Annals of Oncology Advance Access published online on June 6, 2008
Annals of Oncology, doi:10.1093/annonc/mdn374
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Prognostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphomas
1 Department of Pathology
2 Department of Clinical Hematology, Farhat-Hached Hospital of Sousse, Tunisia
* Correspondence to: Dr M. Trimeche, Department of Pathology, Farhat-Hached Hospital, Sousse 4000, Tunisia. Tel: +216-21-311760; Fax: +216-73-210355; E-mail: m_trimech{at}yahoo.fr
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Background: Diffuse large B-cell lymphoma (DLBCL) exhibits heterogeneous clinical features and a marked variable response to treatment.
Patients and methods: We investigated the prognostic significance of the methylation status of DAPK, GSTP1, P14, P15, P16, P33, RB1, SHP1, CDH1, APC, BLU, VHL, TIMP3, and RASSF1A genes in 46 DLBCL specimens from Tunisian patients. Methylation status of each gene was correlated with clinicopathological parameters including the International Prognostic Index (IPI), the germinal center immunophenotype, and response to treatment and survival. Overall survival (OS) and disease-free survival (DFS) rates were calculated by the Kaplan–Meier method and differences were compared with the log-rank test.
Results: Hypermethylation of SHP1 was associated with elevated lactate dehydrogenase level (P = 0.031). P16 and VHL were frequently hypermethylated in patients with high IPI scores (P = 0.006 and 0.004) and a performance status of two or more (P = 0.007 and 0.047). In addition, hypermethylation of P16 was significantly associated with advanced clinical stages and B symptoms (P = 0.041 and 0.012). Interestingly, hypermethylation of DAPK was significantly correlated with resistance to treatment (P = 0.023). With regard to survival rates, promoter hypermethylation of DAPK, P16, and VHL were significantly associated with shortened OS (P = 0.003, 0.001, and 0.017, respectively) and DFS (P = 0.006, 0.003, and 0.046, respectively). In multivariate analysis, hypermethylation of DAPK remains an independent prognostic factor in predicting shortened OS (P = 0.001) and DFS (P = 0.024), as well as the IPI and the germinal center status.
Conclusions: This study demonstrates that DLBCLs with hypermethylated P16, VHL, DAPK, and SHP1 commonly show a biologically aggressive phenotype and worse prognosis. Interestingly, hypermethylation of DAPK was found to be an independent prognostic factor that may be used in conjunction with the conventional prognostic factors such as the IPI and the germinal center status.
diffuse large B-cell lymphomas, hypermethylation, prognosis, tumor suppressor genes
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Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphomas. It displays considerable heterogeneity in clinical presentation, morphology, molecular characteristics, and outcome [1]. The International Prognostic Index (IPI) is useful in predicting the outcome of patients with DLBCL. However, patients with identical IPI still exhibit marked variability in survival, suggesting the presence of significant residual heterogeneity within each IPI category [2]. As new treatments appear, there is an increasing interest in identifying patients in whom conventional therapeutic approach is likely to fail. Many investigators have focused on identification of molecular prognostic factors to improve patients outcome [3]. Recently, application of DNA microarray and tissue array technologies allowed the development of novel diagnostic and prognostic tools capable to improve the current models for outcome prediction [4, 5]. Major subgroups were subsequently identified: germinal center B-cell-like and non-germinal center B-cell-like, the former showing a better prognosis. The prognostic difference between these subgroups has been confirmed by several studies investigating selected germinal center-associated proteins by immunohistochemistry [6–8], although, several other studies showing no difference in survival between these subgroups [9, 10]. The identification of new molecular prognostic markers may therefore help to further stratify patients into different risk groups.
DNA methylation of the CpG island in the promoter region of genes has emerged as an important mechanism of inactivation of tumor suppressor genes, and aberrant promoter hypermethylation has been described for several genes in various malignant diseases [11, 12]. This strongly represents an alternative pathway to gene deletion or mutation for the loss of tumor suppressor genes [12, 13]. There is now an increasing evidence for the relevance of hypermethylation-associated gene silencing in the pathogenesis of human cancers, and markers for aberrant methylation seem to represent a promising avenue for monitoring the onset and progression of malignancies [11, 14, 15]. Interestingly, numerous in vitro experiments have shown that, in contrast to genetic aberrations, the hypermethylation-associated silencing of tumor suppressor genes is a reversible phenomenon [16]. In addition, the therapeutic efficacy of the demethylating agents, such as 5-aza-2'-deoxycytidine or 5-azacytidine, has been demonstrated in several clinical trials [17, 18].
To date, the role of aberrant promoter methylation in DLBCLs has not been investigated exhaustively, and most previous studies have been mainly focused on the cyclin-dependent kinase inhibitors and DNA repair genes [19–22], although other genes have been found to be targeted by aberrant hypermethylation in lymphoid neoplasias [15, 23, 24]. These observations prompted us, in the present study, to investigate the prognostic significance of the methylation status of a large panel of tumor suppressor and -related genes potentially involved in the pathogenesis of B-cell malignancies in a series of 46 cases of DLBCL from Tunisia. The selected genes included DAPK (death-associated protein kinase), GSTP1 (glutathione S-transferase P1), P14, P15, P16, P33, BLU, RB1 (retinoblastoma gene), SHP1 (hematopoietic cell-specific protein tyrosine phosphatase), VHL (Von Hippel-Lindau gene), CDH1 (E-cadherin gene), APC (adenomatous polyposis coli), TIMP3 (tissue inhibitor of metalloproteinase-3), and RASSF1A (RAS association domain family protein 1A) (Table 1).
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patients and tissue samples
Forty-six newly presented and previously untreated patients with DLBCL diagnosed at the Department of Pathology of the Farhat-Hached Hospital (Sousse, Tunisia) between 1996 and 2004 were included in this study. Diagnosis was on the basis of morphology and immunohistochemical analysis according to the criteria of the World Health Organization classification [1]. Cases were selected from our recently reported lymphoma series correlating methylation of many genes to the presence of simian virus 40 [35]. The selection of cases was on the basis of the availability of clinical follow-up data and paraffin-embedded tissue blocks for molecular analyses. All cases were already characterized for the germinal center status by immunohistochemistry [8]. Clinical data were obtained from the patient files and the following data were available for all cases: patient's age and gender, performance status, clinical stage on the basis of the modified Ann Arbor classification system, number of extranodal sites, B symptoms, bulky tumor, bone marrow involvement, serum lactate dehydrogenase (LDH) level, IPI score, response to treatment, and survival.
All patients were uniformly treated at the same institution with standard regimes according to the IPI score and patient's age. Ten (22%) patients have been treated with CHOP, eight (17%) with COP, 10 (22%) with ACVBP, seven (15%) with CVP, and 11 (24%) with mini-CEOP. None of the patients received rituximab or alemtuzumab. Radiotherapy was administered after chemotherapy to six patients who presented with a bulky mass or who had a detectable residual mass after chemotherapy. All patients completed their planned treatment. Clinical outcome was evaluated according to standard international criteria. A complete response was defined as total disappearance of all enhancing tumor. A partial response was defined as a 
50% reduction in the area of the enhancing tumor. Stable disease was defined as no change in the area of the tumor or <50% reduction or <25% increase in the tumoral size. Progressive disease was defined as 25% at least increase in the area of the enhancing tumor or the appearance of new lesions [36].
methylation-specific PCR assays
Genomic DNA was extracted from paraffin-embedded biopsy specimens as described previously [37]. Extracted DNA was then subjected to chemical treatment with sodium bisulfite as previously reported [29]. Briefly, 1–2 µg of DNA was denatured by treatment with NaOH and modified by sodium bisulfite treatment of 18 h at 50°C. DNA samples were then purified using Wizard DNA Clean–UP System (Promega, Madison, WI) according to the manufacturer's instructions, desulphonated by incubating in a final concentration of 0.3 M NaOH, precipitated with ethanol, and resuspended in water. Modified DNA was then stored at –20°C until use.
The presence of bisulfite-modified DNA in each sample was determined by amplification of 133-bp DNA fragment of the β-actin gene using a selected primer set which amplifies bisulfite-modified DNA (but not wild-type DNA), irrespective of the methylation status of the sample [38].
Promoter methylation status was analyzed by methylation-specific PCR (MSP) using methylated and unmethylated gene-specific primers for each gene as described elsewhere by Herman et al. [29]. For all cases, MSP status of DAPK, GSTP1, P14, P15, P16, RB1, SHP1, CDH1, APC, and BLU genes were previously reported [35]. In the currents study, we extended our investigation, by analyzing the promoter methylation status of four other genes: VHL, TIMP3, P33, and RASSFA1. Twenty reactive lymph nodes from patients with nonmalignant diseases and 20 peripheral blood samples from healthy persons were used as control for the methylation status of theses genes in normal lymphocytes. Primers for VHL were 5'-GTTGGAGGATTTTTTTGTGTATGT-3' (sense) and 5'-CCCAAACCAAACACCACAAA-3' (antisense) for the unmethylated reaction and 5'-TGGAGGATTTTTTTGCGTACGC-3' (sense) and 5-GAACCG AACGCCGCGAA-3' (antisense) for the methylated reaction [27]. These primer sets were designed to amplify 165 and 158 bp, respectively. The annealing temperature for both the unmethylated and the methylated reactions was 58°C. Primers for TIMP3 were 5'-TTTTGTTTTGTTATTTTTTGTTTTTGGTTTT-3' (sense) and 5'-CCCCCAAAAACCCCACCTCA-3' (antisense) for the unmethylated reaction and 5'-CGTTTCGTTATTTTTTGTTTTCGGTTTC-3' (sense) and 5'-CCGAAAACCCCGCCTCG-3' (antisense) for the methylated reaction [34]. These primer sets were designed to amplify 119 and 116 bp, respectively. The annealing temperature for both the unmethylated and the methylated reactions was 60°C. Primers for P33 were 5'-TGGATGGTGTAGGTGTGGGAGTT-3' (sense) and 5'-CCAAACACAAACAAAAATAACAACACA-3' (antisense) for the unmethylated reaction and 5'-CGGATGGCGTAGGCGCGGGAGTC-3' (sense) and 5'-CCGAACACGAACGAAAATAAC GACGC-3' (antisense) for the methylated reaction [33]. These primer sets were both designed to amplify 148 bp. The annealing temperature was 58°C for the unmethylated reaction and 60°C for the methylated reaction. Primers for RASSFA1 were 5'-AATTTGTTGTTGTTTTTTAGGTGG-3' (sense) and 5'-AAAAAACCAACAACCCCCACA-3' (antisense) for the unmethylated reaction and 5'-ATTCGTCGTCGTTTTTTAGGC G-3' (sense) and 5'-AAAAACCAACGACCCCCGCG-3' (antisense) for the methylated reaction [25]. These primer sets were designed to amplify 108 and 94 bp, respectively. The annealing temperature was 57°C for the unmethylated reaction and 58°C for the methylated reaction. The PCR amplifications were carried out with treated DNA as template in a total volume of 25 µl containing 0.2 µM of each primer, 200 µM of each dNTP, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 5% DMSO, and 1 U of Taq DNA polymerase (Promega). PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 35 cycles of 30 s at 95°C, for 30 s at the specific temperature, and for 30 s at 72°C. The reaction was finished with a 5-min extension at 72°C. CpG universal methylated DNA (Qbiogene, Carlsbad, CA) was used as the positive control for methylated alleles, and DNA from normal lymphocytes was used as the negative control for unmethylated alleles. Negative controls without DNA were always included in each experiment.
Amplified products were visualized under ultraviolet illumination after electrophoresis in 2% agarose gels containing ethidium bromide using the Gel Doc 2000 System (Bio-Rad, Marnes-la-Coquette, France). For each case, MSP results were scored when a clearly visible band on the electrophoresis gel with the methylated and/or the unmethylatated primers was observed [29]. The presence of a clearly visible band in the MSP using primers for the methylated alleles was considered a positive result for methylation [29, 39]. In the sporadic cases, where only faint bands were observed in both analyses, methylation results were always confirmed by repeat MSP assays after an independently carried out bisulfite treatment.
statistical analysis
Statistical analyses were carried out with SPSS software package, version 11.5. Pairwise correlations among genes and between methylation status of individual genes and clinicopathological parameters were investigated by chi-square test or Fisher's exact test where appropriate. Statistical significance was set at the two-sided 5% comparisonwise (i.e. without correction for multiple comparisons). Overall survival (OS) was defined as the time interval from diagnosis to death from any cause or, for patients remaining alive, the time interval from diagnosis to the last follow-up. Disease-free survival (DFS) was defined as the time interval from treatment to the time of disease progression or recurrence, to the last follow-up, or to death occurrence from any cause. To determine the association between hypermethylation of individual genes and OS and DFS of patients, we used the log-rank test of Kaplan–Meier. The Cox proportional hazard regression model was used to evaluate the effect of multiple variables.
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patient's characteristics
The patients included 28 men and 18 women with a median age of 65 years (range 18–85 years). Twenty-six patients (56%) presented a good performance status (<2). Eight patients (17%) had stage I, 12 (26%) stage II, 10 (22%) stage III, and 16 (35%) stage IV of the disease. An elevated level of serum LDH was observed in 39 patients (85%). Thirty-three patients (72%) exhibited low or low-intermediate IPI scores. Nevertheless, 12 patients (26%) showed B symptoms and four patients (9%) showed a bulky tumor. The number of extranodal sites of the disease at more than one site and the involvement of the bone marrow were observed in seven (15.5%) and nine (20%) patients, respectively. Primary site of tumors was lymph nodes in 33 patients and extranodal organs in 13 patients. Thirty-two cases (70%) had a germinal center immunophenotype and 14 cases (30%) had a non-germinal center immunophenotype. Twenty-six patients (56%) achieved a complete remission at the end of the treatment procedure, while four (9%) were in partial remission and 16 (35%) had stable or progressive disease.
relationship between promoter methylation status and clinicopathological characteristics
The frequencies of hypermethylated genes were 20% (9 of 46) for P33, 44% (20 of 46) for VHL, 15% (7 of 46) for RASSFA1, and 17.5% (8 of 46) for TIMP3. Concerning the other 10 genes, the frequencies of hypermethylation were 78% (36 of 46) for SHP1, 63% (29 of 46) for GSTP1, 59% (27 of 46) for DAPK and CDH1, 52% (24 of 46) for P16, 46% (21 of 46) for APC, 41% (19 of 46) for P14 and P15, 20% (9 of 46) for RB1 and P33, 17.5% (8 of 46) for TIMP3, and 15% (7 of 46) for BLU and RASSFA1. Overall, all DLBCL cases showed at least one hypermethylated gene and 85% (39 of 46) harbored three or more hypermethylated genes. None of the 20 reactive lymph nodes, and the 20 peripheral blood samples has showed evidence of hypermethylation in the promoter of these genes.
Study of correlations between promoter methylation status of each gene and clinicopathological characteristics are summarized in Table 2. Overall, the hypermethylation of SHP1 was present more frequently in patients with elevated LDH level than those with normal LDH level (85% versus 42%; P = 0.031). The hypermethylation of P14 was present more frequently in patients without B symptoms than those with B symptoms (50% versus 17%; P = 0.044). Likewise, patients with methylated CDH1 were significantly older than those with unmethylated CDH1 promoter (77% versus 35%; P = 0.004). Moreover, hypermethylation of P16 was more frequently detected in patients with clinical stage III/IV, high IPI scores, a performance status of two or more, and B symptoms than those with clinical stage I/II, low IPI scores, a performance status of less than two, and without B symptoms, respectively (65.5% versus 35%, 85% versus 39%, 75% versus 35%, and 83% versus 41%; P = 0.041, 0.006, 0.007, and 0.012, respectively). Similarly, hypermethylation of the VHL gene was present more frequently in patients with high IPI scores and a performance status of two or more than those with low IPI scores and a performance status of less than two, respectively (77% versus 30% and 60% versus 31%; P = 0.004 and 0.047, respectively). However, we found no significant associations between the methylation status of DAPK, GSTP1, P15, RB1, P33, BLU, RASSFA1, or TIMP3 and the following clinicopathological features: patient's age and gender, performance status, clinical stage, IPI score, serum LDH level, B symptoms, bone marrow involvement, bulky tumor, and tumor location.
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With regard to response to treatment, only the methylation status of DAPK showed a significant association with therapy response (Table 2). In fact, only 43% of patients who achieved a complete or partial response to treatment showed hypermethylated DAPK promoter, whereas 81% of patients who failed to give any response showed hypermethylated DAPK promoter.
survival analysis
Follow-up data were available for all patients and the median follow-up period was 15 months (range 0–96 months). Two-year survival rates for the entire series group were 55% and 46% for OS and DFS, respectively. In addition, patients with chemoresistant disease had significantly shortened OS and DFS than patients with chemosensitive disease (P = 0.0001 and 0.007; Table 3). Univariate analysis revealed that OS and DFS were also significantly shortened in patients with the following parameters (Table 3): high-intermediate risk or high-risk IPI scores (P = 0.001), clinical stages III/IV (P = 0.011), B symptoms (P = 0.020), a performance status of two or more (P = 0.003), and bulky tumor (P = 0.003). However, no significant differences in OS or DFS rates were seen between patients according to the patient's age and gender, tumor location, serum LDH level, number of extranodal sites, and bone marrow involvement.
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We next carried out survival analysis to investigate the impact of the methylation status of each of the 14 genes on both OS and DFS. Table 4 summarizes the overall associations between individual gene methylation status and OS or DFS. The analysis revealed that the hypermethylation of DAPK, P16, and VHL genes was associated with a significantly poorer OS (P = 0.003, 0.001, and 0.017, respectively; Figure 1) and DFS (P = 0.006, 0.003, and 0.046, respectively; Figure 2). On the other hand, there was a trend towards shorter survival for patients with methylated SHP1 (P = 0.059 for OS and P = 0.065 for DFS, Table 4). However, no significant impact on survival was seen for the other genes (Table 4).
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We also evaluated the predictive value of the methylation status of the investigated genes on OS and DFS according to the two major risk groups defined on the basis of the IPI. This analysis revealed that in the low and low-intermediate risk group, the hypermethylation of DAPK and P16 genes was associated with a significantly poorer OS (P = 0.009 and 0.021, respectively; Figure 3A and C), whereas only the hypermethylation of DAPK was associated with a poor DFS (P = 0.013). In the intermediate-high and high-risk group, only the hypermethylation of DAPK was associated with a significantly poorer OS (P = 0.002; Figure 3B). Among patients with germinal center immunophenotype, hypermethylation of GSTP1, DAPK, and P16 genes was found to be associated with a significantly poor OS (P = 0.013, 0.043, and 0.0005, respectively; Figure 4A, C, and E) and DFS (P = 0.014, 0.049 and 0.001, respectively). However, among patients with non-germinal center immunophenotype, there were no significant correlation between methylation of each of the genes investigated and either OS or DFS (Figure 4B, D, and F).
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We next carried out a multivariate analysis including the methylated genes which have been found to be significant at univariate analysis in conjunction with the IPI score and the germinal center immunophenotype status. The Cox proportional hazard model identified the hypermethylation of DAPK as an independent prognostic factor in predicting survival, in addition to the IPI score and germinal center status (Table 5).
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The aim of this study was to investigate the biological significance and the prognostic relevance of the methylation status of cancer suppressor and -related genes in a series of 46 DLBCL cases from Tunisia. For this issue, 14 cancer-associated genes were considered: SHP1, DAPK, GSTP1, P14, P15, P16, P33, RB1, CDH1, APC, RASSFA1, TIMP3, VHL, and BLU. We found that many of these genes are methylated in lymphoma samples but unmethylated in all nonmalignant lymphoid controls. These findings indicate that this epigenetic alteration is a common phenomenon in DLBCLs and may be an important way to inactivate cancer-related genes in this disease. With regard to clinicopathological and survival data, we found that the methylation of several of the genes analyzed in this study was strongly correlated with various clinicopathological characteristics and patient's survival. It is important to note, however, that since we could investigate multiple non-predefined hypotheses, some significant associations found in our study might not be free from chance fluctuation. Thus, the adjustment for multiple tests would be preferred. However, in our present study, the statistical tests, to identify correlations between methylation status of individual genes and clinicopathological parameters, were conducted without explicit multiple comparison correction (e.g. Bonferroni test) to the significance level of the individual tests. That is, in order for the tests to have reasonable statistical power, no formal effort was made to control the family-wise type I error rate. Therefore, our finding should be interpreted with caution and further independent study is clearly needed to confirm these observations.
The P16 tumor suppressor gene, located on 9p21, encodes a cyclin-dependent kinase inhibitor important for G1 cell cycle arrest [40]. In human cancers, P16 is frequently inactivated by homozygous deletion, point mutations, or methylation of its promoter region [41, 42]. The hypermethylation of P16 has been frequently observed in various human malignant tumors [43–47]. In DLBCLs, hypermethylation of P16 has been found in 27–56% of cases [19, 20, 48–51]. In the present study, we found that 52% of DLBCL cases from Tunisian patients exhibit hypermethylation of the P16. In addition, we found that hypermethylation of P16 was associated with advanced clinical stages, high-intermediate/high IPI scores, a performance status of two or more, and B symptoms (P = 0.040, 0.006, 0.007, and 0.013, respectively; Table 2). Interestingly, we demonstrated a significant correlation between hypermethylation of P16 promoter and shortened OS (P = 0.001) and DFS (P = 0.016).
The prognostic impact of P16 methylation has been previously described by several groups in large variety of human epithelial tumors [44, 45]. In lymphomas, Garcia et al. [47] have showed, in a series of 15 DLBCLs, that hypermethylation of P16 was frequently observed in the high-intermediate and high-risk groups. Pinyol et al. [49] have noted that hypermethylation of P16 was frequently found in patients with aggressive tumors. Sanchez-Beato et al. [48] have found hypermethylation of P16 in 20 (32.2%) of 62 aggressive lymphomas including three (18.7%) of 16 Burkitt lymphomas and 17 (36.9%) of 46 DLBCLs. In addition, cases with hypermethylated P16 have been found more frequently in patients with advanced clinical stages and associated with worse OS. Recently, Shiozawa et al. [20] have showed, in a series of 49 DLBCL cases, that hypermethylation of P16 was a poor prognostic factor in the IPI high-intermediate and high-risk group. Hence, our findings together with other previous works indicate that methylation status of P16 might, in spite of possible variations between techniques used and heterogeneity of patients, provide an interesting prognostic information in patients with DLBCLs that may influence the choice of chemotherapy regimens, especially for the IPI high-intermediate and high-risk group patients.
The VHL gene, which resides on chromosome 3p25, was originally isolated and identified as a tumor suppressor gene responsible for the Von Hippel-Lindau familial syndrome predisposing to the development of retinal angiomas, central nervous system hemangioblastomas, pancreatic tumors, pheochromocytomas, and multiple bilateral clear-cell renal cell carcinomas [51, 52]. The VHL protein is expressed in a wide variety of cells in human tissues including lymphocytes and macrophages [53, 54]. Although it remains unclear why these tissues and cells express the VHL protein, it appears likely that this protein intimately plays fundamental roles in physiological as well as pathological situations in human cell biology [53]. In human cancers, several mechanisms have been showed to lead to loss of VHL function including DNA deletion, mutation, and hypermethylation [27, 51–54].
Aberrant hypermethylation of the VHL gene has been observed mainly in renal cancer but also in other various human carcinomas [27, 55, 56]. In lymphoid malignancies, methylation status of VHL gene has not largely been investigated. In fact, only one study has analyzed the methylation status of VHL in a small series of gastric DLBCLs (n = 12) and has showed methylation of this gene in 25% of cases [57]. In the present study, promoter hypermethylation of VHL gene was observed in 44% of DLBCL cases but not in the nonmalignant lymphoid specimens, and was significantly correlated with high-intermediate/high IPI scores and worse performance status (2) (P = 0.001 and 0.022, respectively; see Table 2). To our knowledge, our current study is the first investigating the prognostic impact of VHL methylation in patients with DLBCLs. Interestingly, we demonstrated a significant association between hypermethylation of VHL and shortened OS and DFS (see Figure 1C and Figure 2C). The hypermethylation of VHL, therefore, seems to be a common event in DLBCLs and may represent an important prognostic biomarker in such a subgroup of patients. However, further studies are required to confirm these observations.
In this study, promoter hypermethylation of DAPK was observed in 59% of DLBCL cases. DAPK is an actin-associated calcium/calmodulin-dependent enzyme with serine/threonine kinase activity [58, 59]. DAPK is involved in TNF-
and FAS-induced apoptosis and has been demonstrated to be an essential mediator in IFN
-induced programmed cell death [59–61]. Recent experiments have established that DAPK functions as a tumor suppressor gene in at least two different stages of tumorigenicity, namely an apoptotic checkpoint functioning early during cell transformation and a second one that occurs later in cancer development (i.e. during metastasis) [61, 62]. The hypermethylation of DAPK has been described in a variety of human tumors including lymphomas [11, 24, 30, 63–69]. In addition, the hypermethylation of the promoter of DAPK has been associated with an unfavorable prognosis in several kinds of human cancers including lymphomas [64–66, 69]. However, its prognostic significance in DLBCLs has not been investigated so far. Interestingly, in the present study, hypermethylation of DAPK was significantly associated with worse OS and DFS (Table 4; Figure 1A and Figure 2A). Furthermore, DAPK methylation-positive status was identified as an independent prognostic factor for predicting overall and disease-free survival in patients with DLBCL in multivariate analyses (Table 5).
Since DAPK is involved in multiple apoptosis pathways, its inactivation could be a key factor in the response to chemotherapy in human cancer [59, 60]. In fact, most chemotherapeutic agents induce cell death in a mitochondria-dependent manner, yet death receptor-mediated signals can also be involved. Of interest, in our present study, we showed a significant correlation between the methylation status of the DAPK and the response to treatment (P = 0.041; Table 2). These data indicate that the block of apoptosis in relation to the reduced DAPK expression caused by the hypermethylation of DAPK could be a mechanism of resistance to chemotherapy-induced apoptosis in DLBCLs. However, because our study is the first reporting the prognostic value of the hypermethylation of DAPK gene in DLBCLs, additional studies for this specific gene with a larger number of cases are mandatory to confirm these observations.
In our present study, 78% of DLBCLs showed hypermethylation of SHP1. The SHP1 gene codes for a phosphotyrosine phosphatase that plays many important roles in regulating immune system cell differentiation and activation [70]. SHP1 acts as a growth inhibitor in B cells by down-regulating the intracellular effects of immunoglobulin binding thus requiring more receptor binding to initiate B-cell activation and proliferation [70, 71]. B lymphocytes with decreased SHP1 activity are thus more likely to proliferate and escape apoptosis [70, 71]. Methylation of SHP1 promoter region appears common across a variety of lymphomas including DLBCLs, with frequencies ranging between 75% and 100% [15, 32, 35, 71, 72]. The high frequency of hypermethylation of SHP1 in B-cell lymphomas/leukemias indicates that SHP1 silencing is one of the critical events to the onset of these diseases as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. Prognostic significance of methylation of SHP1 has not been previously investigated in DLBCLs. In our study, there was a trend towards shorter survival for patients with methylated SHP1 (P = 0.059 for OS and P = 0.065 for DFS; see Table 4). Further studies including larger number of DLBCL cases are needed to confirm these findings.
In summary, this study represents the most extensive research investigating the prognostic impact of the methylation status of a large specter of cancer-associated genes in DLBCLs. We demonstrated that tumors with hypermethylated P16, DAPK, VHL, and SHP1 commonly show a biologically aggressive phenotype and poor prognosis. Interestingly, hypermethylation of DAPK has been shown to be an independent new prognostic factor that could be helpful for predicting shortened survival and resistance to treatment, in conjunction with the conventional prognostic factors such as the IPI score and germinal center status.
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Ministry of Higher Education, Scientific Research, and Technology; Ministry of Public Health of Tunisia.
Received for publication January 31, 2008. Revision received April 29, 2008. Accepted for publication May 2, 2008.
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1. Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Haematopoietic and Lymphoid Tissues (2001) Lyon: International Agency for Research on Cancer Press. 171–174.
2. Armitage JO, Weisenburger DD. New approach to classifying non-Hodgkin's lymphomas: clinical features of the major histologic subtypes. Non-Hodgkin's Lymphoma Classification Project. J Clin Oncol (1998) 16:2780–2795.[Abstract]
3. Lossos IS, Morgensztern D. Prognostic biomarkers in diffuse large B-cell lymphoma. J Clin Oncol (2006) 24:995–1007.
4. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature (2000) 403:503–511.[CrossRef][Medline]
5. Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med (2002) 346:1937–1947.
6. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood (2004) 103:275–282.
7. Chang CC, McClintock S, Cleveland RP, et al. Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma. Am J Surg Pathol (2004) 28:464–470.[Web of Science][Medline]
8. Amara K, Trimeche M, Ziadi S, et al. Presence of Simian virus 40 in diffuse large B-cell lymphomas in Tunisia correlates with germinal center B-cell immunophenotype, t(14;18) translocation, and P53 accumulation. Mod Pathol (2008) 21:282–296.[CrossRef][Web of Science][Medline]
9. Colomo L, Lopez-Guillermo A, Perales M, et al. Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma. Blood (2003) 101:78–84.
10. Moskowitz CH, Zelenetz AD, Kewalramani T, et al. Cell of origin, germinal center versus nongerminal center, determined by immunohistochemistryon tissue microarray, does not correlate with outcome in patients with relapsed and refractory DLBCL. Blood (2005) 106:3383–3385.
11. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med (2003) 349:2042–2054.
12. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev (2002) 3:415–428.
13. Toyota M, Issa JP. The role of DNA hypermethylation in human neoplasia. Electrophoresis (2000) 21:329–333.[CrossRef][Web of Science][Medline]
14. Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med (2001) 134:573–586.
15. Chim CS, Liang R, Kwong YL. Hypermethylation of gene promoters in hematological neoplasia. Hematol Oncol (2002) 20:167–176.[CrossRef][Web of Science][Medline]
16. Paz MF, Fraga MF, Avila S, et al. A systematic profile of DNA methylation in human cancer cell lines. Cancer Res (2003) 63:1114–1121.
17. Issa JP, Garcia-Manero G, Giles FJ, et al. Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies. Blood (2004) 103:1635–1640.
18. Kantarjian HM, O'Brien S, Cortes J, et al. Results of decitabine (5-aza-2'deoxycytidine) therapy in 130 patients with chronic myelogenous leukemia. Cancer (2003) 98:522–528.[CrossRef][Web of Science][Medline]
19. Baur AS, Shaw P, Burri N, et al. Frequent methylation silencing of p15(INK4b)(MTS2) and p16(INK4a) (MTS1) in B-cell and T-cell ymphomas. Blood (1999) 94:1773–1781.
20. Shiozawa E, Takimoto M, Makino R, et al. Hypermethylation of CpG islands in p16 as a prognostic factor for diffuse large B-cell lymphoma in a high-risk group. Leuk Res (2006) 30:859–867.[CrossRef][Web of Science][Medline]
21. Esteller M, Gaidano G, Goodman SN, et al. Hypermethylation of the DNA repair gene O6-methylguanine DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma. J Natl Cancer Inst (2002) 94:26–32.
22. Al-Kuraya K, Narayanappa R, Siraj AK, et al. High frequency and strong prognostic relevance of O6-methylguanine DNA methyltransferase silencing in diffuse large B-cell lymphomas from the Middle East. Human Pathology (2006) 37:742–748.[CrossRef][Web of Science][Medline]
23. Ropero S. Epigenetic pathways in hematological malignancies. Haematol Rep (2006) 2:7–10.
24. Takahashi T, Shivapurkar N, Reddy J, et al. DNA methylation profiles of lymphoid and hematopoietic malignancies. Clin Cancer Res (2004) 10:2928–2935.
25. Wong N, Li L, Tsang K, et al. Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma. J Hepatol (2002) 37:633–639.[CrossRef][Web of Science][Medline]
26. Agathanggelou A, Dallol A, Zochbauer-Muller S, et al. Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers. Oncogene (2003) 22:1580–1588.[CrossRef][Web of Science][Medline]
27. Herman JG, Latif F, Weng Y, et al. Silencing of the VHL tumor suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci USA (1994) 91:9700–9704.
28. Esteller M, Sparks A, Toyota M, et al. Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer. Cancer Res (2000) 60:4366–4371.
29. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA (1996) 93:9821–9826.
30. Katzenellenbogen RA, Baylin SB, Herman JG. Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood (1999) 93:4347–4353.
31. Esteller M, Corn PG, Urena JM, et al. Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res (1998) 58:4515–4518.
32. Oka T, Ouchida M, Koyama M, et al. Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas. Cancer Res (2002) 62:6390–6394.
33. Shen DH, Chan KY, Khoo US, et al. Epigenetic and genetic alterations of p33ING1b in ovarian cancer. Carcinogenesis (2005) 26:855–863.
34. Bachman KE, Herman JG, Corn PG, et al. Methylation associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. Cancer Res (1999) 59:798–802.
35. Amara K, Trimeche M, Ziadi S, et al. Presence of simian virus 40 DNA sequences in diffuse large B-cell lymphomas in Tunisia correlates with aberrant promoter hypermethylation of multiple tumor suppressor genes. Int J Cancer (2007) 121:2693–2702.[CrossRef][Web of Science][Medline]
36. Cheson BD, Horning SJ, Coiffier B, et al. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol (1999) 17:1244–1253.
37. Man Y, Moinfar F, Bratthauer GL, et al. An improved method for DNA extraction from paraffin sections. Pathol Res Pract (2001) 197:635–642.[CrossRef][Web of Science][Medline]
38. Singal R, Ferdinand L, Reis IM, Schlesselman JJ. Methylation of multiple genes in prostate cancer and the relationship with clinicopathological features of disease. Oncol Rep (2004) 12:631–637.[Web of Science][Medline]
39. Roman-Gomez J, Jimenez-Velasco A, Castillejo JA, et al. Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia. Blood (2004) 104:2492–2498.
40. Koh J, Enders GH, Dynlacht BD, Harlow E. Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature (Lond) (1995) 375:506–510.[CrossRef][Medline]
41. Cairns P, Polascik JT, Eby Y, et al. Frequency of homozygous deletion at p16/CDKN2 in primary human tumors. Nat Genet (2001) 11:210–212.[CrossRef]
42. Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res (1995) 55:4525–4530.
43. Kim JR, Kim SY, Kim MJ, Kim JH. Alterations of CDKN2 (MTS1/p16INK4A) gene in paraffin-embedded tumor tissues of human stomach, lung, cervix and liver cancers. Exp Mol Med (1998) 30:109–114.[Web of Science][Medline]
44. Lee M, Han WS, Kim OK, et al. Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer. Pathol Res Pract (2006) 202:415–424.[CrossRef][Web of Science][Medline]
45. Wang J, Lee JJ, Wang L, et al. Value of p16INK4a and RASSF1A promoter hypermethylation in prognosis of patients with resectable non–small cell lung cancer. Clin Cancer Res (2004) 10:6119–6125.
46. Lo KW, Cheung ST, Leung SF, et al. Hypermethylation of the pl6 gene in nasopharyngeal carcinoma. Cancer Res (1996) 56:2721–2725.
47. Garcia MJ, Martinez-Delgado B, Cebrian A, et al. Different incidence and pattern of p15INK4b and p16INK4a promoter region hypermethylation in Hodgkin's and CD30-positive non-Hodgkin's lymphomas. Am J Pathol (2002) 161:1007–1013.
48. Sanchez-Beato M, Saez AI, Navas IC. Overall survival in aggressive B-cell lymphomas is dependent on the accumulation of alterations in p53, p16, and p27. Am J Pathol (2001) 159:205–213.
49. Pinyol M, Cobo F, Bea S, et al. p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas. Blood (1998) 91:2977–2984.
50. Huang Q, Ai L, Zhang ZY, et al. Promoter hypermethylation and protein expression of the p16 gene: analysis of 43 cases of B-cell primary gastric lymphomas from China. Mod Pathol (2004) 17:416–422.[CrossRef][Web of Science][Medline]
51. Latif F, Tory K, Gnarra J, et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science (1993) 260:1317–1320.
52. Linehan WM, Lerman MI, Zbar B. Identification of the von Hippel-Lindau (VHL) gene. Its role in renal cancer. Jama (1995) 273:564–570.
53. Los M, Jansen GH, Kaelin WG, et al. Expression pattern of the von Hippel-Lindau protein in human tissues. Lab Invest (1996) 75:231–238.[Web of Science][Medline]
54. Sakashita N, Takeya M, Kishida T, et al. Expression of von Hippel–Lindau protein in normal and pathological human tissues. Histochem J (1999) 31:133–144.[CrossRef][Web of Science][Medline]
55. Kuroki T, Trapasso F, Yendamuri S, et al. Allele loss and promoter hypermethylation of VHL, RAR-β, RASSF1A, and FHIT tumor suppressor genes on chromosome 3p in esophageal squamous cell carcinoma. Cancer Res (2003) 63:3724–3728.
56. Yu J, Zhang HY, Ma ZZ, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res (2003) 13:319–333.[CrossRef][Web of Science][Medline]
57. Fung MKL, Au WY, Liang R, et al. Aberrant promoter methylation in gastric lymphomas. Haematologica (2003) 88:231–232.
58. Deiss LP, Feinstein E, Berissi H, et al. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon–induced cell death. Genes Dev (2005) 9:15–30.[CrossRef]
59. Cohen O, Kimchi A. DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ (2001) 8:6–15.[CrossRef][Web of Science][Medline]
60. Ng MH. Death associated protein kinase: from regulation of apoptosis to tumor suppressive functions and B cell malignancies. Apoptosis (2002) 7:261–270.[CrossRef][Web of Science][Medline]
61. Inbal B, Cohen O, Polak-Charcon S, et al. DAP kinase links the control of apoptosis to metastasis. Nature (1997) 390:180–184.[CrossRef][Medline]
62. Kim DH, Nelson HH, Wiencke JK, et al. Promoter methylation of DAP-kinase: association with advanced stage in non-small cell lung cancer. Oncogene (2001) 20:1765–1770.[CrossRef][Web of Science][Medline]
63. Mittag F, Kuester K, Vieth M, et al. DAPK promotor methylation is an early event in colorectal carcinogenesis. Cancer Lett (2006) 240:69–75.[CrossRef][Web of Science][Medline]
64. Tozawa T, Tamura G, Honda T, et al. Promoter hypermethylation of DAP-kinase is associated with poor survival in primary biliary tract carcinoma patients. Cancer Sci (2004) 95:736–740.[CrossRef][Medline]
65. Voso MT, Gumiero D, D'Alo' F, et al. DAP-kinase hypermethylation in the bone marrow of patients with follicular lymphoma. Haematologica (2006) 91:1252–1256.
66. Fischera JR, Ohnmachtc U, Riegerb N, et al. Promoter methylation of RASSF1A, RA
and DAPK predict poor prognosis of patients with malignant mesotheliomas. Lung Cancer (2006) 54:109–116.[CrossRef][Web of Science][Medline]
67. Rossi D, Capello D, Gloghini A, et al. Aberrant promoter methylation of multiple genes throughout the clinico-pathologic spectrum of B-cell neoplasia. Haematologica (2004) 89:154–164.
68. Sanchez-Cespedes M, Esteller M, Wu L, et al. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res (2000) 60:892–895.
69. Matsumoto H, Nagao M, Ogawa S, et al. Prognostic significance of death-associated protein-kinase expression in hepatocellular carcinomas. Anticancer Res (2003) 23:1333–1341.[Web of Science][Medline]
70. Pani G, Kozlowski M, Cambier JC, et al. Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling. J Exp Med (1995) 181:2077–2084.
71. Cyster JG, Goodnow CC. Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity (1995) 2:13–24.[CrossRef][Web of Science][Medline]
72. Koyama M, Oka T, Ouchida M, et al. Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation. Lab Invest (2003) 83:1849–1858.[CrossRef][Web of Science][Medline]
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