Aberrant Promoter Hypermethylation in Serum DNA From Patients With Silicosis

7/28/2019 Aberrant Promoter Hypermethylation in Serum DNA From Patients With Silicosis

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Carcinogenesis vol.29 no.9 pp.18451849, 2008doi:10.1093/carcin/bgn169

Advance Access publication July 16, 2008

Aberrant promoter hypermethylation in serum DNA from patients with silicosis

Shigeki Umemura, Nobukazu Fujimoto1,, Akio Hiraki2,Kenichi Gemba1, Nagio Takigawa, Keiichi Fujiwara3,Masanori Fujii, Hiroshi Umemura4, Mamoru Satoh4,Masahiro Tabata, Hiroshi Ueoka5, Katsuyuki Kiura,Takumi Kishimoto6 and Mitsune Tanimoto

Department of Hematology, Oncology and Respiratory Medicine, GraduateSchool of Medicine, Dentistry and Pharmaceutical Sciences, OkayamaUniversity, Okayama 7008558, Japan, 1Department of Respiratory Medicine,Okayama Rosai Hospital, 1-10-25 Chikkomidorimachi, Okayama 702-8055,Japan, 2Division of Epidemiology and Prevention, Aichi Cancer CenterResearch Institute, Nagoya 4648681, Japan,

3Department of Respiratory

Medicine, Okayama Medical Center, Okayama 7011192, Japan,4

Departmentof Molecular Diagnosis, Graduate School of Medicine, Chiba University,Chiba 2608670, Japan,

5Department of Medical Oncology, National Hospital

Organization Sanyo Hospital, Ube 7550241, Japan and6

Department ofInternal Medicine, Okayama Rosai Hospital, 1-10-25 Chikkomidorimachi,Okayama 702-8055, Japan

To whom correspondence should be addressed. Tel: 81 86 2620131;Fax: 81 86 2623391;Email: [email protected]

It is well established that patients with silicosis are at high risk forlung cancer; however, it is difficult to detect lung cancer by chestradiography during follow-up treatment of patients with silicosisbecause of preexisting diffuse pulmonary shadows. The purposeof this study is to evaluate the usefulness of detection of serumDNA methylation for early detection of lung cancer in silicosis.Serum samples from healthy controls (n 5 20) and silicosis pa-tients with (n 5 11) and without (n 5 67) lung cancer were testedfor aberrant hypermethylation at the promoters of the DNA re-pair gene O6-methylguanine-DNA methyltransferase (MGMT),

p16INK4a, ras association domain family 1A (RASSF1A), the apo-ptosis-related gene death-associated protein kinase (DAPK) andretinoic acid receptor b (RARb) by methylation-specific polymer-ase chain reaction. Aberrant promoter methylation in at least oneof five tumor suppressor genes was detected more frequently inthe serum DNA of silicosis patients with lung cancer than in thatof patients without it (P 5 0.006). Furthermore, the odds ratio ofhaving lung cancer was 9.77 (P 5 0.009) for those silicosis pa-tients with methylation of at least one gene. Extended exposureto silica (>30 years) was correlated with an increased methylationfrequency (P 5 0.017); however, methylation status did not cor-relate with age, smoking history or radiographic findings of sili-cosis. These results suggest that testing for aberrant promotermethylation of tumor suppressor genes using serum DNA mayfacilitate early detection of lung cancer in patients with silicosis.

Introduction

Occupational exposure to silica occurs in a large number of industries

and circumstances, including mines and stone quarries as well as inthe production of granite, ceramics, pottery and steel. In the USA, itis estimated that of more than one million workers occupationallyexposed to free crystalline silica dust each year, 59 000 will eventu-ally develop silicosis and $300 will die from the disease (WorldHealth Organization Fact sheet N238, 2000. http://www.who.int/mediacentre/factsheets/fs238/en/). Growing evidence indicates thatthe association between silicosis and lung cancer is causal (14). In1997, the International Agency for Research on Cancer concluded

that inhaled crystalline silica is a human lung carcinogen (5). Chanet al. (6) reported that 33 (2.3%) of 1490 workers diagnosed withsilicosis in Hong Kong died from lung cancer.

Lung cancer spreads gradually throughout the body and has a poorprognosis because it is largely unresponsive to chemotherapy. Only

those patients diagnosed at an early stage of the disease can be curedby complete resection. However, it is difficult to detect lung canceby chest radiography or computed tomography during the follow-uptreatment of patients with silicosis because of preexisting diffusepulmonary shadows (7,8). A reliable clinical marker for early detection of lung cancer in patients with silicosis is thus urgentlyneeded.

Epigenetic changes such as hypermethylation increasingly appeato play a role in carcinogenesis. DNA methylation is one form ofepigenetic variability in mammalian cells. As aberrant hypermethy-lation in the CpG-rich promoter regions of many tumor suppressorgenes interferes with transcription, hypermethylation may contributeto the development and progression of various cancers by abolishingtumor suppressor gene function (911). Aberrant hypermethylation oftumor suppressor genes has been observed in various specimens fromlung cancer patients, including serum, plasma, sputum, lavage fluid

and epithelial brushing (12,13). These findings raise the possibilitythat methylation analysis of such materials may be a useful tool for-cancer detection. A number of studies have reported increased methylation of several tumor suppressor genes in lung cancer cellsincluding the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) (14,15), p16INK4a (16), ras association domain family1A (RASSF1A) (17), apoptosis-associated genes such as deathassociated protein kinase (DAPK) (18) and retinoic acid receptob (RARb) (19); in contrast, methylation of these genes is rarely reportedin non-malignant lung tissue (14). We demonstrated previously thascreening for promoter methylation in these five genes using serum(20) and pleural fluid (21) DNA can be useful for early detection oflung cancer. Thus, detection of tumor suppressor gene promotehypermethylation in serum DNA may facilitate early detection of lungcancer in patients with silicosis. Here, we examined whether promoter

hypermethylation could identify lung cancer in a clinical setting bytesting the promoter methylation status of five tumor suppressor genein serum DNA from 78 silicosis patients with and without lung cancer

Materials and methods

Study population

The subjects included 78 patients with silicosis at Okayama Rosai Hospital(n 5 76) or Bizen City Yoshinaga Hospital (n 5 2) between 2004 and 2006and 20 healthy controls. Of the 78 patients with silicosis, 11 were diagnosedwith lung cancer. The 20 control subjects had no occupational history of silicaexposure and they were matched to the cases by gender, age and smokingstatus. The characteristics of the study population are summarized in Table IPatients with silicosis were defined as those who have occupational historiesfor silica exposure in the industries such as stone quarries, granite productionceramic and pottery industries and steel production and showed profusion rate

from one to four on radiographs based on International Labour Organizationclassification (22). Histological subtypes of lung cancers were based on WorldHealth Organization classification (23). The clinical stage of disease was as-sessed using the International Staging System (24).

Sample collection and DNA extraction

Peripheral blood samples (6 ml) were collected to investigate the methylationstatus of the serum DNA. The serum (2 ml) was isolated by centrifugation a3000 r.p.m. for 10 min and stored at 80C until use. Serum DNA was ex-tracted using a QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germanyaccording to the manufacturers instructions. We also examined methylationstatus of the tumor tissues obtained from surgical resection or autopsy. TumoDNA was extracted from formalin-fixed, paraffin-embedded lung cancer tis-sues using QIAamp DNA Mini Kit (Qiagen) according to the manufacturersinstructions. The researchers were unaware of each patients diagnosis

Abbreviations: CI, confidence interval; DAPK, death-associated protein kinase;MSP, methylation specific polymerase chain reaction; MGMT, O6-methylguanine-DNA methyltransferase; PCR, polymerase chain reaction; RARb, retinoic acidreceptor b; RASSF1A, ras association domain family 1A; SD, standard deviation.

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The institutional review board approved the protocols and written informedconsent was obtained from the subjects.

Methylation specific polymerase chain reaction

Sample DNA was treated with sodium bisulfite using a CpGenome DNAModification Kit (Intergen, Purchase, NY) as described previously (20,21).The primers used for MGMT, p16INK4a, RASSF1A, DAPK and RARb are de-scribed elsewhere (20,21). DNA from SBC-3 (25), a small-cell lung cancer cellline with promoter methylation of all tested genes, was used as a positivecontrol. The polymerase chain reaction (PCR) mixture contained 10 PCRbuffer [100 mM TrisHCl (pH 8.3), 500 mM KCl and 15 mM MgCl2], deox-ynucleotide triphosphates (each at 2.5 mM), 0.5 lM of each primer, 0.75 UHotstar Taq DNA polymerase (Qiagen) and 3 ll of bisulfite-modified DNA ina final volume of 30 ll. An initial denaturation step at 95C for 15 min wasfollowed by 50 cycles of denaturation at 9095C for 20 s, annealing at theappropriate temperature for 30 s and extension at 72C for 30 s with a finalextension at 72C for 10 min. After amplification, each product was electro-phoresed through a 2% agarose gel, stained with ethidium bromide and visu-alized under ultraviolet illumination. The presence of a band was defined asa methylation-positive result, even if it was faint. Each blood sample wasexamined in duplicate. Representative results of our methylation analysis areshown in supplementary Figure 1 (available at Carcinogenesis Online). Quan-titative analysis of the PCR products was conducted using Scion Image soft-ware (http://www.scioncorp.com). The value of the PCR signal of each samplewas calculated as the ratio; PCR signal of each sample to positive control. Thefinal value of the PCR signal was determined as mean value of the ratio in eachgroup.

Quantitative real-time PCR

Quantitative real-time PCR was also performed with locus-specific primersand dual-labeled fluorogenic probes for lung cancer tissue samples. Methyla-tion of p16INK4a and RASSF1A was examined using b-actin as the internalcontrol for DNA quantification. The DNA sequences of primers, which aredifferent from those used in above-mentioned methylation specific polymerasechain reaction (MSP), and probes for these genes were based on published data(26,27). PCR was set up in a reaction volume of 25 ll containing 1 Taqmanuniversal PCR master mix (Applied Biosystems, Foster City, CA), 500 nM ofeach primer, 150 nM of probe and 3 ll bisulfite-treated DNA samples. After aninitial denature step at 50C for 2 min and 95C for 15 min, 50 cycles of 15 s at94C and 1 min at 6064 C were followed. Amplified data were analyzedusing software developed by Applied Biosystems. The methylation ratio wasdefined as the ratio of the fluorescence emission intensity values for methylated

p16INK4a and RASSF1A to those ofb-actin multiplied by 1000.

Statistical analysis

For each patient, the methylation status of the five genes was scored as the totalnumber of methylated genes. The methylation score of those patients withsilicosis plus lung cancer was compared with those with silicosis alone using

a t-test with unequal variance. An unconditional logistic regression model wasapplied to estimate the odds ratios and 95% confidence intervals (CIs) for theoccurrence of lung cancer. Those patients without methylation were used asa referencegroup to estimatethe odds ratios for those patients with one or moremethylated genes. Crude and multivariate models were examined. The factorsconsidered in the multivariate model included age, gender, smoking status,radiological findings and the exposure period. A chi-square or Fishers exacttest was used to examine the distribution in categorical variables. Statisticalsignificance was defined as P , 0.05. All statistical analyses were conductedusing SPSS version 10 (SPSS, Chicago, IL).

Results

Methylation status of five genes and its significance in the detection oflung cancer

We determined the prevalence of MGMT, p16INK4a, RASSF1A, DAPKand RARb methylation in the serum DNA of 78 silicosis patients withor without lung cancer and in 20 healthy controls by MSP. Among thecontrols, methylation was detected at a frequency of 5.0% for MGMT,0.0% for p16INK4a, 0.0% for RASSF1A, 10.0% for DAPKand 5.0% for

RARb (Table II). Among the silicosis patients without lung cancer,methylation was detected at a frequency of 14.9% for MGMT, 3.0%for p16INK4a, 3.0% for RASSF1A, 11.9% for DAPK and 9.0% for

RARb. In contrast, among the silicosis patients with lung cancer,methylation was detected at a frequency of 36.4% for MGMT,

18.2% for p16INK4a

, 9.1% for RASSF1A, 18.2% for DAPK and 0.0%for RARb. The total number of methylations per patient in the fivegenes was 0.82 [standard deviation (SD), 0.603; 95% CI, 0.411.22]for those patients with silicosis plus lung cancer, which was higherthan that for the patients with silicosis alone (0.42; SD, 0.721; 95%CI, 0.240.59; P 5 0.066). Aberrant promoter methylation in at leastone of the five tumor suppressor genes was more frequent in silicosispatients with lung cancer (72.7%) than in those without it (29.9%;P 5 0.006). For those patients with silicosis and methylation in atleast one of the five genes, the sensitivity, specificity, positive pre-dictive values and negative predictive values for the diagnosis of lungcancer were 72.7, 70.1, 28.6 and 94.0%, respectively. Among fivepatients with stage I disease of lung cancer, four patients had meth-ylation in at least one of the five genes. To quantify these PCR results,methylation value was determined using Scion Image software. Meth-

ylation value was higher in silicosis patients with lung cancer thanthose without in four genes tested (Figure 1).Tumor tissues were obtained from 8 of 11 cases with lung cancer,

four from surgically resection and four from autopsy, and sufficientDNA was extracted in six of the eight cases. All the tumor tissuesshowed methylation in at least one gene, and in four of these six cases,methylation was also detected in serum DNA in at least one gene(supplementary Figure 2 is available at Carcinogenesis Online).

To validate our results of MSP, methylation of p16INK4a and RASS-F1A in the tumor tissues was evaluated using quantitative real-timePCR. Methylation of p16INK4a was detected in two cases and RASS-F1A methylation was detected in five cases. The p16INK4a methylationin two cases and RASSF1A methylation in three cases, detected byMSP, were confirmed by this quantitative PCR (supplementary Table1 is available at Carcinogenesis Online).

Methylation status and the likelihood of lung cancer

Given that the silicosis patients with lung cancer exhibited a higherfrequency of aberrant methylation than those without it, we analyzedthe methylation status and risk of lung cancer in patients with silico-sis. Table III shows the results of a crude logistic regression analysisof the correlation between methylation status and the risk of lungcancer. Silicosis patients with at least one methylated gene were6.26 (95% CI, 1.5126.07) times more likely to have lung cancer thanwere silicosis patients with no methylated genes (P 5 0.012). Toconsider the imbalance in the baseline characteristics, we conducteda similar analysis adjusting for age, gender, smoking status, radiologicfindings and the silica exposure period, which were considered asso-ciated with the risk of malignancy. After adjusting for these factors,

Table I. Patient characteristics

Healthycontrols

Silicosis withoutlung cancer

Silicosis withlung cancer

AgeMedian (range) 74 (6085) 71 (5186) 72 (5682)

SexMale/female 17/3 63/4 10/1

Smoking status

S/NS/unknown 14/6/0 47/15/5 9/2/0Exposure period (years)

Median (range) 0 35.5 (147) 33 (1040)0 20 30 22 4.30 40 7Unknown 5 0

Radiographic findingPR 1/2/3/4 11/26/9/21 4/5/0/2

HistologyAd/Sq/Sm 6/4/1

StageI/II/III/IV 5/0/3/3

S, smoker; NS, non-smoker; PR, profusion rate; Ad, adenocarcinoma; Sq,squamous cell carcinoma; Sm, small-cell carcinoma.

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silicosis patients with methylation in at least one gene were still 9.77(1.7853.74; P 5 0.009) times more likely to have lung cancer (TableIII). These results suggest that methylation of these five tumor sup-pressor genes is associated with lung cancer in patients with silicosis.

Methylation status and silicosis

The total number of methylations per patient in the five genes was0.47 (SD, 0.716; 95% CI, 0.310.64) in patients with silicosis, which

tended to be higher than in the healthy controls (0.2; SD, 0.523; 95%CI, 0.000.44; P 5 0.061). We next analyzed methylation status andthe risk of silicosis. Table IV shows the results of a crude logisticregression analysis of the correlation between methylation status andthe risk of silicosis. Patients with at least one methylated gene were3.17 (95% CI, 0.85511.78) times more likely to have silicosis thanwere patients with no methylation (P 5 0.084). We then tested forassociations between the methylation status of the five genes andvarious clinical variables. As shown in Table V, no statistically sig-nificant association between methylation status and clinical variablessuch as age, gender, smoking status or radiologic findings was ob-served; however, we found a statistically significant association be-tween methylation status and the silica exposure period (P 5 0.017).These results suggest that methylation of these five tumor suppressorgenes is associated with silica exposure and might be associated withsilicosis. Among the patients with long exposure to silica (.30 years),85.7% of the patients with lung cancer have at least one gene hyper-methylation compared with 37.5% of those without lung cancer(P 5 0.035) (Table VI).

The altered serum methylation status of two silicosis patients withlung cancer

We describe two cases that methylation status was examined twiceduring follow-up treatment for silicosis (supplementary Figure 3 isavailable at Carcinogenesis Online). Case 1 involved a 69-year-oldwoman who developed adenocarcinoma of the lung. DAPK methyla-tion was detected in her serum at the time of partial resection of theright upper lobe; however, it disappeared 15 months later. The DAPKmethylation was also detected in resected tumor tissue in the case

(sample 4 in supplementary Figure 2 is available at CarcinogenesisOnline). Case 2 involved an 82-year-old man who had received seg-mentectomy for squamous cell carcinoma of the lung. Although nomethylation was observed in his serum while he was disease free

MGMT methylation was detected in his serum at recurrence. Theseresults indicate that the methylated DNA in the serum of these patientwas released from the cancer cells and that methylated DNA in theserum of patients with silicosis may reflect undetected canceroulesions.

Discussion

It is well established that patients with silicosis are at increased riskfor lung cancer (14,28). In the current study, we examined the pro-moter methylation status of five tumor suppressor genes using serumDNA from 78 silicosis patients with or without lung cancer. Promoterhypermethylation in at least one of the five genes was significantlyincreased in silicosis patients with lung cancer than in those without itIn addition, among 11 patients with silicosis that developed lungcancer, eight (72.7%) showed methylation in at least one of the fivegenes tested. In a previous study of the same five genes, almost half ofthe patients (49.5%) with lung cancer had methylation in their serum(20). These results indicate that lung cancer may be associated with anincreased frequency of aberrant methylation if it is accompanied bysilicosis, although the present study should be interpreted carefullydue to the small sample size. We also examined the methylation statusof tumor tissues of these patients with lung cancer and demonstratedthat all the tumor tissues examined showed methylation in at least onegene. These results and the altered methylation profile in two de-scribed cases indicate that the methylated DNA in the serum was re-leased from the cancer cells, though an unexpected result was alsoshown that MGMT methylation was detected in serum from two patients even though tumors did not show the methylation. We have noclear evidences but speculate two possible explanations of the discrepancy: (i) the tumor DNAs were extracted from formalin-fixed, paraffinembedded samples and not from fresh-frozen tissues. So DNA piecesof MGMT sequences might be fragmented and (ii) the methylatedDNA in their serum might come from malignant or premalignanlesions in organs other than the lung. These should be clarified inthe future studies.

The increased incidence of DNA methylation in silicosis patientswith lung cancer compared with those without it motivated us toevaluate serum DNA methylation as a marker for lung cancer. Ouranalysis showed that patients with serum DNA methylation were $10times more likely to have lung cancer. These findings support the useof DNA methylation status as a marker for lung cancer detection insilicosis patients. Early detection of lung cancer in patients with silicosis is a serious clinical problem (14,8,28); thus, a reliable markerfor rapid and accurate diagnosis is urgently needed. In the currenstudy, methylation in serum DNA was detected even in patients withearly stage of lung cancer. These results suggest that aberrant pro-moter methylation of tumor suppressor genes in serum DNA may bea valuable marker for the early detection of lung cancer during thefollow-up treatment of patients with silicosis. Although further eval-uation is essential, these results also suggest that serum DNA meth-

ylation may be worth examining routinely, particularly beforeinvasive procedures.

Before using serum DNA methylation as a marker for lung cancerduring the follow-up of patients with silicosis, several issues must beconsidered. In the present study, we found that the sensitivity, spec-ificity and positive predictive value of methylation in one or moregenes for the diagnosis of lung cancer were 72.7, 70.1 and 28.6%respectively. These results may not be satisfactory for clinical purposes. One strategy for improving the specificity and sensitivity of test-ing for lung cancer during follow-up treatment for silicosis is to usea larger number of genes or to apply a quantitative methylation assay(27,29,30). The sensitivity of the Taqman method is reported to be10-fold higher than conventional qualitative MSP (31). In this studyquantitative PCR was performed for limited samples of lung cancer

Table II. Frequency of methylation in five genes

Gene No. of patients (%)

Healthycontrols,n 5 20

Silicosis withoutlung cancer,n 5 67

Silicosis withlung cancer,n 5 11

MGMT 1 (5.0) 10 (14.9) 4 (36.4)p16INK4a 0 (0.0) 2 (3.0) 2 (18.2)

RASSF1A 0 (0.0) 2 (3.0) 1 (9.1)DAPK 2 (10.0) 8 (11.9) 2 (18.2)RARb 1 (5.0) 6 (9.0) 0 (0.0)

Fig. 1. Methylation value of five genes. Methylation value was determinedas mean value of the ratio: methylated band/positive control quantified withScion Image.

Promoter hypermethylation in silicosi

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tissues and we found inconsistency between results by non-quantitativeMSP and those by quantitative real-time PCR, such as RASSF1A meth-ylation in sample 2. These findings might indicate the higher sensitiv-ity of the Taqman method compared with non-quantitative one.Another strategy is to search for the best combination of genes touse for methylation analysis with or without other diagnostic tests,including low-dose spiral computed tomography (3234). Additionalstudies are warranted.

It is of note that a statistically significant association was detectedbetween methylation status and the silica exposure period, althoughno direct association between methylation status and silicosis wasobserved. A previous study using an animal model demonstrated thatexpression of E-cadherin was significantly reduced by silica-induced

chronic inflammation because of promoter hypermethylation (35).Aberrant promoter methylation has also been reported in precancer-ous lesions, such as in the dysplasias of patients with lung cancer (36).Our finding that long-term silica exposure is correlated with an in-creased frequency of methylation suggests that silicosis may be a so-

called precancerous lesion (1,37). And methylation status did notcorrelate with age, smoking history or radiographic findings of sili-cosis. There are numerous reports that show age-related methylationin a subset of genes (38,39) or association between smoking andmethylation in the lung (4042). We have no clear explanation whymethylation status did not correlate with age or smoking history inthis study, but it might be due to our non-quantitative method formethylation analysis.

We found that the total number of methylations per patient in fivegenes tended to be higher in patients with silicosis than in healthycontrols. These results support the notion that silicosis patients withmethylation in some tumor suppressor genes may already be in anearly stage of lung cancer development or that some proportion ofthese patients already has cancerous lesions. Thus, it will be of greatinterest to investigate whether the methylation-positive patients withsilicosis develop lung cancer in the near future.

In conclusion, we showed that aberrant tumor suppressor gene pro-moter methylation was more frequent in the serum DNA of silicosispatients with lung cancer than in those without it. Our results suggestthat testing for aberrant promoter methylation of these genes in serumDNA may aid in the early detection of lung cancer in patients withsilicosis. Additional studies are warranted to confirm the value of suchanalyses and to determine the most informative combination of genes.

Funding

This research is a part of the research and development and the dis-semination projects related to he 13 fields of occupational injuries andillnesses of the Japan Labour Health and Welfare Organization.

Table V. Univariate analysis of clinical variables and methylation status

Variable n Methylation status P valuea

Negative Positiveb

Age,70 39 25 14 0.51!70 59 42 17

GenderMale 90 61 29 0.999Female 8 6 2

Smoking statusNon-smoker 23 16 7 0.999Smoker 70 48 22

Radiologic findings (PR)0 20 17 3 0.1511, 2 46 31 153, 4 32 19 13

Silica exposure period (years)0 20 17 3 0.01730 26 21 5.30 47 26 21

PR, profusion rate.aChi-square test.b

Patients with at least one gene hypermethylation.

Table III. Methylation status and risk of lung cancer in patients with pulmonary silicosis

Silicosis withoutlung cancer

Silicosis withlung cancer

Model 1 Model 2

Odds ratio 95% CI P valuea

Odds ratio 95% CI P valuea

No. of methylations0 47 3 1 1.00!1 20 8 6.26 1.50526.071 0.012 9.77 1.77653.74 0.009

Age (,70 to !70) 1.45 0.3236.482 0.630

Sex (female to male) 0.86 0.05613.329 0.914Smoking status (never to smoker) 1.17 0.1608.465 0.88Radiologic finding (0 versus 1, 2 versus 3, 4) 4.89 0.78730.346 0.089Exposure period to silica (30 versus .30) 0.38 0.0702.099 0.269

aLogistic regression model.

Table IV. Methylation status and the risk of silicosis

No. ofmethylations

Healthycontrols

Silicosispatients

Oddsratio

95% CI P valuea

0 17 50 1.00!1 3 28 3.17 0.85511.78 0.084

aLogistic regression model.

Table VI. Comparison of methylation frequency in patients with long silicaexpose (.30 years)

Gene No. of patients (%) P valuea

Without lungcancer, n 5 40

With lungcancer, n 5 7

MGMT 8 (20.0) 2 (28.6)p16INK4a 2 (5.0) 2 (28.6)RASSF1A 1 (2.5) 0 (0.0)DAPK 6 (15.0) 2 (28.6)RARb 4 (10.0) 0 (0.0)At least one gene 15 (37.5) 6 (85.7) 0.035

aChi-square test.

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Acknowledgements

We wish to thank Drs Kenji Ogino, Toshiyuki Kozuki, Eiki Ichihara, KatsuyukiHotta and Shinichi Toyooka for their support and comments on our analysis.

Conflict of Interest Statement: None declared.

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Received February 6, 2008; revised July 9, 2008; accepted July 10, 2008

Promoter hypermethylation in silicosi

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Aberrant Promoter Hypermethylation in Serum DNA From Patients With Silicosis

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