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Gene Promoter Hypermethylation in Tumors and Lymph Nodes of Stage I Lung Cancer Patients

Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research [S. V. H., Y. T., W. H. W., D. S.] and Departments of Pathology [W. H. W.], Oncology/Biostatistics [S. G.], and Thoracic Surgery [S. C. Y.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21206-2198, and Department of Surgery, University of Rochester Medical Center, Rochester, New York [S. A. A.]

	ABSTRACT

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Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells and a promising marker for cancer detection. We tested five gene promoters [CDKN2A (p16), O⁶-methylguanine-DNA-methyltransferase, glutathione S-transferase P1 (GSTP1), adenomatous polyposis coli (APC), and death-associated protein kinase (DAPK)] by real-time methylation-specific PCR in primary tumors from 90 stage I lung cancer patients for aberrant DNA methylation. We then used the presence of tumor methylation as a marker to investigate the presence of occult metastasis in corresponding histologically negative lymph nodes. Of the primary tumors, 73 of 90 (81%) displayed promoter hypermethylation in at least one of the genes studied: 17% (15 of 90) at p16 (CDKN2A); 16% (14 of 90) at O⁶-methylguanine-DNA-methyltransferase; 8% (7 of 90) at GSTP1; 72% (65 of 90) at APC; and 17% (15 of 90) at DAPK. Squamous histology was predictive of worse overall survival (P = 0.074, log-rank test). APC methylation and GSTP1 methylation in the primary tumor were both correlated with nonsquamous histology (P = 0.02 and P = 0.01 likelihood ratio respectively). The presence of both APC methylation and DAPK methylation in the primary tumor predicted a worse outcome, with 7 of 13 (54%) deaths in this group compared with 21 of 77 (27%) deaths in cases without both genes methylated (P = 0.229, log-rank test). The same methylation pattern was detected in DNA from at least one of the corresponding lymph nodes in 11 of 73 (15%) cases. Five of 11 (45%) patients with occult metastasis detected by methylation analysis have died compared with 17 of 62 (27%) patients with negative lymph nodes, although survival analysis did not reach statistical significance (P = 0.632, log-rank test). Promoter hypermethylation is common in lung cancer and represents a promising marker for the molecular staging of lung cancer patients. Although this study showed important trends, a larger prospective study is required to better understand the value of methylation analysis in detecting occult metastasis.

	INTRODUCTION

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Lung cancer is the leading cause of cancer-related death in the United States (1) . Surgical resection is the most effective form of treatment for NSCLC,² although at the time of diagnosis, only one-third of patients will have early-stage disease amenable to curative surgery. In addition, a large percentage of patients with early-stage disease undergoing surgical resection ultimately die of recurrent disease, suggesting the presence of occult metastatic disease at the time of diagnosis (2) . Several studies have used a variety of molecular techniques in a range of different tumors to detect occult metastatic spread (3, 4, 5, 6) . We have previously looked for occult metastasis to lymph nodes in lung cancer using p53 and K-ras mutations as markers of tumor DNA (7) . However, the presence of p53 mutation in the primary tumor was such a strong predictor of poor prognosis that detection of occult spread did not appear to affect outcome.

Silencing of tumor suppressor or other cancer-associated genes by hypermethylation of CpG islands within the promoter and/or 5'-regions of many genes is a common feature of human cancer and is often associated with a transcriptional block and loss of the relevant protein (8, 9, 10, 11) . Moreover, a number of gene promoters have been found to be hypermethylated in NSCLC (12 , 13) . In addition to the functional implications of gene inactivation in tumor development, the presence of epigenetic methylation has been shown to be useful as a molecular target for tumor cell detection in serum, plasma, and bronchioaveolar lavage fluid from NSCLC patients (12 , 14 , 15) . We sought to detect occult metastasis by using a panel of genes known to be frequently hypermethylated in lung tumors. To detect the presence of neoplastic DNA with a sensitivity of 1 cell in 1000 normal cells, we used real-time QMSP. This PCR approach is more sensitive than conventional PCR and more specific due to the use of an internally binding, fluorogenic hybridization probe (16 , 17) . Using QMSP, we analyzed the promoter hypermethylation pattern of the p16, MGMT, GSTP1, APC, and DAPK genes in the tumor DNA of 90 stage I primary lung cancers. The methylation patterns found in the tumors were then used as molecular markers for cancer cell detection in the paired lymph node DNA.

	MATERIALS AND METHODS

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Sample Collection and DNA Extraction.
Primary tumor and lymph nodes were collected prospectively from 90 patients undergoing surgical resection of stage I NSCLC by a single surgeon at The Johns Hopkins Hospital or the Johns Hopkins Bayview Medical Center from 1995–1999. The samples were promptly frozen and stored at -80°C after initial gross pathological examination.

This research protocol was approved by the Joint Committee on Clinical Investigation of the Johns Hopkins School of Medicine in accord with an assurance filed with the United States Department of Health and Human Services. Written informed consent was obtained from all patients.

The 90 cases consisted of 50 males and 40 females with a median age of 67 years (range, 40–87 years). There were 44 stage IA and 46 stage IB cases (33 squamous cell carcinomas, 36 adenocarcinomas, 11 bronchioaveolar carcinomas, and 10 large cell/unspecified non-small cell carcinomas). The revised International System determined pathological stage for Staging Lung Cancer (18) .

Ninety primary tumor and 311 lymph node samples were cut into 12-µm sections and placed in a mixture of 1% SDS and proteinase K (0.5 mg/ml) at 48°C overnight. In addition, 5-µm sections were taken every 15 slices and stained with H&E for examination by light microscopy. Tumors with <70% tumor cells were microdissected to remove areas of normal tissue. DNA was then extracted from all samples with phenol/chloroform and precipitated with ethanol (19) . In addition, 8 primary tumor and 44 lymph node samples from stage III patients were processed to document the accuracy of the assay.

Bisulfite Treatment and Real-Time MSP.
Chemical modification of unmethylated (but not methylated) cytosines to uracil within CpG islands using sodium-bisulfite treatment was performed as described previously (20) . Two µg of tumor or lymph node DNA were used for the sodium-bisulfite treatment. DNA samples were then purified using the Wizard purification resin (Promega, Madison, WI), treated again with sodium hydroxide, precipitated with ethanol, and resuspended in 120 µl of water.

The modified DNA was used as a template for real-time fluorogenic MSP. Primers and probes were designed for the five genes of interest. In addition, primers and a probe were designed to amplify an internal reference gene, ACTB. These were located in areas without CpG nucleotides, thus amplifying the modified ACTB gene independently of the methylation status of CpG nucleotides. PCR was performed in separate wells for each primer/probe set. To determine the relative levels of methylated promoter DNA in each sample, the values of the gene of interest were compared with the values of the internal reference gene to obtain a ratio that was then multiplied by 100 to give a percentage value (gene of interest/reference gene x 100). Tumors were considered positive if a percentage value of 1% was obtained. This cutoff was chosen for being clinically relevant and also to exclude very low-level background readings that can occur in certain individuals for certain genes (21) .

In all cases, the first primer is the forward PCR primer, the second is the reverse PCR primer, and the third is the TaqMan probe. The primer sequences were as follows: (a) APC (74-bp amplicon; position 761–834; GenBank accession number U020509), 5'-GAACCAAAACGCTCCCCAT-3', 5'-TTATATGTCGGTTACGTGCGTTTATAT-3', and 6FAM5'-CCCGTCGAAAACCCGCCGATTA-3'TAMRA; (b) DAPK (98-bp amplicon; position 5–102; GenBank accession number X76104), 5'-GGATAGTCGGATCGAGTTAACGTC-3', 5'-CCCTCCCAAACGCCGA-3', and 6FAM5'-TTCGGTAATTCGTAGCGGTAGGGTTTGG-3'TAMRA; (c) GSTP1 (140-bp amplicon; position 1033–1172; GenBank accession number M24485), 5'-AGTTGCGC-GGCGATTTC-3', 5'-GCCCCAATACTAAATCACGACG-3', and 6FAM5'-CGGTCGACGTTCGGGGTGTAGCG-TAMRA; (d) p16 (150-bp amplicon; position 25–174; GenBank accession number U12818), 5'-TTATTAGAGGGTGGGGCGGATCGC-3', 5'-GACCCCGAACCGCGACCGTAA-3', and 6FAM5'-AGTAGTATGGAGTCGGCGGCGGG-3'TAMRA; (e) MGMT (122-bp amplicon; position 1029–1150; GenBank accession number X61657), 5'-CGAATATACTAAAACAACCCGCG-3', 5'-GTATTTTTTCGGGAGCGAGGC-3', and 6FAM5'-AATCCTCGCGATACGCACCGTTTACG-3'TAMRA; and (f) ACTB (133-bp amplicon; position 390–522; GenBank accession number Y00474), 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3', 5'-AACCAATAAAACCTACTCCTCCCTTAA-3', and 6FAM5'-ACCACCACCCAACACACAATAACAAACACA-3'TAMRA.

Fluorogenic PCR was carried out in a reaction volume of 20 µl. Each PCR reaction mixture consisted of 600 nM of each primer (Invitrogen, Carlsbad, CA); 200 nM probe (Applied Biosystems, Foster City, CA); 0.75 unit of platinum Taq polymerase (Invitrogen); 200 µM each of dATP, dCTP, dGTP, and dTTP; 16.6 mM ammonium sulfate; 67 mM Trizma; 6.7 mM magnesium chloride (2.5 mM for p16); 10 mM mercaptoethanol; and 0.1% DMSO. Five µl of treated DNA solution were used in each real-time MSP reaction for p16, MGMT, and GSTP1, and 3 µl were used for APC and DAPK. Thermal cycling was initiated with a first denaturation step of 95°C for 2 min. The thermal profile for the PCR was 95°C for 15 s and 60°C for 1 min. Data obtained during 50 cycles of amplification were analyzed.

Amplifications were carried out in 384-well plates in a 7900 Sequence detector (Applied Biosystems). All samples were run in duplicate and repeated in duplicate again if they did not match. Each plate included multiple water blanks, a negative control, and serial dilutions of a positive control for constructing the calibration curve on each plate. Leukocyte DNA from a healthy individual was used as the negative control for all genes. The same leukocyte DNA was methylated in vitro with excess SssI methyltransferase (New England Biolabs Inc., Beverly, MA) to generate completely methylated DNA at all CpGs and used as the positive control for all genes.

Statistical Analysis.
Contingency tables were analyzed using Fisher’s exact tests, and log-rank tests were used for survival curve comparisons.

	RESULTS

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QMSP in Tissue.
To document the accuracy of the assay, we first tested 44 lymph nodes from eight patients with stage III NSCLC; 31 of the 44 lymph nodes were histologically positive. QMSP correctly identified all positive lymph nodes corresponding to the aberrant methylation pattern of the primary tumor (data not shown).

We used QMSP to test for the presence of promoter methylation of five genes in 90 primary stage I NSCLC tumors. We found aberrant methylation of at least one gene in 73 of 90 (81%) primary tumors (Table 1) . MGMT was methylated in 14 of 90 (16%) primary tumors, p16 was methylated in 15 of 90 (17%) primary tumors, GSTP1 was methylated in 7 of 90 (8%) primary tumors, APC was methylated in 65 of 90 (72%) primary tumors, and DAPK was methylated in 15 of 90 (17%) primary tumors. A single gene was methylated in 41 of 90 (46%) primary tumors, two genes were methylated in 22 of 90 (24%) primary tumors, three genes were methylated in 8 of 90 (9%) primary tumors, and four genes were methylated in 2 (2%) primary tumors.

Clinical Follow-up.
Follow-up data were obtained from our research database and the Social Security Death Index and further confirmed by patient and physician phone calls. Length of patient follow-up ranged from 1 to 81 months, with a median of 43 months. Survival data were stratified by histology, stage (IA versus IB), tumor methylation (overall, DAPK, APC), and lymph node methylation molecular upstaging (Fig. 1, A–C) . The presence of both DAPK methylation and APC methylation in the primary tumor displayed a trend toward worse survival (P = 0.229, log-rank test; P = 0.1011, Fisher’s exact test). In particular, of the 13 cases positive for both genes, 7 (54%) patients subsequently died, compared with 21 of 77 (27%) deaths in the rest of the patients. Patients with squamous cell carcinoma had significantly worse overall survival than patients with other histologies (P = 0.0074, log-rank test) in this study. Of the five markers, APC (P = 0.02) and GSTP1 (P = 0.01) correlated with nonsquamous histology.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Survival curves. A, overall survival curves by tumor histology. B, overall survival curves by combined DAPK and APC methylation in the primary tumor. C, overall survival curves by methylated promoter DNA detected in lymph nodes from 73 cases with primary tumor methylation.

	DISCUSSION

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Using real-time MSP, we found the presence of promoter hypermethylation in at least one of five genes in 81% of primary stage I NSCLC. Several studies have suggested that the presence of DAPK or APC methylation in the primary tumor is associated with worse outcome (14 , 22) . Our study supports these findings because the number of deaths doubled in the cases positive for both genes compared with the rest of our group (54% versus 27%). Although the presence of APC or DAPK methylation alone was not a predictor in this study, the relatively small numbers of events within the marker groups meant that the confidence intervals on these relationships (data not shown) were wide, and therefore these data are still compatible with moderate effects on survival. The impact of DAPK and APC methylation in the primary tumor on outcome was not related to histology. In this prospective trial, we were able to detect the presence of occult metastasis to lymph nodes at the time of initial surgery in a subset of stage I lung cancer patients. This is the first time that methylation was used as a marker for detection of occult metastasis to lymph nodes in lung cancer. Of the 73 cases harboring promoter hypermethylation, 11 harbored an identical methylation pattern in the lymph nodes, and these cases had a worse outcome. There were 45% deaths in this group compared with 27% deaths in the rest of the cases. Although survival comparisons were not statistically significant, the small number of upstaged patients severely underpowered the study. It is possible that a much larger study (n = 300) may show a statistically significant difference in survival. However, an alternative explanation is that presence of occult metastasis in hilar/bronchial lymph nodes that are resected en bloc with the primary tumor does not greatly alter outcome. Rather, the presence of occult spread to mediastinal lymph nodes may be more directly relevant for prediction of tumor spread and survival.

The study also highlighted important technical issues for the use of methylation assays in clinical samples with a high background of normal cells. We designed our assay to detect the presence of tumor DNA at low level in a background of normal tissue. In view of this, high levels of input DNA were used to quantitative DNA values down to a level over 1000 times lower than total input DNA, as assessed by ß-actin amplification. We found that virtually every single lymph node of the 311 lymph nodes analyzed showed a low-level background level of DAPK methylation (0.01–5% relative to reference gene). We saw the same positive results using conventional MSP to 35 cycles and using a different DAPK primers/probe set. We have sequenced the PCR product and found that the amplified sequence is methylated at all relevant sites, attesting to the specificity of the probe (data not shown). This low-level methylation does not show up as prominently if conventional MSP is only run to 30 cycles, but this is not surprising because the threshold cycle number for all these results is between 30 and 40 using real-time analysis. The low-level positivity of DAPK in lymph nodes relative to total input DNA varies over 100-fold from individual to individual (range,0.01–5%; median value, 0.5%). Tumor positivity, however, only ranged from 1% to 8%, and there were many cases where tumor level was the same as the lymph node levels. We therefore only classified a tumor as positive for DAPK methylation if the level was at least 3-fold greater than that in the lymph nodes from the same patient. In practice, this excluded all tumors with a ratio of <1% relative to ACTB, but it did not directly correlate with the highest absolute levels. Some groups have used an absolute cutoff criterion of 4% relative to ACTB for calling tumors positive for methylation, others have argued that methylation in <20% of cells is unlikely to play a functional role. Setting a threshold for QMSP is feasible for primary tumor analysis, but it sets tough limits on using methylation as a marker for low-level detection in tissues such as lymph nodes or tumor margins. We believe activated lymphocytes may methylate DAPK to avoid apoptosis, potentially accounting for the low-level methylation in lymphocyte compartments.³ Contamination of serum DNA by lysed lymphocytes could similarly cause false positive results for this marker. Fortunately, this limit to low-level detection is not an issue for other critical markers.

Our results using methylation of multiple genes to detect occult metastasis compared well with our previous study using p53 and K-ras mutation analysis in lymph nodes from lung cancer patients (7) . Adjusting for the cases uninformative for methylation analysis due to low-level background positivity, similar rates of occult metastasis were detected in the 30 cases of overlap between the two studies, suggesting that both the genetic and epigenetic assays have similar sensitivities.

Thus, QMSP is a powerful indicator of occult metastasis, but some methylation markers may have limited use, particularly for lymphocyte-rich samples. Larger prospective trials and mediastinal lymph node sampling are clearly needed to further establish the use of this assay for molecular staging.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research, The Johns Hopkins University School of Medicine, 818 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. Phone: (410) 502-5153; Fax: (410) 614-1411; E-mail: dsidrans{at}jhmi.edu

² The abbreviations used are: NSCLC, non-small cell lung cancer; MGMT, O⁶-methylguanine-DNA-methyltransferase; GSTP1, glutathione S-transferase P1; APC, adenomatous polyposis coli; DAPK, death-associated protein kinase; MSP, methylation-specific PCR; QMSP, quantitative MSP; FAM, 6 carboxyfluoroscein; TAMRA, 5(6) carboxy-tetramethylrhodamine.

³ A. N. Reddy, W. W. Jiang, N. Benoit, S. Harden, W. Koch, D. Sidransky, and J. Califano. DAPK1 promoter hypermethylation in a subset of lymphocytes, manuscript in preparation.

Received 9/ 9/02; revised 12/27/02; accepted 1/10/03.

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