Abstract
Background: DNA methylation is one of major factors in cancer progression. We observed multiple genes involved in cancer-related signaling and focused on patients with advanced non-small cell lung cancer (NSCLC) and evaluated methylation in relation to various clinical parameters. Patients and Methods: Thirty genes were examined in 121 NSCLC patients using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method. Correlations to gender, smoking status, tumor subtype, disease stage and EGFR/KRAS mutation status were performed by chi-square test. Results: 90% of tumors exhibited methylation of at least one gene. Most frequently methylated were cadherin-13 (CDH13), Ras associated domain-containing protein (RASSF1A), Wilms' tumor protein (WT1), adenomatous polyposis coli protein (APC), paired box protein Pax-5 (PAX5), estrogen receptor (ESR1), an inhibitor of cyclin-dependent kinase p15 (CDKN2B), paired box protein Pax-6 (PAX6), transcription factor GATA-5 (GATA5) and cell adhesion molecule 4 (IGSF4). Overall methylation (any gene) was increased in adenocarcinomas (p=0.0329), unrelated to gender or disease stage. Several genes exhibited variable methylation with gender (CDH13, p<0.001; GATA5, p=0.02; PAX6, p=0.01 and ESR1, p=0.03), smoking (CDH13, p=0.002), or epidermal growth factor receptor (EGFR) mutation status [Von Hippel-Lindau disease tumor supresor (VHL), p=0.001; CDKN2B, p=0.02; CDH13, p=0.02; APC, p=0.04 and ESR1, p=0.04]. Conclusion: Differences in gene methylation associated with gender, smoking and EGFR mutation suggest potential for prediction in relation to management of tyrosine kinase inhibitor therapy.
DNA hypermethylation is a key epigenetic mechanism regulating gene transcription in living cells. DNA of cells in normal tissue exhibits methylation of all cytosines adjacent to glycines. The methylated cytosine-guanine sequences (termed CpG islands) prevent binding of most transcription factors resulting in effective repression of the transcription of most genes (1-3). In tumor cells, methylation-associated silencing of vital tumor-supressor genes results in facilitation of tumor development. Methylation-associated silencing may play a role in cellular systems responsible for (i) cell cycle control, (ii) tumor cell proliferation and differentiation, (iii) cell adhesion, invasion, metastasis and (iv) regulation of apoptosis, as well as (v) DNA repair gene transcription and detoxification of DNA adducts, such as those induced by cancer chemotherapy.
Studies of DNA methylation in lung cancer are aiming at several clinical applications including a) screening for cancer predisposition and primary diagnostics, b) monitoring of disease progression and early detection of recurrence, and c) prediction of response to anticancer therapy. Several reports of methylation of various genes in lung carcinomas have previously been reported (4-6). The most frequently reported genes include cadherin-13 (CDH13), retinoic acid receptor (RARB), Ras associated domain-containing protein (RASSF1A), adenomatous polyposis coli protein (APC), tumor protein p16 (CDKN2A), O6-methyltransferase-DNA methylguanin (MGMT), and E3 ubiquitin-protein ligase (CHFR) (7). Studies investigating various genes have proven that the methylation of more than three genes is associated with a 6.5-fold greater likelihood of carcinoma presentation (8). In addition, patients with concurrent methylation of more genes have greater risk of relapse (9). Aberrant methylation of 14-3-3Σ (SFN) gene was found to increase sensitivity to cisplatin plus gemicitabine therapy in patients with advanced-stage non-small cell lung cancer (NSCLC) (10). Recently, the methylation status of selected genes has also been examined on cell-free tumor DNA, circulating in peripheral blood of lung cancer patients (11).
The objective of this study was to analyze DNA methylation of an extensive panel of genes in cytology samples from NSCLC tumors in patients with locally advanced disease. In addition to investigating the spectrum of methylation, we sought for correlation between the methylation status and clinically relevant parameters of tumor subtype, disease stage, smoking and epidermal growth factor receptor (EGFR)/c-Kirsten-ras protein (KRAS) mutation status.
Patients and Methods
We investigated patients with morphologically proven progressive NSCLC who underwent cancer treatment in the course of their disease. The cohort consisted of 121 patients (71 males and 50 females) aged between 28 and 83 years with a median age of 63 years. Of these, 99 were smokers and 22 non-smokers. There were 75 adenocarcinomas, 35 squamous cell carcinomas, 6 anaplastic carcinomas and 5 unspecified/non-differentiated carcinomas of stage III (47) and IV (76). Eleven tumors (9.7%) were EGFR mutation positive and 17 tumors (14%) were KRAS positive.
Tissue samples from patients with clinically confirmed NSCLC having progressed on chemotherapy and targeted therapy were collected and processed as either cytology slides or formalin fixed paraffin embedded (FFPE) sections. Genomic DNA for methylation analysis was extracted by standard spin-column method using JetQuick tissue isolation kit (Genomed, G.m.b.H, Loehne, Germany). Hypermethylation was evaluated by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) using a combination two commercial kits (SALSA ME001-C1 and ME002-A1, MRC Holland, Amsterdam, the Netherlands). The evaluated panel consisted of 30 genes listed in Table I.
Mutual relations between methylation of genes and sex, cancer type and stage, smoking status and response to therapy were studied in detail. Statistical analysis was performed using contingency tables, chi-square test, Wilcoxon test and Kruskal-Wallis analysis. In addition, we evaluated the effect of hypermethylation in regard to the above factors after segregating the tumors according to EGFR and KRAS mutation status.
Results
The typical result from methylation-specific restriction approach by MS-MLPA approach is shown in Figure 1. The upper trace (A) shows a sample without hypermethylation, displaying uncleaved intact fragments for all tested genes. The botton trace (B) shows the same set of tested fragments, but in addition, several extra peaks (CDH13, Wilms' tumor protein (WT1) and paired box protein Pax-6 (PAX6) in this case), can be observed as products of methylation-specific restriction. The intensity of the extra peaks is directly related to the methylation level – i.e. the content of cells bearing methylated DNA in the studied sample.
Overall methylation rates. A total of 109 out of 121 (90%) tumor samples exhibited methylation of at least one gene. In adenocarcinomas, the number of samples with methylation was 93% (70 out of 75), which is significantly higher compared to squamous cell carcinomas, with 77% of methylated samples (27 out of 35; chi-square=4.549, p=0.0329). EGFR-mutated tumors exhibited methylation in al cases (11 out of 11) and KRAS-mutated samples in 88.2% cases (15 out of 17). When comparing males vs. females, no significant difference in overall methylation rates was found, with 64% vs. 65% (males vs. females, respectively). Similarly, overall methylation rates were not related to the disease stage, with 96% in stage III and 87.5% in stage IV.
Methylation of specific genes. As shown in Figure 2, the most frequently methylated genes, with frequency of at least 10%, were CDH13 (48%, 58/120), RASSF1A (32%, 36/114), WT1 (31%, 28/89), APC (30%, 35/115), paired box protein Pax-5 (PAX5) (27%, 24/89), ESR1 (25%, 28/111), CDKN2B (25%, 27/107), PAX6 (19%, 17/90), transcription factor GATA-5 (GATA5) (14%, 13/91) and IGSF4 (12%, 14/119). In the majority of tumors, the methylation affected more than a single gene with the most common combinations of CDH13 with WT1, APC with WT1, CDH13 with PAX6 and APC with CDH13.
Methylation of CDH13 was more frequent in females compared to males (67% vs. 36%; 33/49 vs. 25/69; chi-square=0.98, p<0.001), in non-smokers compared to smokers (80% vs. 43%; 16/20 vs. 42/98; chi-square=0.96, p=0.002) and in EGFR-positive tumors compared to EGFR-negative ones (82% vs. 46%; 9/11 vs. 49/107; chi-square=0.88, p=0.02). Several other genes were also more frequently methylated in females compared to males, including GATA5 (25% vs. 7%; 9/36 vs. 4/55; chi-square=0.89, p=0.02), PAX6 (32% vs. 11%; 11/34 vs. 6/55; chi-square=0.91, p=0.01) and ESR1 (36% vs. 18%; 16/44 vs. 12/67; chi-square=0.87, p=0.03). EGFR-mutated tumors also had higher methylation rates of APC (7/11 vs. 34/103; chi-square=0.83, p=0.04), CDKN2B (6/11 vs. 21/98; chi-square=0.90, p=0.02), ESR1 (5/10 vs. 23/111; chi-square=0.85, p=0.04) and VHL (1/11 vs. 0/110; chi-square=0.87, p=0.001) compared to tumors without EGFR mutation (64% vs. 33%, 55% vs. 21%, 50% vs. 23% and 9% vs. 0%, respectively).
Discussion
Our observations of the most hypermethylated genes in NSCLC may be correlated with similar results which were described by other authors (4, 12-16). On the studied gene set, the average cumulative methylation rate was 90%, leaving only 10% of lung tumors with none of the 30 studied genes methylated. In a separate project, we only found methylation of CDKN2B gene in normal tissue (data not shown). It is known that methylations rarely occur in normal non-cancerous lung tissue (14), and is therefore seen as an early sign of malignant transformation, especially in smokers (17). The high frequency of methylated genes found in our study only further supports such a concept. We also correlated the results of methylation with clinical parameters such as sex, smoking status, disease stage, duration of overall survival and disease-free period. Several conclusions arose from these correlations. We confirmed previous reports that there are differences in the frequency of methylation among the particular histological types of lung cancer (18, 19) and between specific subtypes of NSCLC. In our samples, we detected both the individual and combined gene methylation more frequently in adenocarcinomas, which correlates with results of other authors (20).
List of genes evaluated for methylation status in non-small cell lung cancer samples.
Result of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis showing a sample without methylation (A) and a sample with hypermethylation of cadherin-13 (CDH13), Wilms' tumor protein (WT1) and paired box protein PAX-6 (PAX6) genes. The uncleaved fragments can be observed in both samples (connected by dotted lines), but only a sample with methylation reveals the additional fragments as a result of the methylation-specific cleavage.
Differences in frequency of gene methylation were also found depending on the gender of patients. In our case, however, other genes were methylated [more frequent methylation of CDH13, GATA5, PAX6 and ESR1 in women and caspase-8 (CASP8) in men] unlike in literature, where more frequent methylation of RASSF1A (21) and RARB (22) are described in men and death-associated protein kinase 1 (DAPK1) gene methylation in women (23).
The occurence of methylation changes and the presence of EGFR and KRAS mutations are interesting from the perspective of the now widely used biological therapy in locally advanced and metastatic non-small cell carcinomas (24). Despite the significant efficacy of therapy, there is recurrence or progression of originally tyrosine kinase inhibitor (TKI) sensitive EGFR-mutated tumors within months or even years. In addition to the TKI-resistant EGFR and KRAS mutations (25, 26), it can be assumed that the failure of treatment may also be caused by epigenetic factors, such as DNA methylation. In our study, we related methylation analysis of 30 selected genes with the analysis of EGFR and KRAS mutations, which may also have a negative prognostic significance under certain circumstances (27). Methylation was more frequent in carcinomas containing mutations in the EGFR gene. Patients with EGFR mutations had a greater percentage of methylation of APC, CDKN2B, ESR1 and VHL genes.
Genes with the most frequent methylation found in a set of 111 non-small cell lung cancer samples (See Table I for gene names).
We observed methylation of several genes to be linked to factors such as female gender, smoking status and EGFR mutation, which have positive predictive and prognostic value in relation to therapy by TKIs. Among them, methylation of CDH13 was found to be associated with all three factors. This gene was found to be more frequently methylated in nonsmokers (p<0.01).
The above findings only further suggest a complex interaction between genetic and epigenetic changes in the process of lung adenocarcinoma tumorigenesis. In view of the fact that methylation is, as opposed to DNA mutation, a reversible event, it is possible to consider the use of demethylating agents in the treatment for patients with methylation-positive tumors. For example, 5-aza-2’-deoxycytidine (Decitabine) or hydralazine represent such demethylators. Properly chosen inhibitors would therefore represent a complementary form of chemotherapy in the future in the effective treatment of solid tumors.
Acknowledgements
The Authors would like to thank Hana Mataseje for administration of patients, Gabriela Krakorova, Radka Bittenglova and Ondrej Fiala for help corellating clinical data and Barbora Belsanova and Andrea Krajcova for technical assitance in the laboratory. This work was supported by the Czech Ministry of Health grant no. NS9718. This is contribution no. 4 from CEGES (OPPK CZ.2.16/3.1.00/22213).
- Received October 14, 2011.
- Revision received November 14, 2011.
- Accepted November 15, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
