J Cancer Res Clin Oncol

DOI 10.1007/s00432-009-0614-4

ORIGINAL PAPER

RASSF1A, APC, ESR1, ABCB1 and HOXC9, but not p16INK4A, DAPK1, PTEN and MT1G genes were frequently methylated in the stage I non-small cell lung cancer in China

Qiang Lin · Junfeng Geng · Kelong Ma · Jian Yu · Jinfeng Sun · Zhenya Shen · Guoliang Bao · Yinming Chen · Hongyu Zhang · Yinghua He · Xiaoying Luo · Xu Feng · Jingde Zhu

Received: 18 March 2009 / Accepted: 25 May 2009© Springer-Verlag 2009

AbstractPurpose To identify the DNA methylation biomarkers forthe detection of the stage I non-small cell lung cancer(NSCLC).Materials and methods The methylated state ofp16INK4A, ESR1, HOX9, RASSF1A, DAPK1, PTEN,ABCB1, MGMT, APC and MT1G genes that have been

reported frequently methylated in lung cancer was deter-mined using methylation-speciWc PCR in four lung cancercell lines, 124 cancer tissues of the stage I NSCLC and 26non-cancerous disease tissues.Result The RASSF1A (53/124, 42.74%), APC (49/123,39.52%), ESR1 (37/124, 29.84%), ABCB1 (31/124,24.19%, MT1G (25/124, 20.16%) and HOXC9 (17/124,13.71%) genes were more frequently methylated in the lungtissue from the stage I NSCLC than the non-cancerouslesion patients (2/26, 7.69%, P < 0.01; 2/26, 7.69%,P < 0.01; 2/26, 7.69%, P < 0.05; 1/26, 3.85% P < 0.01;0/26 0%, P value: <0.01; 0/26, 0%, P < 0.05, respectively).p16INK4A was methylated in 28/124 (22.56%) of cancertissues and 2/26 (7.69%) of non-cancerous tissues (P value>0.05). No signiWcant association between the methylatedstate of the genes and the smoking, age or the pathologictypes (squamous carcinoma, adenoma and the mixed types)was found. However, p16INK4A methylation was morefrequently detected in the male (23/80, 28.75%) than thefemale (5/44, 11.36%, P > 0.05) patients. MGMT wasbarely methylated: 1/67, 1.49%), while DAPK1 and PTENwere not at all methylated in the cancer groups.Conclusions Methylation analysis in tissue of RASSF1A,APC, ESR1, ABCB1 and HOXC9 genes conWrmed 79.8%of the existing diagnosis for the stage I NSCLC at speciWc-ity: 73.1%. The insuYciency of predicting disease onsetin China, using the previously recommended targets(MGMT, DAPK1 and PTEN) in the United States reXectsa potential disease disparity between these two popula-tions. Alternatively, methylated state of this set of genesmay be more speciWc to the late rather than the early stageof NSCLC.

Keywords DNA methylation · Methylation-speciWc PCR · Non-small cell lung cancer · Stage I · Diagnosis

Q. Lin, J. Geng, K. Ma and J. Yu have contributed equally to this work.

Q. Lin · J. GengDepartment of General Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiaotong University, 200030 Shanghai, China

K. Ma · J. Yu · J. Sun · Y. Chen · H. Zhang · Y. He · X. Luo · J. Zhu (&)Cancer Epigenetics and Gene Therapy Program, The State-Key Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiaotong University, LN 2200/25, Xietu Road, 200032 Shanghai, Chinae-mail: zhujingde@gmail.comURL: http://www.shsci.org/eyjz3.htm

Z. ShenThe First Hospital AYliated to Suzhou University, 215006 Suzhou, Jiangsu, China

G. BaoDivision of Basic Research, Shanghai Chest Hospital, Shanghai Jiaotong University, 200030 Shanghai, China

X. FengDepartment of Urology, Guangxi Medical University, Tumor Hospital, Nanning 530022, China

J. ZhuCancer Epigenetics Laboratory, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China

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AbbreviationsAPC Adenomatous polyposis coliDAPK1 Death-associated protein kinase 1p16INK4A Cyclin-dependent kinase inhibitor 2APTEN Phosphatase and tensin homologRASSF1A RAS association family 1AABCB1 ATP-binding cassette, subfamily B, member 1MGMT O(6)-methylguanine-DNA-methyltransferaseESR1 Estrogen receptor alphaHOXC9 Homeobox C9MT1G Metallothionein 1G

Introduction

Lung cancer is the leading cause of cancer death in menand women in the United States and Western Europe. Over160,000 Americans die of this disease every year. The5-year survival rate is 15%—signiWcantly lower than thatof other major cancers (Jemal et al. 2007). Owing to the rapidindustrialization and increase in the smoking consumptionin society, lung cancer presents as the number 1 cancer typeof threats in China (Song et al. 2008) (Globoscan 2002,http://www-dep.iarc.fr/). The non-small cell lung cancer(NSCLC) is the most common type, accounting for 85–90% of the total cases (Molina et al. 2008) and its current 5-year survival rate varies within the range from 2 to 47% fordiVerent stages and diVerent pathological classes (squa-mous carcinoma versus adenocarcinoma) (Risch and Plass2008). Its early detection is crucial to the prolonged sur-vival of the disease. Imaging and cytology-based screeningstrategies have been employed for its early detection formany years (Anglim et al. 2008), but little progress, judgedfrom whether and how much in the reduction of mortality,has been made. A better understanding of both genetic andepigenetic mechanisms underpinned with the disease initia-tion and progression should aid in the development of thebetter diagnosis, clinical management and outcome predic-tion of this devastating malignancy. DNA hypermethyla-tion is recognized as an important mechanism for tumorsuppressor gene inactivation that underlies the tumor-spe-ciWc biological proWle, as well as the promising biomarkersfor the early detection of various types of cancer, includingNSCLC.

Altered DNA methylation is a common hallmark forhuman cancer (Esteller 2008; Zhu and Yao 2009), reXect-ing the epigenetic disturbance that is likely to contribute tothe changed patterns of the gene expression and, therefore,the unique phenotypes of cancer cells. The hypermethy-lated state of the promoter CGI of the genes, encoding theproteins that negatively regulate the cell proliferation andmaintain the genome stability, such as tumor suppressorand DNA-repairing mediators, correlates well with the

transcriptional silencing state of their expression in cancercells. Therefore, this set of the targets has been intensivelytested for their utility in cancer diagnosis and prognosis.Some of them have been reported speciWc to NSCLC fromthe studies in the patients outside of the mainland of China.The environmental factors that vary drastically with coun-tries exert a great impact to both carcinogenesis and diseasecharacteristics, which is believed to eVect chieXy on theepigenetic makeup of cell. Aiming to identify the valuableDNA methylation biomarkers for the early detection of thistype of cancer, we have methylation proWled, in the lung tis-sue of cohort of the stage I non-small cell lung cancer (124diseases) and a cohort of the non-cancerous lesion patients(24 cases), ten methylated genes “informative: for NSCLCthe United States: APC (Yanagawa et al. 2003), DAPK1(Liu et al. 2007), p16INK4A (Nakata et al. 2006), PTEN(Marsit et al. 2005), RASSF1A (Nakata et al. 2006),ABCB1 (Anglim et al. 2008), MGMT (Liu et al. 2006),ESR1 (Tsou et al. 2005), HOXC9 (Anglim et al. 2008) andMT1G (Anglim et al. 2008). Although a signiWcant associa-tion of the methylated RASSF1A (53/124, 42.74%), APC(49/123, 39.52%), ESR1 (37/124, 29.84%), ABCB1 (31/124, 24.19%, MT1G [25/124, 20.16%) and HOXC9 (17/124, 13.71%)] with the stage I disease was found, theremaining were either found little (p16INK4A) or no associ-ation with the stage I NSCLC (MGMT, DAPK1 and PTEN).

Materials and methods

Sample collection and genome DNA isolation

Lung cancer cell lines A549 (carcinoma, ATCC No: CCL-185TM),NCI-H460 (large cell cancer, ATCC No: HTB-177TM), NCI-H446 (small cell lung cancer, ATCC No:HTB-171TM) and SPC-A-1 (lung adenoma, Cell Bank inShanghai No:TCHu 53) were cultured in L-DMEM mediumcontaining 10% FBS at 37°C in a 95% air, 5% CO2 humid-iWed incubator to the log phase of proliferation stage beforecell collection. BJ cell line (human Wbroblast from skin,ATCC No: CRL-2522TM) was used as the control for thenormal human cells.

With the informed consents obtained from all eligiblepatients and approval of the ethics committee, 124 tissuesamples from the Stage I NSCLC and 26 from the non-can-cerous respiratory diseases were collected from ShanghaiChest Hospital, China (Table 1 for the patient proWles,including the cell types, ages, sex and smoking etc). Thetumor-node-metastasis (TNM) staging/classiWcation of thepatient cohorts were performed according to the WHO clas-siWcation (Sobin and Wittekind 2002; Travis et al. 2004).The genomic DNA was prepared from both cell lines andthe frozen clinical tissues using conventional proteinase

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J Cancer Res Clin Oncol

K/organic extraction methods as previous described (Yuet al. 2007, 2002)

BisulWte treatment of DNA and methylation-speciWc PCR (MSP)

The primer pairs for MSP analysis were directly taken fromthe literatures for APC (Esteller et al. 2000), CDKN2(Rosas et al. 2001), DAPK1 (Zochbauer-Muller et al.2001), PTEN (Yu et al. 2002), ESR1 (Wang et al. 2008)and RASSF1A (Lo et al. 2001) or designed for ABCB1,MGMT, HOXC9 and MT1G with the assistance of tworelevant software (http://www.ebi.ac.uk/emboss/cpgplot/index.html) and (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Table 2). The bisulfate conversion andPCR analyses were done as described previously (Yu et al.2007; Yu et al. 2002), followed by sequencing validation ofthe representative PCR products that have been T cloned.

Statistics

The association between gene methylation and clinicalpathological variables was statistically tested. The hyper-

methylation frequency with a 95% conWdence interval(95% CI; � = 0.05) was calculated using SPSS software.The hypermethylated incidence in non-small cell lung can-cer versus the respiratory system lesion patients was com-pleted by a 2X2 Fisher’s exact test and tested by the Rstatistical package. The receiver operating characteristics ofboth speciWcity and sensitivity of the sets was constructedas previously described (Yu et al. 2007).

Results

MSP proWling in lung cancer cell lines and the normal human cell line

Established cancer cell lines are expected to share the mostbiological features and the underlying molecular mecha-nisms, including the DNA methylation with the cancer insitu. Therefore, methylation proWling by MSP was carriedout in four lung cancer cell lines: A549 (lung carcinoma),NCI-H460 (large cell carcinoma), NCI- H446 (non-smallcell carcinoma) and SPC-A-1 (lung adenoma), the identityof the PCR products was conWrmed by sequencing. BJ

Table 1 Clinical proWle of the lung cancer patients and controls

Non-small cell lung cancer cases (n = 124)

Non-cancerous lung lesions (n = 26)

Gender

Female 44 7

Male 80 19

31–40 3 0

41–50 12 5

51–60 42 10

61–70 43 6

70– 24 5

Range 32–79 42–75

Median 61.13 59.48

Stage category I 124

Squamous 38

Adenocarcinoma 70

Adenosquamous 11

Others 5

Pulmonary tuberculosis 6

Bronchiectasis 4

Pulmonary abscess 5

Organizing pneumonia 2

Pulmonary sclerosing hemangioma 3

Pulmonary giant lymph node hyperplasia 1

Pulmonary hamartoma 2

Pulmonary sequestration 1

Pulmonary inXammatory pseudotumor 2

a According to the World Health Organization Guidelines for the conduct of tobacco-smoking sur-veys among health professionals (WHO 1984)

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J Cancer Res Clin Oncol

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J Cancer Res Clin Oncol

human Wbroblast cell line, as the control for the normaldiploid cells, was found in a full unmethylated state of eachof all the ten genes analyzed (Fig. 1 and data not shown).As shown in Table 3 and Fig. 1, APC was fully hyperme-thylated in both A549 and SPC-A-1 and heterozygouslymethylated in both NCI-H460 and NCI- H446 cell lines.RASSF1A was fully hypermethylated in both A549 andNCI-H446 cell lines, but unmethylated in both NCI-H460and SPC-A-1 cell lines. ESR1 was heterozygously methyl-ated in both A549 and SPC-A-1, but unmethylated in bothNCI-H460 and NCI- H446 cell lines. p16INK4A was het-erozygously methylated in both NCI-H460 and SPC-A-1cancer cell lines. PTEN was heterozygously methylated inSPC-A-1, but was unmethylated in A549, NCI-H460 andNCI- H446 cancer cell lines. ABCB1 was heterozygouslymethylated in both A549 and SPC-A-1 cancer cell lines,but unmethylated in both NCI-H460 and NCI-H446 cancercell lines. MT1G was heterozygously hypermethylated inboth NCI-H460 and SPC-A-1, but unmethylated in bothA549 and NCI- H446 cancer cell lines. HOXC9 was het-erozygously methylated in NCI-H446, while unmethylatedin A549, NCI- H460 and SPC-A-1 cancer cell lines. BothDAPK1 and MGMT were unmethylated in all cancer celllines.

MSP proWling in NSCLC and non-cancerous lesion control groups

We MSP proWled each promoter CGI in the cancer tissuesfrom a subset of the stage I NSCLC patient cohort (67cases) as well as the lung tissues of a subset of the non-can-cerous disease cohort (13 cases) (Table 1). The following,then, were proWled in the remaining cases of both NSCLCand control cohorts: RASSF1A, APC, ABCB1, MT1G,ESR1 and HOXC9.

Death-associated protein kinase 1 (DAPK1) functionsas a positive mediator of apoptosis and associated withAlzheimer’s disease. DAPK1 gene promoter methyla-tion frequency in these tumors was 32.8% (40/122) anddid not diVer according to the patients’ smoking status,tumor histology or tumor stage (Liu et al. 2007). DAPK1was unmethylated in all four lung cancer cell lines(Fig. 1; Table 3) as well as in both the cancerous andnon-cancerous tissues (Fig. 2; Table 4).The tumor sup-pressor gene, PTEN encodes a lipid phosphatase thatnegatively regulates the phosphatidylinositol 3-kinase/AKT cell survival pathway, underlying the cell growthand invasion. Methylation of PTEN was previouslyfound in 26% (39/151 cases) of NSCLC (Marsit et al.2005), but was unmethylated in the cancer tissues(Table 4; Fig. 2).

MGMT encodes a DNA repair protein O6-methylgua-nine-DNA methyltransferase that removes alkyl adducts

from the O6 position of guanine. Its inactivation by the pro-mote CGI methylation was Wrst reported in colo-rectal can-cer, mechanistically underlying the increase in G–Amutation (Esteller et al. 1999). It was also methylated ashigh as 30.3% (27/126 cases) in NSCLC (Liu et al. 2006).However, it was only methylated in 1 of 67 cases (1.49%)of the cancer tissues (Table 4).

It has been previously established that transcriptionalsilencing of the tumor suppressor gene, RASSF1A was fre-quently mediated by the promoter CGI methylation in thehuman cancer (Lo et al. 2001), including NSCLC (Marsitet al. 2005) where 71 of 146 cases were reported hyperme-thylated. In this study, it was methylated at a compatiblefrequency: in the previous study in the patient cohort in theUnited States, in 53 of 124 cases (42.74) of cancer tissuesand only 2 of 26 cases the non-cancerous tissues (7.69%),displaying a signiWcant cancer state speciWcity (P < 0.01).Similarly, the promoter CpG island (CGI) hypermethyla-tion/transcription silencing of APC was Wrst reported incolo-rectal cancer (Esteller et al. 2000) and then in lungcancer. For instance, the increased level of DNA methyla-tion at the APC promoter CGI in the non-small cancer tis-sues was found in 37% (28/75) of the cases tested(Yanagawa et al. 2003). In this study, the methylated APCwas detected in 49 of 124 cases (39.52%) of cancer tissuesand 2 of 26 cases (7.69%) of the non-cancerous controls,showing a signiWcant cancer state association (P < 0.01).ESR1 (short for estrogen receptor alpha) encodes the mainmediator of estrogen eVect in breast epithelia and has alsobeen shown to be activated by epidermal growth factor(EGF), methylation of which was found in 43% (3/7 cases)of lung adenocarcinoma tissue (Tsou et al. 2005). In thisstudy, ESR1 methylation was also speciWc to the stage INSCLC (P value <0.05) is, in 37 of 124 cases (29.84%),and in 2 of 26 cases (7.69%) of the non-cancerous tissues.ABCB1, short for ATP-binding cassette, subfamily B,member 1, encodes a protein that actively pumps the smallmolecule out from the cell. Its hypermethylated state hasbeen frequently reported in various types of human cancer,including the squamous cell lung cancer where it was meth-ylated in 100% cases in a small cohort (Anglim et al. 2008).We found that it was methylated in 31 of 124 cancercases (24.19%) and in 1 of 26 cases (3.69%) of the non-cancerous tissues, showing a tight cancer state association(P < 0.001).

MT1G encodes a key member of the metallothioneinprotein, loss of which expression has been regarded instru-mental to cancer formation and progression (Ferrario et al.2008). The hypermethylated state of this gene was found in95% cases of squamous cell lung cancer (Anglim et al.2008). However, its methylated state was detected in20.16% (25/124 cases and in none of 26 non-cancerouscases, exhibiting a clear speciWcity to the stage I disease

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Fig. 1 The MSP proWle and sequencing veriWcation. The MSP proWleand sequencing veriWcation of both methylated and unmethylated tar-geted regions of the ten informative genes in each of four lung cancercell lines: A549, NCL-H460, NCL-H446 and SPC-A-1. Both the elec-trophoretic patterns of the representative MSP data and the sequencingveriWcation are shown. SssI is the positive control with the DNA of the

normal liver tissue in vitro methylation modiWed. BJ is the bisulfateDNA from BJ, a normal Wbroblast cell line. H2O the no templatecontrol. The target identity of each panel is indicated. The wild-typesequence is aligned with the sequences of the T-vector cloned with therepresentative PCR product

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(P < 0.01). HOXC9, encoding C9 member of the homeo-box superfamily, was found hypermethylated in the squa-mous lung cancer frequently: 66%(Anglim et al. 2008).Its methylated state was found in 17 of 124 cancer cases(13.71%) and in none of 26 non-cancerous tissue controlshowing a signiWcant cancer state association.

p16INK4A is a classic tumor suppressor, frequentlymethylated and lost in function in most types of cancerstudied, including lung cancer. For instance, its methylatedstate was reported in 49 of 224 cases (21.9%) of NSCLC(Nakata et al. 2006). It was hypermethylated in 28 of 124cases (22.58%) in cancer tissues. But, its methylated statewas also found in 2 of 26 cases (7.69%) of the non-cancer-ous tissues and its cancer state association fails to surviveby the stringent statistic test (Table 4). A plausible explana-tion for this observation may suggest that the methylation

of p16INK4A may be speciWc to the premalignant state ofthe cancer, as proposed from the correlation of its methyla-tion in the sputum DNA with the late onset of lung cancer(Palmisano et al. 2000).

Discussion

DNA methylation is a promising biomarker for cancerdetection (Esteller 2008; Zhu and Yao 2009). DNA methyl-ation proWle underlines the gene expression pattern for thecancerous behaviors at the present time and reXects the pasthistory of the interaction between the epigenomic interfaceof cells and the environment factors (Zhu and Yao 2009).Its value as the biomarker for cancer detection, especiallyfor the early stage of the disease has been highly suggested.

Table 3 Methylation proWle from lung cancer cell lines

1 in the white background, the unmethylated in both alleles, 2 in gray background one allele methylated and the other unmethylated, and 3 in thedark background methylated in both alleles

A549 NCI-H460 NCI-H446 SPC-A-1 BJ

RASSF1A 3 1 3 1 1

APC 3 2 2 3 1

ESR1 2 1 1 2 1

ABCB1 2 1 1 2 1

MT1G 1 2 1 2 1

HOXC9 1 1 2 1 1

p16INK4A 1 2 1 2 1

MGMT 1 1 1 1 1

DAPK1 1 1 1 1 1

PTEN 1 1 1 2 1

Gene symbolCell line

Fig. 2 The occurrence of the methylated genes in non-small cell lung cancer and non-cancer-ous disease control. The methyl-ation frequency (%; Y axis) of each gene (X axis) in NSCLC patients (column 2, Table 4) and non-cancerous disease control (column 3, Table 4) was plotted. The hypermethylation frequency with a 95% conWdence interval (� = 0.05) was calculated and indicated. P value for each gene is presented as vertical line in the plot. The positions of P values <0.01 and <0.05 in the plot are shown

42.74

39.52

24.19

20.16

29.84

13.71

22.58

7.69 7.69

3.85

0

7.69

0

7.69

0

5

10

15

20

25

30

35

40

45

50

RASSF1A APC ABCB1 MT1G ESR1 HOXC9 p16INK4A

Met

hyla

tion

freq

uenc

y (%

)

Gene symbol

NSCLC(n=124)

Non-cancerous disease(n=26)

p>0.05p<0.05p<0.01

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To determine the value of the set of the methylated genesthat are “informative” for lung cancer in the United States(Table 5), both the stage I NSCLC (124 cases) and the non-cancerous lesion patient groups in China were recruited forMSP analysis.

RASSF1A, APC, ESR1, ABCB1, MT1G and HOXC9were found methylated at a frequency no less than 13.71%(17 of 124 cases) of the stage I disease and no more than7.69% (2 of 26 cases) of the non-cancerous tissues(Table 3), exhibiting a signiWcant association with the stageI NSCLC. A comparable frequency of the methylated formsof both RASSF1A and APC were detected, while the meth-ylated state other four informative genes (ESR1, ABCB1,MT1G and HOXC9) was found at a signiWcantly lowerincidence in this study than that in the previous reports(Table 4). This may indicate that the methylation of thelater four genes may more speciWc to the late stage ofdisease. No signiWcant association of the methylated stateof any single target or six targets in combination with the

age, gender, cell types and smoking habit (Table 1) hasbeen identiWed. The p16INK4A methylation seems moretightly associated with the male (23/80, 28.75%) thanfemale (5/44, 11.36%, P < 0.05), the implication of thisobservation to the clinical management of this diseaseremains to be assessed.

Methylation proWling of RASSF1A, APC, ESR1, HOXC9 and ABCB1, conWrms 79.8% (99/124 cases) of the existing diagnosis of the stage I NSCLC at a speciWcity of 73.1%

Assaying a single gene conWrmed no more than 43.74%(RASSF1A) of the stage I cancer (Tables 4, 5). However,the methylation analysis of multiple genes that werehypermethylated in the cancer at a statistically signiW-cantly higher frequency than the non-cancerous controls(4 genes with P < 0.01: RASSF1A, APC, ABCB1 andMT1G and 2 gene: ESR1 and HOXC9, with P < 0.05)(Fig. 2; Table 4) should conWrm more cases by the existing

Table 4 MSP proWles of stage I non-small cell lung cancer and controls

Gene symbol

Non-small cell lung cancer/frequency (%) (n = 124)

Non- cancerous disease(s)/frequency (%) (n = 26)

Fisher exact test, right tail P value

RASSF1A 53/42.74 2/7.69 <0.01

APC 49/39.52 2/7.69 <0.01

ABCB1 16/24.19 1/3.85 <0.01

MT1G 25/20.16 0 <0.01

ESR1 37/29.84 2/7.69 <0.05

HOXC9 17/13.71 0 <0.05

P16INK4A 28/22.58 2/7.69 >0.05

(n = 67) (n = 14)

MGMT 1/1.49 0 >0.05

DAPK1 0 0 1

PTEN 0 0 1

Table 5 Receiver operating characteristics of the informative sets for non-small cell lung cancer detection

Both sensitivity (%), TP/(TP + FN) (column 5, Table 5) and speciWcity (%), TN/(TN + FP) (column 6, Table 5) of each gene sets were calculatedand plotted

TP true positive, FN false negative, FP false positive, TN true negative, NB inclusion of MT1G does not improve the detectability and thereforeeliminated

No. Gene TP/FN (124) FP/TN (26) Sensitivity (%) SpeciWcity (%)

TP/(TP + FN) TN/(TN + FP)

1 RASSF1A 53/71 2/24 42.7 92.3

2 RASSF1A APC 79/45 4/22 63.7 84.6

3 RASSF1A APC ESR1 93/31 6/20 75.0 76.9

4 RASSF1A APC ESR1 HOXC9 97/27 6/20 78.2 76.9

5 RASSF1A APC ESR1 HOXC9 ABCB1 99/25 7/19 79.8 73.1

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diagnosis. However, four genes in this set were alsomethylated at a frequency no more than 2 of 26 cases(7.69%) of the non-cancerous control group, inclusion ofwhich as the informative sets of the methylated targetsfor cancer detection would reduce the speciWcity for thecancer detection. The receiver operating characteristic(ROC) of both speciWcity and sensitivity was constructedfor the sets consisting of two up to Wve genes(RASSF1A, APC, ESR1, ABCB1 and HOXC9) (Fig. 3;Table 5). The sensitivity was 42.7.3% (53/124) forRASSF1A alone, 63.7% (79/124) for RASSF1A andAPC, 75% (93/124) for RASSF1A, APC, ESR1 andHOXC9, 78.2% (97/124) as well as 79.2%(99/124) forRASSF1A, APC, ESR1, HOXC9 and ABCB1. It wasnoticed that a further recruitment of MT1G would notimprove the detectability of the set. As expected, thespeciWcity by methylation proWling to detect the stage 1disease was reduced from 92.3, 84.6, 76.9, 76/9 and73.1% for the sets ranging from one to Wve targets,respectively. The clinical utility of the suggested set(s) ofthe targets for the stage 1 disease that are recommendedfrom this study remains to be evaluated in large cohorts ofNSCLC in a near future.

The possible disease disparity between the NSCLC in China and its US counterparts

The environmental factors that vary drastically with coun-tries exert a great impact to both carcinogenesis and theclinical proWle of the disease, which is believed to eVectchieXy on the epigenetic makeup of cell. Three of ten genesthat have been reported frequently methylated in NSCLCpatients in the United States were either unmethylated[DAPK1 (Liu et al. 2007) and PTEN (Marsit et al. 2005)],or barely methylated [MGMT (Liu et al. 2006)] (Table 6),implicating the disease disparity in the NSCLC populationsbetween the mainland of China and the United States.

Alternatively, the methylated state of these three genes maynot be associated with the early stage NSCLC.

This report has provided a good set of the candidate tar-gets for methylation analysis to detect the stage I NSCLC inChina. It is desirable to further validate this set of informa-tive targets in larger patient cohorts as well as assess theirmethylation status in the plasma DNA of the same clinicalsetting.

Acknowledgments This work is supported to J. Zhu by ShanghaiScience Foundation grant: 07JC14074, National Science Foundationgrants 30570850 and 90919024, National Research Program forBasic Research of China grants 2004CB518804, 2009CB825606 and2009CB825607, European 6th program grant LSHB-CT-2005-019067and supported to J. Yu by National Science Foundation grant:30872963. Thanks are due to Q. Li for the statistic analysis.

Fig. 3 Receiver operating characteristics of the informative sets fornon-small cell lung cancer detection. Both the sensitivity (%), TP/(TP + FN) (column 5, Table 5) and speciWcity (%),TN/(TN + FP)(column 6, Table 5) of each gene sets were calculated and plotted.TPtrue positive, FN false negative, FP false positive, TN true negative.Inclusion of MT1G does not improve the detectability and thereforeeliminated

1

2

3

4

5

60.0

70.0

80.0

90.0

100.0

40.0 50.0 60.0 70.0 80.0 90.0

Spe

cific

ity (

%)

Sensitivity (%)

Table 6 Methylation frequency in the literature and in this study

Gene symbol Cases/frequency (%) Specimen In this study (%) References

RASSF1A 71/146 (49%) Non-small cell lung cancer 42.74 (Marsit et al. 2006)

APC 28/75 (37%) Non-small cell lung cancer 39.52 (Yanagawa et al. 2003)

ABCB1 100% Squamous cell lung cancer 24.19 (Anglim et al. 2008)

MT1G 95% Squamous cell lung cancer 20.16 (Anglim et al. 2008)

ESR1 3/7 (43%) Lung adenocarcinoma 29.84% (Tsou et al. 2005)

HOXC9 66% Squamous cell lung cancer 13.71% (Anglim et al. 2008)

p16INK4A 49/224 (21.9%) Non-small cell lung cancer 22.58% (Nakata et al. 2006)

MGMT 37/122 (30.3%) Non-small cell lung cancer 1.49% (Liu et al. 2006)

DAPK1 40/122 (32.8%) Non-small cell lung cancer 0.00% (Liu et al. 2007)

PTEN 39/151 (26%) Non-small cell lung cancer 0.00% Marsit et al. (2005a, b)

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ConXict of interest statement We declare that we have no conXictof interest.

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