ANTICANCER RESEARCH 29: 363-370 (2009)

Promoter Methylation Primarily Occurs in Tumor Cells of Patients with Non-small Lung

WOUTER K. DE JONG1, GONDA F. VERPOOTEN2, HENK KRAMER1, JOOST LOUWAGIE2 and HARRY J.M. GROEN1

1Department of Pulmonary Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2Oncomethylome Sciences SA, Liège, Belgium

Abstract. Background: The distribution of promoter silencing of tumor suppressor genes, and is among the methylation throughout the lungs of patients with non-small earliest and most frequent alterations in cancer. The varying cell (NSCLC) is unknown. In this explorative levels of tumor suppressor gene methylations among study, we assessed the methylation status of the promoter different malignancies can be measured with a sensitive region of 11 genes in brush samples of 3 well-defined methylation-specific polymerase chain reaction (MSP), endobronchial locations in patients with NSCLC and in which seems a promising tool for early cancer detection. brushes of former and current smokers without NSCLC. Aberrant methylation is common in non-small cell lung Materials and Methods: The methylation status of RASSF1A, cancer (NSCLC) (2). GATA4, GATA5, SFRP1, RARβ2, DAPK, MGMT, p16, p14, Promoter methylation of multiple genes has not only been CHFR and APC2 was determined in all samples using real- observed in NSCLC tumor tissue and NSCLC cell lines (3, time methylation-specific PCR. Results: Ten patients with 4), but also in histologically tumor-free lung tissue of NSCLC and 18 non-NSCLC controls were included. Eight patients with resected NSCLC (5, 6), lymph nodes (7, 8), patients had one or more methylated genes in their tumor tumor brushes (9), serum (10), bronchoalveolar lavage brush. Promoter methylation of genes in proximal or (BAL) (11-14) and sputum (15, 16) of patients with contralateral locations was much less frequent than in tumor NSCLC. In addition, promoter methylation was also detected brushes, and almost exclusively occurred in normal tissue if in sputum and bronchial brushes of heavy smokers (9, 17, the same gene was also methylated in the tumor brush. 18). The detection of promoter methylation in sputum or Conclusion: Promoter methylation almost exclusively brushes of cancer-free heavy smokers proved to be a risk occurred in tumor cells of patients with NSCLC. factor for the subsequent development of cancer (9, 15, 19), highlighting the potential of methylation assays as lung DNA-methylation is one of the most well-known epigenetic cancer screening tool. Evidence for a possible association processes (1) and refers to the binding of a methyl group to between the presence of promoter methylation and smoking cytosine nucleotides in the DNA sequence. Methylated status and/or number of packyears is conflicting (12, 13, 17, cytosines are found in relatively high contents in CpG 18, 20-23). Altogether, methylation assays offer new islands. These CpG islands are distributed in a non-random possibilities in studying the pathogenesis, diagnosis, and manner across the humane genome, including the promoter eventually the treatment of NSCLC. regions of many tumor suppressor genes. Aberrant DNA- One could hypothesize that aberrant promoter methylation methylation of CpG islands may lead to the transcriptional occurs randomly in bronchial epithelium that is exposed to tobacco smoke. Local recurrences or second primary tumors after first-line treatment may develop from premalignant lesions other than the lesion responsible for the first tumor. Correspondence to: W.K. de Jong, MD, Ph.D., Department of The presence of random promoter methylation throughout Pulmonary Diseases, University Medical Center Groningen, P.O. the lungs could therefore be a sign of the presence of Box 30.001, NL-9700 RB Groningen, the Netherlands. Tel: +31 random, multiple lesions with genetic damage caused by 503616161, Fax: +31 503619320, e-mail: [email protected] tobacco smoke. Key Words: Promoter methylation, non-small cell lung cancer, In contrast with the occurrence of multiple, random methylation-specific PCR, bronchial epithelial cells, clonal field lesions, one could as well hypothesize that molecular and expansion, endobronchial brush. subsequent histological abnormalities first arise at one,

0250-7005/2009 $2.00+.40 363 ANTICANCER RESEARCH 29: 363-370 (2009) unique location. Synchronous abnormalities observed in Controls were former and current smokers who had no history of other locations, and also subsequent local recurrences and lung cancer, in order to match patients and controls as well as second primary tumors, could all originate from this primary possible. Eighteen controls (15 males) were included, with a median age of 59 (range, 23 to 77 years). Eight controls were former tumor by means of clonal expansion (24). This concurs with smokers (1 to 39 years since quitting) and 10 were current smokers. the field cancerization theory, originally proposed by The median number of packyears was 23 (range, 1 to 70). Slaughter in 1953 (25). Obviously, the field cancerization The study protocol was approved by the Medical Ethics theory was based on histological parameters and not on Committee of the University Medical Center Groningen. All patients methylation events. According to the field cancerization and controls gave informed consent. theory, promoter methylation detected in tissue sampled from locations other than the primary tumor originates from the Sample collection. Flexible fiberoptic bronchoscopy (Olympus, primary tumor by either clonal expansion or cellular Tokyo, Japan) via nasal introduction was performed under local or general anesthesia. In controls, one bronchial brush was obtained contamination. The clonal expansion theory predicts a from the normal-appearing main bronchus. In patients, brushes were decreasing number of promoter methylation events with sampled of three well-defined endobronchial locations. One brush increasing distance from the primary tumor. was collected from the tumor, a second brush from the normal- A bronchial brush is an excellent tool for obtaining a large appearing main bronchus 2-3 cm proximal from the tumor, and a sample of bronchial epithelial cells of well-defined locations third from the normal appearing main bronchus of the contralateral in the bronchial tree. These brushes are widely used for the lung. To avoid contamination of the bronchoscope with tumor cells, cytological diagnosis of pulmonary abnormalities, such as the contralateral brush was collected first, followed by the proximal brush and finally the tumor brush. Moreover, all brushes were NSCLC. Bronchial brushes generally contain more than one sheathed cytology brushes (Cellebrity Endoscopic Cytology Brush; million cells, 80-90% of which are epithelial cells (26, 27). Boston Scientific, Boston, MA, USA). One study reported that 32% of former smokers had one or After sampling, brushes were stored in a solution of 50% ethanol more genes with promoter methylation in epithelial cells and 2% polyethylene glycol at room temperature until further obtained with a carinal bronchial brush (18). Zochbauer- processing. Muller et al. observed promoter methylation in up to 20% of bronchial brushes of smokers (17). In another study, DNA isolation and modification. Cells attached on the bronchial brush promoter methylation of p16 was observed in 16% and 8% were collected by centrifugation at 960 relative centrifugal forces for 10 minutes and by an additional washing with phosphate buffered saline in bronchial brushes of patients with lung cancer versus (PBS) and a centrifugation step. Genomic DNA was then extracted smokers without lung cancer, respectively(9). using the classic SDS/protein K digestion and phenol-chloroform To evaluate the distribution of promoter methylation, extraction, and resuspended in 50 μl Lo TE (3 mM Tris, 0.2 mM EDTA; bronchial epithelial cells of three well-defined endobronchial pH 8.0). The DNA was quantified using the Picogreen dsDNA locations in patients with NSCLC were collected and the quantification kit (Molecular Probes, Invitrogen, Breda, the Netherlands) presence of promoter methylation of eleven genes [RAS- following the manufacturer’s recommendations. The average yield of association domain family 1 isoform A (RASSF1A), GATA- DNA was 19.2 μg (range, 0.5 to 87.7 μg). Up to 1.5 μg of genomic DNA were modified using sodium bisulfite to deaminate selectively binding protein 4 (GATA4), GATA-binding protein 5 (GATA5), unmethylated cytosine residues to uracil, while 5-methyl cytosine secreted frizzled-related protein 1 (SFRP1), retinoic acid residues were not modified. The bisulfite modification was performed receptor beta-2 (RARβ2), death-associated protein kinase using the EZ DNA Methylation Kit™ (Zymo Research, Orange, CA, (DAPK), O6-methylguanine-DNA-methyltransferase (MGMT), USA), which includes successive steps of conversion, desalting and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16 and p14), desulfonation. At the end of the procedure, the modified DNA was checkpoint with forkhead and ring finger domains (CHFR), eluted in 50 μl of 1mM Tris-HCl (pH 8.0), and then stored at –80˚C and adenomatous polyposis coli 2 (APC2)] at those three until used for real-time MSP. locations in the bronchial tree was quantitatively determined. Real-time MSP. Briefly, for each gene of interest and beta-actin (ACTB), methylation was determined in real-time MSP using bisulfite- Materials and Methods modified DNA as previously described by Herman et al. (28). ACTB was used as an independent reference gene. The promoter methylation Patients. Patients who were scheduled for a diagnostic bronchoscopy status of RASSF1A, GATA4, GATA5, SFRP1, RARβ2, DAPK, MGMT, during the work-up for suspected lung cancer were asked to p16, p14, CHFR, and APC2 was calculated as the ratio of the gene of participate in the study. Ten patients were included (6 females, 4 interest and ACTB according to the following equation: (copies males), with a median age of 54 years (range, 40 to 70 years). Six methylated gene/copies ACTB) ×1,000. This ratio is a measure of the patients were current smokers; four patients were former smokers, 1 relative level of methylation in an individual sample. to 12 years since quitting. The median number of packyears was 33 (range, 20 to 60). Five patients had adenocarcinoma, four had large Statistical analysis and cut-off value. Promoter methylation was cell carcinoma and one patient had a neuroendocrine carcinoma. At considered present if the ratio was above a certain cut-off value. The diagnosis, one patient had stage IA disease, two had stage IIIB and relative level of methylation varied significantly among the 11 genes seven patients had stage IV disease. and therefore cut-off points were studied for each gene individually.

364 De Jong et al: Promoter Methylation in NSCLC

Table I. Promoter methylation status of eleven genes in endobronchial epithelial cells from 10 patients with NSCLC and 18 controls.

Gene Mean ratio gene/ACTB Mean ratio gene/ACTB Percentage of controls with Cut-off value ×1,000 in patients (range) ×1,000 in controls (range) a ratio gene/ACTB ×1,000 > 0 (based on 100% specificity)

RASSF1A 70.8 (0-393) 0.18 (0-2.5) 33 10 GATA4 21.9 (0-217) 0.03 (0-0.2) 39 1 GATA5 1.1 (0-11) 0.06 (0-0.3) 28 0.5 SFRP1 1.7 (0-17) 0.10 (0-0.8) 28 5 RARβ2 1.1 (0-6) 0 0 0.5 DAPK 0.08 (0-0.5) 0.07 (0-0.5) 39 2 MGMT 13.9 (0-138) 0.02 (0-0.2) 28 5 p16 0.63 (0-4.6) 0.01 (0-0.1) 17 1 p14 0.02 (0-0.2) 0.05 (0-0.6) 11 1 CHFR 55.0 (0-549) 0 0 0.5 APC2 5.4 (0-30) 0.77 (0-2.57) 94 2.6

The gene-specific cut-off value was defined as the highest ratio patient had 5 genes with promoter methylation, one patient 4, observed in controls (visualized in scatter plots). In this way, the one patient 3, three patients 2, and two patients only one. specificity of a single gene was set at 100% . Promoter methylation was most frequent in APC2 and The presence of promoter methylation at the tumor, proximal, RASSF1A (in 5 and 4 patients, respectively). For DAPK and and contralateral locations was visualized. In addition, the overall p14, no promoter methylation was observed in these 10 level of methylation was determined using the methylation index (MI) (29). The MI is defined as the ratio of number of genes with patients. promoter methylation to the number of genes tested ×100% . Overall, in 10 NSCLC patients using a panel of 11 Associations between promoter methylation and demographic and different genes we identified an MI of only 18% (20/110) clinical characteristics of patients and controls were calculated using (Figure 2) in tumor cells and 2.7% in normal epithelial chi-square and Mann-Whitney tests, where appropriate. A p-value of cells. Epithelial cells obtained from the proximal and <0.05 was considered statistically significant. contralateral brush did not differ in the number of genes Results with promoter methylation. No random pattern of promoter methylation in normal bronchial epithelium at the proximal Exploring cut-off values using normal controls and tumor and contralateral localizations was observed. The highest brushes. Patients and controls were well-matched regarding overall number of methylated genes was observed in patient smoking status, number of packyears and age. The control 8. His p16/ACTB ratios ×1,000 for tumor, proximal and group consisted of fewer females than the patient group. contralateral brushes were 4.6, 6.0 and 1.3, respectively, In epithelial cells from bronchial brushes of 18 controls above the cut-off value of 1. Patient 8’s APC2/ACTB ratios and 10 patients with NSCLC, the ratios of expression of 11 ×1,000 were 11.2, 12.0, and 3.7, respectively, with a cut- methylated genes as compared to the expression of ACTB off value of 2.6. APC2 was also methylated in the proximal ranged from 0 to 71 (Table I). The mean ratio of methylated brush of patient 1 (3.4) and in the contralateral brush of genes versus ACTB was higher in patients than in controls patient 6 (3.2). for all eleven genes except p14 (Table I). For DAPK and p14, the highest ratio observed in controls was higher than Correlation between promoter methylation and clinical in patients with NSCLC; for the other nine genes at least one characteristics. The presence of promoter methylation in ratio in patients was higher than the highest ratio observed tumor brushes was not associated with exposure to smoking in controls (Figure 1). The percentage of controls with a (p=0.53 for number of packyears) or acute smoking effects ratio >0 but below our gene-specific cut-off value ranged (p=0.2 for current versus former smokers). In this study, age from 94% (DAPK) to 0% (Table I). and gender were also not associated with the presence of methylation (p=0.53 and p=0.75, respectively). Finally, the Distribution of promoter methylation in bronchial epithelium. histological subtype of NSCLC was not associated with Eight out of ten patients with NSCLC had at least one gene methylation (p=0.29). The two patients without any promoter with promoter methylation in the tumor brush and none of methylation were a 51-year-old female, a current smoker with the 18 controls using our cut-off value, resulting in an overall 35 packyears, and a 69-year-old male, a current smoker with sensitivity of 80% and an accuracy of 93% (Figure 2). One 40 packyears, both with adenocarcinoma of the lung.

365 ANTICANCER RESEARCH 29: 363-370 (2009)

Figure 1. Scatter plots of methylation ratios of 11 genes/ACTB levels ×1,000 (10log scale) in endobronchial brushes of 18 controls and 10 NSCLC patients. The bar in the NSCLC column represents the cut-off value based on 100% specificity. *Number of patients or controls with undetectable levels of the methylated allele.

Discussion The overall frequencies of promoter methylation in proximal and contralateral brushes were substantially lower The present exploratory study is the first study to evaluate the than in tumor brushes (Figure 2). In the present study, no presence of promoter methylation at different endobronchial random pattern of distribution of promoter methylation in locations in patients with NSCLC. Promoter methylation was the proximal and contralateral endobronchial locations was observed in tumor brushes of up to 80% of patients with observed; methylation at these locations was, in all but one NSCLC, whereas it was uncommon in proximal and case, only present if the tumor brush also contained contralateral brushes, and almost exclusively present if the promoter methylation of the same gene. This observation same gene was also methylated in the tumor brush. favors the idea that promoter methylation is primarily

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Figure 2. Distribution of gene promoter methylation at three endobronchial locations (tumor, 2-3 cm proximal of tumor, contralateral lung) in 10 patients using a cut-off value based on 100% specificity in 18 controls. Grey denotes unmethylated genes, black methylated genes. The right column provides a summary with the number of methylated genes at a single location. present in tumor cells, and subsequently may spread from is claimed that 1 positive marker in 50,000 negative markers the primary tumor to adjacent and even further locations in can be detected (15). The prognostic and clinical value of the bronchial epithelium, as was predicted with the theory one positive signal among many negative signals is difficult of clonal field expansion (24). Exposure to tobacco smoke to interpret; we think that ratios are much more clinically causes genetic damage to the entire bronchial epithelium, as meaningful. Apart from technical differences between our was recently demonstrated by the construction of a study and other studies, the reported methylation frequencies biomarker panel that was able to predict the presence of in other studies may be due to patient selection, for instance lung cancer in normal airway epithelium of smokers with presence of sputum atypia (17), or use of controls with a suspected lung cancer (30). This panel has no overlap with history of cancer (18). In addition, our strategy differed from the genes we tested. In our study, smoking did not result in others in that we set our design and test condition in such a a random development of promoter methylation in the entire way as to have the highest specificity for NSCLC. bronchial epithelium in patients with lung cancer. The To our knowledge, only one study reported on promoter absence of methylated genes in our controls, who all had a methylation assays (using quantitative MSP) on brushes of long history of exposure to tobacco smoke, is in line with patients with lung cancer (9). In brushes from either the this finding. main bronchus or tumor of patients, a 16% methylation In contrast, several other studies reported the detection of frequency was observed for p16, dropping to 8% for chronic promoter methylation in exfoliative bronchial material in 5- smokers without lung cancer. Real-time MSP has not been 15% of heavy smokers without NSCLC (6, 9, 11-15, 18-20, performed on bronchial epithelial cells collected by sheathed 31-37). Some of these studies applied quantitative brushes before. In our study, single gene methylation methylation-specific PCR (11, 13, 14, 35, 37). For most of frequencies of tumor brushes ranged from 0-50% , with an the genes in our panel, several of our controls had detectable overall methylation index of 18% . ratios (Table I), but these levels were generally well below The quantitative results of a methylation-specific real- our chosen cut-off values. Our findings with real-time PCR time PCR are often dichotomized for comparative purposes are different from the reported results using quantitative (11, 35, 37-39). Different methods for defining the optimal PCR. These quantitative (nested) PCRs are very sensitive: it cut-off values for dichotomization exist. One could define

367 ANTICANCER RESEARCH 29: 363-370 (2009) cut-off values based on methylation frequencies of genes Conflict of Interest in tumor tissue. These frequencies can be derived from previously published studies (38) and the cut-off values No financial or other potential conflicts of interest are declared for are adjusted to match the expected methylation Drs de Jong and Kramer. Drs Verpooten and Louwagie are frequencies. It is essential for this method that consistency employees of Oncomethylome Sciences SA. Dr Groen is consultant to Oncomethylome Sciences SA and received a research grant of is observed in independently established promoter Oncomethylome Sciences SA. methylation frequencies of tumor tissues for individual genes (38). Yet the methylation frequencies in exfoliative Acknowledgements samples are generally lower than those in tumor tissue (17). Moreover, it is known that median methylation ratios The authors thank Alexandre Meunier for his technical assistance. were observed to be higher in tumor tissue than in other tissues (11, 37). This is partially explained by the fact that References brushes and sputum contain relatively fewer bronchial epithelial tumor cells than tumor tissue due to 1 Herman JG and Baylin SB: Gene silencing in cancer in contamination with other cells. Therefore, we chose to association with promoter hypermethylation. N Engl J Med define a specific cut-off value for brushes based on the 349: 2042-2054, 2003. ratios observed in controls, with specificity set at 100% . 2 Belinsky SA: Gene-promoter hypermethylation as a This means that cut-off points specifically determined for biomarker in lung cancer. Nat Rev Cancer 4: 707-717, 2004. brushes are more appropriate for the use of methylation 3 Tsou JA, Hagen JA, Carpenter CL et al: DNA methylation analysis: a powerful new tool for lung cancer diagnosis. assays in the early detection of cancer in exfoliative Oncogene 21: 5450-5461, 2002. samples than tissue-based cut-off values. 4 Shames DS, Girard L, Gao B et al: A genome-wide screen In our study, methylation was much more pronounced in for promoter methylation in lung cancer identifies novel tumor cells as compared to non-tumor cells from the same methylation markers for multiple malignancies. PLoS Med 3: individual. This finding suggests that the tumor field effect e486, 2006. may be limited in terms of distance from the tumor and/or 5 Guo M, House MG, Hooker C et al: Promoter percentage of affected cells in the vicinity of the tumor. In hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res 10: 5131-5136, 2004. addition, if methylation tests are to be used as a screening 6 Zochbauer-Muller S, Fong KM, Virmani AK et al: Aberrant tool for lung cancer, our results suggest that the value of promoter methylation of multiple genes in non-small cell such a test is highly dependent on the presence of tumor cells lung . Cancer Res 61: 249-255, 2001. in the samples to be screened. Whether or not the presence of 7 Pellise M, Castells A, Gines A et al: Detection of lymph promoter methylation was correlated to the cytological node micrometastases by gene promoter hypermethylation in features of the samples (e.g. normal epithelium, ) samples obtained by endosonography-guided fine-needle was not determined in our study. aspiration biopsy. Clin Cancer Res 10: 4444-4449, 2004. We did not observe any association between smoking 8 Harden SV, Tokumaru Y, Westra WH et al: Gene promoter hypermethylation in tumors and lymph nodes of stage I lung history, age, gender, or even tumor type, in agreement with cancer patients. Clin Cancer Res 9: 1370-1375, 2003. most studies (11-13, 17, 18, 20, 23, 35, 40). In contrast, 9 Kersting M, Friedl C, Kraus A et al: Differential frequencies some studies reported that the frequency of methylation is of p16(INK4a) promoter hypermethylation, p53 mutation, higher in current smokers versus former or never smokers and K-ras mutation in exfoliative material mark the (12, 14, 21, 41), in males versus females (6), or is dependent development of lung cancer in symptomatic chronic smokers. on tumor (41, 42). Our results are not necessarily J Clin Oncol 18: 3221-3229, 2000. discrepant with results of other groups observing methylation 10 Esteller M, Sanchez-Cespedes M, Rosell R et al: Detection of aberrant promoter hypermethylation of tumor suppressor in smokers without lung cancer and in cancer-free tissue. genes in serum DNA from non-small cell lung cancer The difference may be due to the use of cut-off values, as patients. Cancer Res 59: 67-70, 1999. described earlier, differences in specimens, our sample size, 11 Topaloglu O, Hoque MO, Tokumaru Y et al: Detection of or other undefined factors. promoter hypermethylation of multiple genes in the tumor In conclusion, promoter methylation was almost and bronchoalveolar lavage of patients with lung cancer. Clin exclusively observed in endobronchial tumor cells and was Cancer Res 10: 2284-2288, 2004. very infrequent or absent in cancer-free samples. Whether 12 Kim H, Kwon YM, Kim JS et al: Tumor-specific methylation the promoter methylation observed by other groups in in bronchial lavage for the early detection of non-small cell lung cancer. J Clin Oncol 22: 2363-2370, 2004. cancer-free tissue and in controls without lung cancer, in 13 Grote HJ, Schmiemann V, Geddert H et al: Aberrant contrast with our results, is a result of contamination with promoter methylation of p16(INK4a), RARB2 and SEMA3B tumor cells or is due to methodological issues such as the in bronchial aspirates from patients with suspected lung high specificity of real-time MSP is not known. cancer. Int J Cancer 116: 720-725, 2005.

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