencing of PTPRG in lymphoid cells has not yet been established and demands further investigation.

THBS4, hypermethylated in 11 (52%) of 21 CTCL samples, encodes an extracellular calcium-binding protein involved in proliferation, adhesion, and migration. Promoter hypermethylation of THBS4, as well of THBS1, has been reported in colon carcinoma, but as yet not in lymphoma.18,47

The promoter of MGMT was methylated in all CTCL samples, but also in three of six control samples, precluding its use as a marker for malignancy in CTCL. MGMT encodes a DNA repair enzyme that acts through removal of alkylating lesions at the guanine base protecting against mutagenesis and malignant transformation, loss of which increases sensitivity to treatment with alkylating agents. The region amplified for BSA was located in the promoter of the MGMT gene, stretching 279 to 508 nucleotides upstream of the transcription start site and 365 nucleotides upstream of the region reported to be methylated in B-cell lymphomas and gliomas (accession number X61657).35,48,49 The observation of MGMT promoter methylation in T cells of healthy subjects is analogous to the finding of promoter methylation of the tumor suppressor gene DAPK in benign as well as malignant B cells.50

The p73 gene promoter was hypermethylated in 11 (48%) of 21 CTCL patient samples. Epigenetic inactivation of the p73 gene appears to be particularly relevant in the pathogenesis of CTCL, since activation-induced cell death in T cells is dependent on the activity of p73.51 Activation-induced cell death is a mechanism essential in preventing unrestricted clonal expansion of activated T cells, such as occurs in CTCL. Although hypermethylation of p73 has been described in nodal B-cell lymphomas and natural-killer cell lymphomas, its methylation had not been previously reported in lymphomas of T-cell origin.35,52,53

The promoter of the CHFR gene, which encodes a protein regulating the mitotic checkpoint pathway governing the transition to metaphase, was hypermethylated in a minority of CTCL patients (19%). Its epigenetic inactivation, previously shown in colon and gastric cancer, may contribute to chromosomal instability.38 Consistent with results of other research groups, we showed hypermethylation of the p15 and p16 promoter in MF patient samples in 18% and 28%6,7 of MF patients. Promoter hypermethylation of p16 was additionally found in CD30+ LTCL. Methylation of MLH1, previously found in a subset of MF patients with demonstrated microsatellite instability, could not be detected in patients included in this study.9

Our results provide evidence for epigenetic instability and widespread promoter methylation in CTCL associated with inactivation of several tumor suppressor genes, that may lead to cell cycle dysregulation (p15, p16, p73), defective DNA repair (MGMT), disruption of apoptosis signaling (TMS1, p73), and chromosomal instability (CHFR). In this study, the occurrence of promoter hypermethylation has been analyzed only in advanced tumor stage MF with blast cell transformation. In previous studies, promoter hypermethylation of the p16 and MLH1 gene has also been detected in the patch/plaque stage of MF, suggesting that these epigenetic alterations may arise early in the development of this T-cell malignancy.6,7,9 The demethylating agents 5-aza-2'-deoxycytidine, zebularine, and MG98, which can reverse silencing due to promoter hypermethylation, are currently tested in clinical trials for treatment of various malignancies and the first agent has already proven to be efficacious in chronic myeloid leukemia.54-56 The finding of promoter hypermethylation in CTCL not only gives insight into the molecular pathogenesis of these malignancies, but in addition provides a rationale for treatment of CTCL patients with demethylating agents.


   Authors' Disclosures of Potential Conflicts of Interest
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
REFERENCES
 
The authors indicated no potential conflicts of interest.


   Acknowledgment
 
We thank Joseph Liu (Division of Human Cancer Genetics, Comprehensive Cancer Center, Ohio State University) for technical assistance related to production of CpG island microarrays, Eddy Wierenga (Department of Cell Biology and Histology, AMC) for providing DNA from T cells of healthy subjects, Judith Boer (Department of Human and Clinical Genetics, LUMC) for advice concerning microarray data analysis, and Jan Willem Dierssen and Tom van Wezel (Department of Pathology, LUMC) for advice on measurement of gene expression in cells treated with 5-aza-2'-deoxycytidine.


   NOTES
 
R. van Doorn was supported by a Genomics Fellowship, awarded by the Netherlands Organization for Scientific Research.

Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.

Authors' disclosures of potential conflicts of interest are found at the end of this article.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
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Submitted January 11, 2005; accepted April 4, 2005.




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