Experimental and Molecular Pathology 84 (2008) 256–261
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Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / yex m p
Aberrant gene promoter methylation in plasma cell dyscrasias Paloma Martin a, Monica Garcia-Cosio b, Almudena Santon b, Carmen Bellas a,⁎ a b
Laboratory of Molecular Pathology, Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain Laboratory of Molecular Pathology, Department of Pathology, Hospital Ramon y Cajal, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 18 October 2007 Available online 7 March 2008 Keywords: Methylation Myeloma Monoclonal gammopathy of undetermined signiﬁcance Plasmacytoma
a b s t r a c t The aberrant methylation of promoter CpG island is known to be a major inactivation mechanism of tumourrelated genes. To determine the clinicopathological signiﬁcance of gene promoter methylation in monoclonal gammopathies, we analysed the methylation status of 6 tumour suppressor genes and their association with loss of gene function. Methylation status of the genes p14, p15, p16, hMLH1, MGMT, and DAPK was determined by methylation-speciﬁc PCR in 52 cases: 30 MM, 13 MGUS, and 9 plasmacytomas, comparing them with their protein expression by immunohistochemistry, and association between methylation status, protein expression, and clinical characteristics was assessed. The methylation frequencies were 50% for p16, 17% for p15, 10% for hMLH1, 23% for MGMT and 30% for DAPK in MM samples, and 38%, 15%, 8%, and 15% for p16, p15, MGMT and DAPK respectively in MGUS samples. In plasmacytomas samples we found methylation of p16 in 55%, p15 in 22%, MGMT in 67% and DAPK in 44%. hMLH1 was unmethylated in all cases of MGUS and plasmacytomas. Immunohistochemistry showed that gene methylation was closely associated with a loss of protein expression. Our study demonstrates that methylation-mediated silencing is a frequent event in monoclonal gammopathies: 83% of MM, 46% of MGUS and 77% of plasmacytomas have at least one gene methylated, affecting different molecular pathways involved in cell cycle, DNA repair and apoptosis. This high prevalence of aberrant promoter hypermethylation suggests that monoclonal gammopathies carry a CpG island methylator phenotype, therefore the development of new DNA demethylation agents may be a potential therapeutic use in this disease. © 2008 Elsevier Inc. All rights reserved.
Introduction Plasma cell dyscrasias are characterised by monoclonal proliferations of plasma cells composed of a number of clinicopathologic entities, including monoclonal gammopathy of undetermined signiﬁcance (MGUS), solitary plasmacytoma, multiple myeloma (MM), and plasma cell leukaemia (PCL). MM is a prototypical clonal B-cell malignancy with a terminally differentiated plasma cell phenotype. MM ranks as the second most frequently occurring haematological malignancy after non-Hodgkin lymphoma, and is characterised by multi-organ dysfunction as a result of bone marrow inﬁltration by malignant cells, and the systemic damage of monoclonal circulating protein (Kyle and Rajkumar, 2004). MGUS is a stable pre-malignant plasma cell tumour that can progress to malignant MM or a related malignant condition at a rate of 1k per year (Kyle and Rajkumar, 2006). Molecular genetic events associated with this transformation have not been identiﬁed. Although molecular studies have largely focused on genetic alterations in MM, during the last few years there has been growing evidence that, in addition to genetic aberrations, epigenetic processes
⁎ Corresponding author. Laboratory of Molecular Pathology, Department of Pathology, Hospital Universitario Puerta de Hierro, C/ San Martin de Porres 4, Madrid 28035, Spain. Fax: +34 913445909. E-mail address: [email protected]
(C. Bellas). 0014-4800/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2008.02.003
play a major role in carcinogenesis (Jones and Baylin, 2002). DNA methylation, catalysed by DNA methyltransferase, involves the addition of a methyl group to the carbon 5 position of the cytosine ring in the CpG nucleotide, leading to a conversion to methylcytosine. In many cancers, the CpG islands of selected genes are aberrantly methylated (hypermethylated), resulting in transcriptional repression of these genes. Tumour-speciﬁc methylation changes in different genes have been identiﬁed, and the methylation proﬁle helps us to distinguish tumour types and, perhaps, their response to chemotherapeutic agents (Alizadeh et al., 2000; Choy et al., 2002; Murata et al., 2005; Takahashi et al., 2004: Yu et al., 2004). Hypermethylation affects different cellular pathways such as cell cycle regulation, DNA repair, and apoptosis, and has relevant consequences in human cancer (Das and Singal, 2004). The INK4A-ARF locus, situated at 9p21, contains two tumour suppressor genes, p14ARF and p16INK4A. p14ARF protein interacts physically with MDM2 and stabilises the p53 protein in the nucleus by blocking its cytoplasmic transport and MDM2-mediated degradation, indicating that it acts as an upstream regulation of p53 function, hence p14 inactivation is an alternate mechanism of disrupting the p53 pathway (Quelle et al., 1995). p14 is inactivated in various cancers through homozygous deletion, promoter methylation or mutations, however the latter is rare (Zheng et al., 2000). p15INK4b and p16INK4a proteins are cell cycle regulators involved in the inhibition of G1 phase progression. Both proteins associate with cyclindependent kinases 4 and 6 (CDK4, CDK6), and inhibit their kinase activities (Le and Yang, 2003). Homozygous deletion, point mutation, or
P. Martin et al. / Experimental and Molecular Pathology 84 (2008) 256–261 Table 1 Clinical and laboratory characteristics of 52 patients with monoclonal gammopathies Characteristics
No. of patients
Median patient age (range), years Gender (n = 52) Male Female Paraprotein isotype IgA IgG IgM Light chain only Non-secretory Plasmacytomas (n = 9) Extramedullary Solitary bone Stage MGUS I II III Plasma cell leukaemia (n)
68 (39–97) 24 28 16 20 1 5 1 5 4 13 5 5 19 1
methylation can inactivate the p16INK4A and p15INK4B genes. Hypermethylation of the p16 promoter has been detected across many tumour types such as colorectal cancer, lung, and breast carcinomas and lymphomas, whereas hypermethylation of p15 is only observed in haematological malignancies (Esteller et al., 2001). DNA repair systems act to maintain genome integrity in the presence of replication errors, environmental insults, and the cumulative effects of ageing (Fink et al., 1998). hMLH1 and MGMT are two enzymes implicated in DNA repair, and we have recently shown that inactivation of the repair pathway plays an important role in genetic instability in plasma cell dyscrasias (Martin et al., 2006). hMLH1 is an MMR gene responsible for correcting insertion/deletion loops and single base–base mismatched pairs that arise during normal DNA replication, especially in the repeated sequence motifs, such as microsatellites (Velangi et al., 2004; Jiricny and Nystrom-Lahti, 2000). MGMT (O6-methylguanine DNA methyl-transferase) encodes a DNA repair protein that removes mutagenics and cytotoxic adducts at
Table 2 Primers used for the analysis of methylation-speciﬁc PCR
O6 of guanine and, therefore, plays an important role in maintaining normal cell physiology and genomic stability (Gerson, 2004). MGMT is closely related with cellular sensitivity to alkylating agents, and is inactivated by promoter hypermethylation in several human cancers. The presence of MGMT methylation is associated with better overall survival in diffuse large B-cell lymphoma (Esteller et al., 2002), and is involved in response to treatment in glioblastomas (Hau et al., 2007; Paz et al., 2004; Hiraga et al., 2006). Death-associated protein kinase (DAPK) is a pro-apoptotic serine/ threonine kinase involved in the extrinsic pathway of apoptosis, initiated by γ-interferon FAS ligand and tumour necrosis factor-α (Ng, 2002; Cohen et al., 1999). Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, especially in B-cell malignancies (Katzenellenbogen et al., 1999), providing a selective growth advantage during tumour progression (Tang et al., 2000). To explore the role of DNA methylation in plasma cell disorders we selected six tumour-related genes frequently silenced by aberrant methylation, and examined the methylation status for the following genes by methylation-speciﬁc PCR (MSPCR): p16, p14, p15, DAPK, hMLH1 and MGMT. We also analysed protein expression of p15, p16, hMLH1 and MGMT using immunohistochemistry (IHC). Materials and methods Human tissue samples A total of 54 cases was included in the study: 29 MM, 13 MGUS, 1 PCL, 2 polyclonal plasmacytosis and 9 plasmacytomas. Bone marrow aspirates (n = 45) and bone marrow biopsies (n = 29) from the posterior iliac crest were collected from 45 consenting patients during routine clinical assessment. Patients were classiﬁed according to the current WHO classiﬁcation. Analyses of age, gender, haemoglobin levels, presence of lytic bone lesions, creatinine, serum calcium levels, LDH, β2 microglobulin, paraprotein subtype, percentage and morphology of plasma cells and stage of disease were performed for all patients. Mononuclear cell suspensions (MCS) were prepared from bone marrow aspirates by Ficoll-Paque plus gradient centrifugation (Amersham Pharmacia AB, Uppsala, Sweeden), and plasma cell isolation from MCS was performed by immunomagnetic bead selection with CD138 using the Automacs system (Miltenyi, CA). Enriched fractions were assessed for purity by CD138-ﬂuoresceina isotiocyanate (FITC) monoclonal antibody labelling (BB4, Cytognos). CD138, also known as syndecan-1, is expressed on plasma cells, but not on circulating B cells, T cells, or monocytes (Wijdenes et al., 1996). The purity of plasma cells obtained by this method was more than 90% as conﬁrmed by ﬂow cytometry (FAC sort: Becton Dickinson, San José) using the Paint a Gate program. Plasma cell leukaemia and plasmacytomas were not puriﬁed. Frozen and parafﬁn embedded plasmacytomas were obtained from the Spanish National Tumour Bank Network, coordinated by the Spanish National Cancer Centre (CNIO), following the technical and ethical procedures of the network. DNA was extracted from CD138 positive cells using Tri-Reagent (from Becton Dickinson, San José) according to the manufacturer's instructions.
Primer sequence 5′ to 3′
TTA ATA GGA AGA GTG GAT AGT G TCT ATA AAT TAC TAA ATC TCT TCA TTA ATA GGA AGA GCG GAT AGC CTA TAA ATT ACT AAA TCT CTT CG TTT GTG TTT TGA TGT TTG TAG GTT TTT GT AAC TCC ACA CTC TTC CAA AAA CAA AAC A TTT CGA CGT TCG TAG GTT TTC GC GCA CTC TTC CGA AAA CGA AAC G GGA GGA TAG TTG GAT TGA GTT AAT GTT CAA ATC CCT CCC AAA CAC CAA GGA TAG TCG GAT CGA GTT AAC GTC CCC TCC CAA ACG A TTA TTA GAG GGT GGG GTG GAT TGT CAA CCC CAA ACC ACA ACC ATA A TTA TTA GAG GGT GGG GCG GAT CGC GAC CCC GAA CCG CGA CCG TAA TGT GAT GTG TTT GTA TTT TGT GGT T CCA TAC AAT AAC CAA ACA ACC AA GCG TTC GTA TTT TGC GGT T CGT ACA ATA ACC GAA CGA CCG A TTT TTG GTG TTA AAG GGT GGT GTA GT CAC AAA AAC CCT CAC TCA CAA GTG TTA AAG GGC GGC GTA GC AAA ACC CTC ACT CGC GAC GA
Ta 55 °C
Bisulphite modiﬁcation and methylation-speciﬁc PCR
Ta 55 °C
Ta 62 °C
Bisulphite conversion was performed as previously described (Herman et al., 1996). DNA (1 μg) was denatured by NaOH. The denatured DNA was treated with sodium bisulphite to chemically modify the unmethylated cytosines to uracil. Modiﬁed DNA was puriﬁed using the Wizard DNA Clean-up system (Promega, Madison, WI). Samples
Ta 62 °C
Ta 60 °C
Ta 60 °C
Ta 61 °C
Us Ua Ms Ma Us Ua Ms Ma Us Ua Ms Ma Us Ua Ms Ma Us Ua Ms Ma Us Ua Ms Ma
Ta 67 °C
Table 3 Results of methylation and immunohistochemical expression p16
MM and PCL MSPCR (n = 30) IHC neg (n = 16)
15 (50%) 11 (69%)
5 (17%) ND
3 (10%) 8 (50%)
7 (23%) 7 (44%)
9 (30%) ND
MGUS MSPCR (n = 13) IHC neg (n = 11)
5 (39%) 7 (64%)
2 (15%) ND
1 (8%) 4 (36%)
2 (15%) ND
Plasmacytomas MSPCR (n = 9) IHC neg (n = 9)
5 (55%) 6 (67%)
2 (22%) 5 (55%)
0 2 (22%)
6 (67%) 6 (67%)
4 (44%) ND
T 62 °C
Ta 62 °C
Ta 60 °C
Ta 60 °C
Us: unmethylated sense; Ua: unmethylated antisense; Ms: methylated sense; Ma: methylated antisense; Ta: annealing temperature.
MSPCR: methylation-speciﬁc PCR; ND: Not done; IHC neg: loss of immunohistochemical protein expression.
P. Martin et al. / Experimental and Molecular Pathology 84 (2008) 256–261 Stained slides were evaluated for the presence of p15, p16, MGMT or hMLH1 expression in the tumour by two independent observers (P.M and M.G-C). Statistical analysis The methylation status of the genes p14, p15, p16, MGMT, hMLH1, and DAPK was compared with clinicopathologic characteristics, including age, gender, type of paraprotein, type of light chain, tumour stage, renal insufﬁciency, LDH, β2-microglobulin, C-reactive protein, albumin, and level of M-component using the chi-square test. Survival probability curves were analysed according to the method of Kaplan and Meier and were compared using the log-rank test.
Fig. 1. Percentage methylation of ﬁve selected genes in patients with multiple myeloma, monoclonal gammopathy of undetermined signiﬁcance, and plasmacytoma.
Results Clinical and histological ﬁndings
were eluted in 50 μl of water, and modiﬁcation was completed by NaOH, precipitated with ethanol and resuspended in 25 μl of water. This process converts unmethylated cytosine residues to uracil, and methylated cytosine residues remain unchanged. MSPCR was then performed to examine the methylation status of six cancer related genes involved in: cell cycle regulation (p14, p15, p16), repair of DNA (hMLH1, MGMT), and apoptosis (DAPK). DNA from peripheral blood lymphocytes taken from healthy donors was used as the unmethylated control, whereas universal methylated DNA (CpGenome™ Universal Methylated Control DNA, Chemicon) was used as the positive control. Additionally, control experiments without DNA accompanied every ampliﬁcation reaction. The sequences of the primers and annealing temperatures are summarized in Table 1. Each PCR product was loaded directly onto 3% agarose gel stained ethidium bromide and directly visualised under UV illumination. Immunohistochemistry Parafﬁn-embedded sections from bone marrow biopsies and plasmacytomas were stained with p15, p16, hMLH1 and MGMT. Antigen retrieval was achieved by heating in a pressure cooker for 4 min in 10 mM citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked for 20 min in 0.3% H2O2 in methanol. The sections were incubated for 1 h at 37 °C with the primary antibodies. We used monoclonal mouse antihuman hMLH1 (clone G-168-15, PharMingen International, CA) at a dilution of 1:50, MGMT (clone MT3.1, Dako) 1:100 and immunodetection was performed with the Dako EnVision+™ DAB system. Normal laryngeal epithelium and reactive lymphadenitis were used as positive controls for hMLH1 and MGMT, respectively. hMLH1 and MGMT were interpreted as positive when there were more than 30% of positive cells (Liu et al., 2003; Kitajima et al., 2003). p16 (clone E6H4, Dako) and p15 (clone 15PO6, Laboratory Vision, Newmarket, U.K.) were used at a dilution of 1:25. These antibodies were detected using the LSAB Visualization System (Dako, Glostgrup, Denmark). p15 and p16 were interpreted as positive when there were more than 25% of positive plasma cells. The positive control for p16 marker was a case of squamous carcinoma of the uterine cervix, and reactive lymph node was used for p15. Counterstaining of the nuclei was performed with hematoxylin. Only nuclear staining was regarded as positive staining.
The clinicopathologic data for the cases are summarized in Table 2. Patients were classiﬁed as MGUS (n = 13), MM (n = 29), PCL (n = 1) and 9 plasmacytomas according to the World Health Organization classiﬁcation system. Of the 13 MGUS patients, 5 were male and 8 were female. The MGUS patients had a median age of 71 years (range: 52–85 years). The monoclonal serum protein was IgG kappa in 6 cases, IgG lambda in 4 cases, IgA lambda in 1, IgA kappa in 1, and IgM lambda in 1 case. The 29 MM patients (14 male and 15 female) had a median age of 68 years (range: 39–85 years) and the PCL corresponds to a 77-year-old man. The monoclonal serum protein was IgG kappa in 8 cases, IgG lambda in 2 cases, IgA lambda in 5, IgA kappa in 9 (including the PCL). Five cases were light chains only, and one was MM non-secretor. According to the recently developed International Staging System (Greipp et al., 2005), myeloma patients were distributed as follows: ﬁve patients had stage I disease, ﬁve patients had stage II, and nineteen patients had stage III. One patient had PCL with a previous history of MM. Of the nine plasmacytomas (4 male, 5 female), four were extramedullary plasmacytomas and ﬁve were solitary bone plasmacytomas. The patients with plasmacytomas had a median age of 60 years (range: 39–97). Frequency of promoter hypermethylation of tumour-related genes in monoclonal gammopathies The methylation proﬁles of patients are shown in Table 3. From 52 plasma cell disorders, the methylation percentages were as follows: of the 30 MM samples hypermethylation of p15, p16, hMLH1, MGMT, and
Fig. 2. Representative methylation-speciﬁc PCR assays for p14, p15, p16, hMLH1, MGMT, and DAPKinase in six samples. M: reactions with methylation-speciﬁc primer set, U: reactions with unmethylation-speciﬁc primer. C−: peripheral blood mononuclear cells were used as control for unmethylated alleles. C+: commercial positive control for methylated alleles, Mw: Molecular weight marker, B: blank.
P. Martin et al. / Experimental and Molecular Pathology 84 (2008) 256–261
DAPK occurred in ﬁve (16.7%), ﬁfteen (50%), three (10%), seven (23.3%), and nine (30%) respectively. In the MGUS group (n = 13) we found methylation of p15 in two samples (15.4%), p16 in ﬁve (38.5%), MGMT in one case (7.7%), and DAPK in two cases (15.4%). Two cases (22.2%) out of 9 plasmacytomas showed methylation of p15, ﬁve cases (55.5%) of p16, six cases (66.6%) of MGMT and four cases (44.4%) of DAPK. None of the patients with MGUS or plasmacytomas had methylation of hMLH1, and none of the 52 cases had p14 methylated. Representative results of the MSPCR are shown in Figs. 1 and 2. At least one hypermethylated gene was found in 83% (25 / 30) of the multiple myeloma, while 10% (3 / 30) of MM harboured more than two methylated genes. At least one gene was found methylated in 46% (6 / 13) of MGUS samples, and in 77% (7 / 9) of plasmacytoma samples. Polyclonal cases were unmethylated for the six genes studied. Positive and negative controls worked appropriately in each PCR reaction round. Immunohistochemical analysis We investigated the expression of p16, MGMT and hMLH1 protein in 36 samples (16 MM, 11 MGUS, and 9 plasmacytomas) using IHC analysis
(Table 3, Fig. 3). Eleven cases of MM (68.7%), seven cases of MGUS (63.6%) and six plasmacytomas (66.6%) showed loss of p16 expression. Half of the myeloma cases (8 cases) and two (22.2%) plasmacytomas showed loss of hMLH1 expression. All samples of MGUS showed hMLH1 protein expression. Seven MM samples (43.7%), four MGUS samples (36.4%), and six plasmacytomas (66.6%) lacked MGMT protein expression. For technical reasons, expression of p15 was only performed in plasmacytomas: ﬁve cases (55.5%) had lost its expression. Relation between methylation, immunohistochemistry, and clinicopathological parameters Methylation results were analysed for potential correlations with clinicopathological parameters; we did not ﬁnd statistically signiﬁcant correlations between methylation status and overall survival, neither with clinicopathologic characteristics. We observed a trend toward poorer survival for patients who had hMLH1 methylated. Methylated samples have failed to express the respective protein by immunohistochemistry. We have found lack of expression in some unmethylated samples, suggesting that an additional mechanism could be involved in gene inactivation.
Fig. 3. Photomicrographs of bone marrow biopsies from cases with plasma cell dyscrasia A–C; MGMT expression in: MM with low plasma cell inﬁltration (A), MM sample MGMT negative (B), PCL with nuclei strongly positive (C). D–E; hMLH1 expression in MM (D) and in MGUS (E), both cases were positive. F–G; Loss of p16 expression in MM (F) and MGUS (G). H; Positive staining for p15 in plasmacytoma sample.
P. Martin et al. / Experimental and Molecular Pathology 84 (2008) 256–261
Discussion In the current study, the prevalence of hypermethylation of a panel of tumour-associated genes was investigated in a group of monoclonal gammopathies. The ﬁndings show that genes p15, p16, MGMT and DAPK are frequently methylated throughout the clinicopathologic spectrum of monoclonal gammopathies. We found methylation and lack of p16 protein expression in all stages of the disease and, like others authors, we did not ﬁnd any correlation between p16 methylation and clinical and laboratory parameters (Seidl et al., 2004; Guillerm et al., 2001; Gonzalez-Paz et al., 2007). These characteristics allow us to suggest that G1 cell phase deregulation is an early event that might contribute to the immortalization of plasma cells rather than malignant transformation (from MGUS to MM) or disease progression. Discrepant results about methylation of p15 in MM can be found in scientiﬁc literature (Seidl et al., 2004; Chim et al., 2004; Galm et al., 2004). Our results are similar to those reported by Seidl et al., 2004; approximately 15% of cases were methylated in MGUS and MM, and no association was found between p15 methylation and clinical data. The immunohistochemical analysis showed that more than 50% of cases of MM, MGUS, and plasmacytomas had lost expression of p16, and 55.5% of plasmacytomas were negative for p15. Recently Sarasquete et al. (2006) suggested that MM patients with high levels of tumour suppressor genes (p16 and p15) may have a relatively indolent form of the disease, with good prognostic features and long overall survival. They used gene expression (real time quantitative polymerase chain reaction) to assess the expression of p14, p15, and p16, in puriﬁed plasma cells, and they observed that their expression was higher in smouldering MM, and this overexpression helps to arrest cell cycling and leads to the disease behaving less aggressively. Frequency of p16 methylation in plasma cell dyscrasias has been described by other groups (Guillerm et al., 2001; Ng et al., 1997; Mateos et al., 2002; Uchida et al., 2001; Gonzalez et al., 2000), and more recently by Chim C.S. (Chim et al., 2007), however the role of p16 methylation in monoclonal gammopathies pathogenesis is still unclear, and further studies should be undertaken to understand its association with transcriptional silencing and its impact on clinical outcome. p14 promoter hypermethylation has been described in primary tumours such as colorectal, gastric, bladder, lung and breast carcinomas (Dominguez et al., 2003), but little is known about the role of p14 in haematopoietic malignancies; aberrant methylation of p14 is found in 40% of accelerated phases of chronic myeloid leukaemia (Nagy et al., 2003), whereas a recent study shows that p14 was completely unmethylated in chronic lymphoid leukaemia samples (Chim et al., 2006). We did not ﬁnd hypermethylation of p14 in any of the study samples; hence we believe that p14 promoter methylation is not an oncogenic mechanism in plasma cell disorders, as it happens in follicular lymphoma (Hayslip and Montero, 2006). Inactivation of DAP-kinase due to promoter hypermethylation is a common event in B-cell diseases and MM (Ng et al., 2001). Its inactivation may provide cells with a resistance mechanism against different apoptotic stimuli. Voso et al. (2006) reported that DAPK hypermethylation may be used as a potential residual disease marker in B-cell lymphomas, and could identify a subset of follicular lymphoma patients with a negative prognostic proﬁle. In our study, we observed that DAPK hypermethylation was more frequent in MM and plasmacytomas than in MGUS, and this fact suggests that DAPK could be implicated in the malignant progression of monoclonal gammopathies. Additional studies including a larger number of cases and longer follow-up will be needed to conﬁrm its prognostic signiﬁcance in plasma cell malignancies. Alterations in the expression of hMLH1 and MGMT have been detected in several haematological (Rossi et al., 2003) and solid tumours. The ﬁnding that none of the MGUS or plasmacytomas cases had a loss of hMLH1 expression, whereas half the MM cases lacked it,
leads us to suggest that the loss of hMLH1 plays a role in the malignant transformation of plasma cells (Martin et al., 2006). We know that the MMR system is involved in signalling the presence of DNA damage to the apoptotic machinery conferring resistance to chemotherapy (Jiricny and Nystrom-Lahti, 2000), consequently, the silencing of hMLH1 may be one of the causes of drug-resistance, relapse and refractory disease characteristic in MM. MGMT inactivation is an important mechanism in lymphomagenesis, MGMT-knockout mice develop lymphoma with high frequency (Sakumi et al., 1997). MGMT methylation and loss of protein expression have been found in all stages of the disease, especially in plasmacytomas. In the survival analysis, we only found a trend to poorer survival in cases with hMLH1 methylated, supporting the work of Kotoula et al. (2002), who described low expression or absence of hMLH1 in MM samples with extensive bone marrow inﬁltration. The methylation status of other genes had no prognostic impact in our series. Although there are different reports that describe the frequency of methylation in monoclonal gammopathies, to our knowledge this is the largest series that study methylation in plasmacytomas. In this study, we have showed that absent of MGMT, p16, and p15 protein expression is frequent in plasmacytomas, nearly 70% were negative for p16 and MGMT, and 55% were negative for p15. Hence we have found a high rate of methylation of p15, p16, MGMT and DAPK in plasmacytomas supporting that aberrant gene methylation is a common phenomenon in the multistep transformation process of plasma cells. We have not found differences of methylation between single bone and extramedullary plasmacytomas. In summary, our results indicate that the hypermethylation of several genes, especially p16, MGMT, and DAPK, is a frequent event in plasma cell dyscrasias, and supports the hypothesis that a variety of genes regulating different pathways may be affected by aberrant methylation in malignant plasma cell disorders. Methylation changes are already present in the pre-malignant MGUS condition, and the frequency of methylation is higher in myeloma and plasmacytomas, suggesting that epigenetic mechanisms could contribute to the malignant transformation in these diseases, presenting the possibility of using demethylating therapeutics as an adjuvant regimen. Acknowledgments This work has been partially supported by projects from the Spanish Ministry of Health (FIS G03/179) and from Fundacion Mutua Madrileña. PM is a recipient of research grant from the Fondo de Investigaciones Sanitarias. The authors wish to thank Martin HadleyAdams for his assistance with the English language and preparation of the manuscript. The study was conducted according to good clinical and laboratory practice rules, and the principles of the Declaration of Helsinki. Parts of our study had been presented at the XIII Meeting of the European Association for Haematopathology, Vienna 2006. References Alizadeh, A.A., Eisen, M.B., Davis, R.E., Ma, C., Lossos, I.S., Rosenwald, A., Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L., Marti, G.E., Moore, T., Hudson Jr., J., Lu, L., Lewis, D.B., Tibshirani, R., Sherlock, G., Chan, W.C., Greiner, T.C., Weisenburger, D.D., Armitage, J.O., Warnke, R., Levy, R., Wilson, W., Grever, M.R., Byrd, J.C., Botstein, D., Brown, P.O., Staudt, L.M., 2000. Distinct types of diffuse large B-cell lymphoma identiﬁed by gene expression proﬁling. Nature 403, 503–511. Chim, C.S., Kwong, Y.L., Fung, T.K., Liang, R., 2004. Methylation proﬁling in multiple myeloma. Leuk.Res. 28, 379–385. Chim, C.S., Fung, T.K., Wong, K.F., Lau, J.S., Liang, R., 2006. Frequent DAP kinase but not p14 or Apaf-1 hypermethylation in B-cell chronic lymphocytic leukemia. J. Hum. Genet. 51, 832–838. Chim, C.S., Lyang, R., Leung, M.H., Kwong, Y.L., 2007. Aberrant gene methylation implicated in the progression of monoclonal gammopathy of undetermined signiﬁcance to multiple myeloma. J. Clin. Pathol. 60, 104–106. Choy, K.W., Pang, C.P., To, K.F., Yu, C.B., Ng, J.S., Lam, D.S., 2002. Impaired expression and promotor hypermethylation of O6-methylguanine-DNA methyltransferase in retinoblastoma tissues. Invest. Ophthalmol. Vis. Sci. 43, 1344–1349.
P. Martin et al. / Experimental and Molecular Pathology 84 (2008) 256–261 Cohen, O., Inbal, B., Kissil, J.L., Raveh, T., Berissi, H., Spivak-Kroizaman, T., Feinstein, E., Kimchi, A., 1999. DAP-kinase participates in TNF-alfa and Fas-induced apoptosis and its function requires the death domain. J. Cell Biol. 146, 141–148. Das, P.M., Singal, R., 2004. DNA methylation and cancer. J. Clin. Oncol. 22, 4632–4642. Dominguez, G., Silva, J., Garcia, J.M., Silva, J.M., Rodríguez, R., Muñoz, C., Chacon, I., Sanchez, R., Carballido, J., Colas, A., España, P., Bonilla, F., 2003. Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors. Mutat. Res. 530, 9–17. Esteller, M., Corn, P.G., Baylin, S.B., Herman, J.G., 2001. A gene hypermethylation proﬁle of human cancer. Cancer Res. 61, 3225–3229. Esteller, M., Gaidano, G., Goodman, S.N., Zagonel, V., Capello, D., Botto, B., Rossi, D., Gloghini, A., Vitolo, U., Carbone, A., Baylin, S.B., Herman, J.G., 2002. Hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma. J. Natl. Cancer Inst. 94, 26–32. Fink, D., Nebel, S., Norris, P.S., Aebi, S., Lin, T.P., Nehme, A., Christen, R.D., Haas, M., McLeod, C.L., Howel, S.B., 1998. The effect of different chemotherapeutic agents on the enrichment of DNA mismatch repair-deﬁcient tumour cells. Br. J. Cancer 77, 703–708. Galm, O., Wilop, S., Reichelt, J., Jost, E., Gehbauer, G., Herman, J.G., Osieka, R., 2004. DNA methylation changes in multiple myeloma. Leukemia 18, 1687–1692. Gerson, S.L., 2004. MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev., Cancer 4, 296–307. Gonzalez, M., Mateos, M.V., Garcia-Sanz, R., Balanzategui, A., Lopez-Perez, R., Chillon, M.C., Gonzalez, D., Alaejos, I., San Miguel, J.F., 2000. De novo methylation of tumor suppressor gene p16/INK4a is a frequent ﬁnding in multiple myeloma patients at diagnosis. Leukemia 14, 183–187. Gonzalez-Paz, N., Chng, W.J., McClure, M.F., Blood, E., Oken, M.M., Van Ness, B., James, C.D., Kurtin, P.J., Henderson, K., Ahmann, G.J., Gertz, M., Lacy, M., Dispenzieri, A., Greipp, P.R., Fonseca, R., 2007. Tumor suppressor p16 methylation in multiple myeloma: biological and clinical implications. Blood 109, 1228–1232. Greipp, P.R., San Miguel, J., Durie, B.G., Crowley, J.J., Barlogie, B., Blade, J., Boccadoro, M., Child, J.A., Avet-Loiseau, H., Kyle, R.A., Lahuerta, J.J., Ludwig, H., Morgan, G., Powles, R., Shimizu, K., Shustik, C., Sonneveld, P., Tosi, P., Turesson, I., Westin, J., 2005. International staging system for multiple myeloma. J. Clin. Oncol. 23, 3412–3420. Guillerm, G., Gyan, E., Wolowiec, D., Facon, T., Avet-Loiseau, H., Kuliczkowski, K., Bauters, F., Fenaux, P., Quesnel, B., 2001. p16INK4a and p15INK4b gene methylations in plasma cells from monoclonal gammopathy of undetermined signiﬁcance. Blood 98, 244–246. Hau, P., Stupp, R., Hegi, M.E., 2007. MGMT methylation status: the advent of stratiﬁed therapy in glioblastoma? Dis. Markers 23, 97–104. Hayslip, J., Montero, A., 2006. Tumor suppressor gene methylation in follicular lymphoma: a comprehensive review. Mol. Cancer 5, 44. Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., Baylin, S.B., 1996. Methylationspeciﬁc PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U. S. A. 93, 9821–9826. Hiraga, J., Kinoshita, T., Ohno, T., Mori, N., Ohashi, H., Fukami, S., Noda, A., Ichikawa, A., Naoe, T., 2006. Promoter hypermethylation of the DNA repair gene O6methylguanine-DNA methyltransferase and p53 mutation in diffuse large B-cell lymphoma. Int. J. Hematol. 84, 248–255. Jiricny, J., Nystrom-Lahti, M., 2000. Mismatch repair defects in cancer. Curr. Opin. Genet. Dev. 10, 157–161. Jones, P.A., Baylin, S.B., 2002. The fundamental role of epigenetics events in cancer. Nat. Rev., Genet. 3, 415–428. Katzenellenbogen, R.A., Baylin, S.B., Herman, J.G., 1999. Hypermethylation of the DAPkinase CpG island is a common alteration in B-cell malignancies. Blood 93, 4347–4353. Kitajima, Y., Miyazaki, K., Matsukura, S., Tanaka, M., Sekiguchi, M., 2003. Loss of expression of DNA repair enzymes MGMT, hMLH1, and hMSH2 during tumor progression in gastric cancer. Gastric Cancer 6, 86–95. Kotoula, V., Hytiroglou, P., Kaloutsi, V., Barbani, S., Kouidou, S., Papadimitriou, C.S., 2002. Mismatch repair gene expression in malignant lymphoproliferative disorders of B-cell origin. Leuk. Lymphoma 43, 393–399. Kyle, R.A., Rajkumar, S.V., 2004. Multiple myeloma. N. Engl. J. Med. 351, 1860–1873. Kyle, R.A., Rajkumar, S.V., 2006. Monoclonal gammopathy of undetermined signiﬁcance. Br. J. Haematol. 134, 573–589. Le, M.H., Yang, H.Y., 2003. Regulators of G1 cyclin-dependent kinases and cancer. Cancer Metastasis Rev. 22, 435–449. Liu, K., Zuo, C., Luo, Q.K., Suen, J.Y., Hanna, E., Fan, C.Y., 2003. Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma. Diagn. Mol. Pathol. 12, 50–56. Martin, P., Santon, A., Garcia-Cosio, M., Bellas, C., 2006. hMLH1 and MGMT inactivation as a mechanism of tumorigenesis in monoclonal gammopathies. Mod. Path. 19, 914–921.
Mateos, M.V., Garcia-Sanz, R., Lopez-Perez, R., Moro, M.J., Ocio, E., Hernandez, J., Megido, M., Caballero, M.D., Fernandez-Calvo, J., Barez, A., Almeida, J., Orfao, A., Gonzalez, M., San Miguel, J.F., 2002. Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival. Br. J. Haematol. 118, 1034–1040. Murata, H., Khattar, N.H., Gu, L., Li, G.M., 2005. Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer. Cancer Lett. 223, 143–150. Nagy, E., Beck, Z., Kiss, A., Csoma, E., Telek, B., Konya, J., Olah, E., Rak, K., Coth, F.P., 2003. Frequent methylation of p16INK4A and p14ARF genes implicated in the evolution of chronic myeloid leukaemia from its chronic to accelerated phase. Eur. J. Cancer 39, 2298. Ng, M.H., 2002. Death associated protein kinase: from regulation of apoptosis to tumour suppressive functions and B-cell malignancies. Apoptosis 7, 261–270. Ng, M.H., Chung, Y.F., Lo, K.W., Wickman, N.W., Lee, J.C., Huang, D.P., 1997. Frequent hypermethylation of p16 and p15 genes in multiple myeloma. Blood 89, 2500–2506. Ng, M.H., To, K.W., Lo, K.W., Chan, S., Tsang, K.S., Cheng, S.H., Ng, H.K., 2001. Frequent death-associated protein kinase promoter hypermethylation in multiple myeloma. Clin. Cancer Res. 7, 1724–1729. Paz, M.F., Yaya-Tur, R., Rojas-Marcos, I., Reynes, G., Pollan, M., Aguirre-Cruz, L., GarciaLopez, J.L., Piquer, J., Safont, M.J., Balana, C., Sanchez-Cespedes, M., GarciaVillanueva, M., Arribas, L., Esteller, M., 2004. CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas. Clin. Cancer Res. 10, 4933–4938. Quelle, D.E., Zindy, F., Ashmun, R.A., Sherr, C.J., 1995. Alternative ready frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducting cell cycle arrest. Cell 83, 993–1000. Rossi, D., Gaidano, G., Gloghini, A., Deambrogi, C., Franceschetti, S., Berra, E., Cerri, M., Vendramin, C., Conconi, A., Viglio, A., Muti, G., Oreste, P., Morra, E., Paulli, M., Capello, D., Carbone, A., 2003. Frequent aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase and death-associated protein kinase genes in immunodeﬁciency-related lymphomas. Br. J. Haematol. 123, 475–478. Sakumi, K., Shiraishi, A., Shimizu, S., Tsuzuki, T., Ishikawa, T., Sekiguchi, M., 1997. Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice. Cancer Res. 57, 2415–2418. Sarasquete, M.E., Garcia-Sanz, R., Armellini, A., Fuertes, M., Martín-Jiménez, P., Sierra, M., Chillón, M.C., Alcoceba, M., Balanzategui, A., Ortega, F., Hernández, J.M., Sureda, A., Palomara, L., González, M., San Miguel, J.F., 2006. The association of increased p14ARF/p16INK4a and p15INK4b gene expression with proliferative activity and the clinical course of multiple myeloma. Haematologica 91, 1551–1554. Seidl, S., Ackermann, J., Kaufmann, H., Keck, A., Nösslinger, T., Zielinski, C.C., Drach, J., Zöchbauer-Müller, S., 2004. DNA-methylation analysis identiﬁes the E-cadherin gene as a potential maker of disease progression in patients with monoclonal gammopathies. Cancer 100, 2598–2606. Takahashi, T., Shivapurkar, N., Reddy, J., Suzuki, M., Miyajima, K., Zhou, X., Bekele, B.N., Gazdar, A.F., Wistuba, I.I., 2004. DNA methylation proﬁles of lymphoid and hematopoietic malignancies. Clin. Cancer Res. 10, 2928–2935. Tang, X., Khuri, F.R., Lee, J.J., Kemp, B.L., Liu, D., Hong, W.K., Mao, L., 2000. Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I nonsmall-cell lung cancer. J. Natl. Cancer Inst. 92, 1511–1516. Uchida, T., Kinoshita, T., Ohno, T., Ohashi, H., Nagai, H., Saito, H., 2001. Hypermethylation of p16INK4A gene promoter during the progression of plasma cell dyscrasia. Leukemia 15, 157–165. Velangi, M.R., Matheson, E.C., Morgan, G.J., Jackson, G.H., Taylor, P.R., Hall, A.G., Irving, J.A., 2004. DNA mismatch repair pathway defects in the pathogenesis and evolution of myeloma. Carcinogenesis 25, 1795–1803. Voso, M.T., Gumiero, D., D'Alo', F., Guida, F., Mansuelo, G., Di Febo, A.L., Massini, G., Martín, M., Larocca, L.M., Hohaus, S., Leone, G., 2006. DAP-kinase hypermethylation in the bone marrow of patients with follicular lymphoma. Haematologica 91, 1252–1256. Wijdenes, J., Vooijs, W.C., Clement, C., Post, J., Morard, F., Vita, N., Laurent, P., Sun, R.X., Klein, B., Dore, J.M., 1996. A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1. Br. J. Haematol. 94, 318–323. Yu, J., Zhang, H., Gu, J., Lin, S., Li, J., Lu, W., Wang, Y., Zhu, J., 2004. Methylation proﬁles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma. BMC Cancer 4, 65. Zheng, S., Chen, P., McMillan, A., Lafuente, A., Lafuente, M.J., Ballesta, A., Trias, M., Wiencke, J.K., 2000. Correlations of partial and extensive methylation at the p14ARF locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors. Carcinogenesis 21, 2057–2064.