DNA Methylation in Serum and Tumors of Cervical Cancer Patients (original) (raw)

Purpose: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease.

Experimental Design: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique.

Results: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1.

Conclusions: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

INTRODUCTION

Cancer of the uterine cervix is an important cause of death in women worldwide (1). Many studies have investigated clinical and histopathological characteristics as prognostic factors for cervical cancer. Uni- and/or multivariate analysis have revealed that stage, pelvic lymph node metastasis, tumor volume, vascular invasion, and depth of invasion can be prognostic factors for recurrent disease (2, 3). However, new molecular and biochemical approaches for the recognition and treatment of high-risk patients are needed to improve survival and avoid overtreatment of low-risk patients. Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasias (4). It has been increasingly recognized over the past 4–5 years that the CpG islands of a large number of genes that are unmethylated in normal tissue are methylated to various degrees in multiple types of human cancer (4, 5). Aberrant methylation of CpG islands within the promoter regions of several genes such as p16, DAPK (death-associated protein-kinase), MGMT (O 6 _-_methylguanin-DNA-methyltransferase), _E_-cadherin, and _RAR-_β (retinoic acid receptor β) has been identified in cervical cancer (6, 7). Recently, we identified five additional genes, namely CALCA (calcitonin-related polypeptide α), hTERT (telomerase reverse transcriptase), MYOD1, (myoblast determination protein 1), PGR (progesterone receptor), and TIMP3, as being methylated significantly more frequently in cervical cancer than in normal cervical tissue.3 The presence of abnormally high DNA concentrations in the serum of patients with various malignant diseases was described years ago (8, 9). The discovery that cell-free DNA can be shed into the bloodstream has generated great interest. Numerous studies have demonstrated tumor-specific alterations in DNA recovered from plasma or serum of patients with various malignancies, a finding that has potential for molecular diagnosis and prognosis. The nucleic acid markers described in plasma and serum include oncogene mutations, microsatellite alterations, gene rearrangements, and epigenetic alterations, such as aberrant promoter hypermethylation (10, 11). On the basis of these observations, we examined the methylation status of CALCA, hTERT, MYOD1, PGR, and TIMP3 genes in serum samples of cervical cancer patients and compared it with clinicopathological characteristics and outcome of the disease.

MATERIALS AND METHODS

Patients and Samples.

A total of 93 patients with invasive cervical cancer (ages 26–96 years; median, 52 years), all treated at the Department of Obstetrics and Gynecology, Innsbruck University Hospital, between 1990 and 1998, were included in this study. Serum samples were taken on the date of diagnosis and before initial treatment. These serum samples were taken from a prior study investigating the presence of serum human papillomavirus DNA in cervical cancer patients (12). Major clinical and histopathological characteristics of patients are given in Table 1. In 19 cases, the corresponding cervical cancer tissue samples were available for analysis. These tissue samples were analyzed in a prior study investigating the methylation status of 25 genes in 65 cervical cancer tissues and 14 normal cervical tissues.3 All of the patients were followed up after primary treatment at our department at intervals, increasing from 3 months to 1 year, until death or end of the study. The follow-up period ranged from 1 month to 12.4 years (mean, 4.4 years).

DNA Isolation and Methylation Analysis.

Genomic DNA from cervical cancer specimen was isolated using the QIAmp tissue kit (Qiagen, Hilden, Germany). Serum samples were treated with SDS and proteinase K at 55°C overnight, followed by phenol/chloroform extraction and ethanol precipitation of DNA. After sodium bisulfite conversion, the methylation analysis was performed by the fluorescence-based, real-time PCR assay MethyLight as described previously (13, 14). Briefly, three sets of primers and probes, designed specifically for bisulfite-converted DNA, were used: a methylated set for the gene of interest and two reference sets, β-actin (ACTB) and collagen (COL2A1), to normalize for input DNA. Specificity of the reactions for methylated DNA was confirmed separately using _Sss_I (New England Biolabs)-treated human genomic DNA (heavily methylated). Two separate percentage-of-fully-methylated-reference (PMR) values (separately calculated for ACTB and COL2A1) were calculated. The percentage of fully methylated molecules at a specific locus was calculated by dividing the GENE:ACTB ratio of a sample by the GENE:ACTB ratio of _Sss_I-treated human genomic DNA and multiplying by 100. The abbreviation PMR indicates this measurement. The same calculation was done for the GENE:COL2A1 ratio. The average of both PMR values (calculated for ACTB and COL2A1) was used as the final PMR. A gene was deemed methylated if the PMR value was >0. To verify the reproducibility of each assay, the normalized value (Gene:ACTB) of the standard sample was compared between the different PCR runs. The following primers and probes were used for the MethyLight reactions: (a) hTERT: 5′-GCGTCGGAGGTTAAGGTTGTT-3′ (forward primer), 5′-CTCTCCAAAATTACCGTACGCG-3′ (reverse primer), 5′-6FAM-AACTCGCTCGCCCGCCGAA-BHQ-1-3′ (probe); and (b) PGR: 5′-TTATAATTCGAGGCGGTTAGTGTTT-3′ (forward primer), 5′-TCGAACTTCTACTAACTCCGTACTACGA-3′ (reverse primer), 5′-6FAM-ATCATCTCCGAAAATCTCAAATCCCAATAATACG-BHQ-1-3′ (probe). The nucleotide sequences of the primers and probes used for the MethyLight reactions for CALCA, MYOD1, and TIMP3 were described elsewhere (14).

Statistical Analysis.

Associations between categorical variables were tested with Pearson’s χ2 test. Differences in median of PMR values were examined with the Mann-Whitney U test. The Kaplan-Meier method was used for univariate survival analysis, and the log-rank test was used to assess the difference between survival curves. Cox’s proportional hazards analysis was used to estimate the prognostic effects of various variables. A P of less then 0.05 was considered statistically significant. These statistical calculations were performed using SPSS, version 11.0, for Windows.

RESULTS

In a prior study, we investigated the methylation status of 25 genes in 65 cervical cancer tissues and 14 normal cervical tissues.3 Five genes, namely CALCA, hTERT, MYOD1, PGR, and TIMP3, were found to be methylated significantly more frequently in cervical cancer than in normal cervical tissue. Comparison of methylation (PMR values) between normal and cancer tissue revealed the most significant results for these five genes (Fig. 1). In the present study, we searched for the presence of promoter hypermethylation of these five genes in serum samples of cervical cancer patients taken before initial treatment. Aberrant promoter hypermethylation of any of the genes studied was detected in 87% (81 of 93) of the investigated serum samples. Sixty (74%) of the 81 methylation-positive serum samples showed epigenetic changes in more than one of the genes tested. Promoter hypermethylation of the PGR was detected most frequently (79%, 73 of 93) of all investigated genes, especially in serum samples of patients with adenocarcinomas and adenosquamous carcinomas, whereas methylation of hTERT was not observed in any of the examined serum samples (Table 1). In advanced stage, methylation of MYOD1 was detected at a significantly higher frequency than in early-stage cervical cancer (P < 0.03). TIMP3 and MYOD1 were methylated only in sera of patients with squamous cell carcinoma (Table 1). In all serum samples from patients with a carcinoma classified as tumor grade 3, at least one gene was methylated.

Distant metastases at the date of diagnosis were detected in five patients, and aberrant methylation in serum DNA was observed in all of these cases. Of patients who experienced recurrence with distant metastases (n = 13), DNA was methylated in at least one gene in 11 cases, whereas only 2 patients showed no detectable methylation changes in serum DNA. In these 11 cases, DNA methylation was detected in one, two, and three genes in 4, 2, and 5 cases, respectively.

In 19 cases, the corresponding cervical cancer tissue samples were available for analysis. CALCA, MYOD1, and PGR were methylated in all tissue samples, whereas hTERT was unmethylated in six cases and TIMP3 in one case. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except one patient who revealed _TIMP3_-methylated serum DNA but no methylation of the corresponding tissue sample. Comparison of cervical cancer tissue methylation (PMR values) between unmethylated and methylated serum samples for each investigated gene revealed no significant results (Fig. 2).

To determine whether the methylation status in serum samples taken at the date of diagnosis has prognostic value, we compared serum DNA methylation of the investigated genes with the clinical outcome of the patients. Fifty-three patients (57%) experienced a recurrence and 51 (55%) died. Median overall survival of all patients was 4.4 years. CALCA, hTERT, PGR, and TIMP3 methylation status revealed no prognostic significance. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free and overall survival in comparison with patients with methylated MYOD1 (Fig. 3). Median survival was 1.9 and 6.1 years for MYOD1 methylation-positive and -negative patients, respectively. To assess for independent prognostic significance, a Cox proportional hazard model analysis was performed. The logistic regression model included tumor stage, histology, grade of differentiation, age, and MYOD1 methylation status. Only International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.0001) was of independent prognostic significance for both disease-free and overall survival.

DISCUSSION

Previous studies have described the importance of DNA methylation in human cancers. Recently, an aberrant methylation pattern was found during the multistage pathogenesis of cervical cancer with an increasing trend to methylation with increasing pathological changes (7). Promoter hypermethylation of various genes is a frequent epigenetic event in cervical carcinoma (6, 7, 15).3 Epigenetic alterations have been successfully used as indicators of neoplastic serum DNA in patients with various carcinomas (16). To date, no studies have been undertaken to investigate the methylation status of various genes in serum samples of cervical cancer patients. Recently, we identified five genes, namely CALCA, hTERT, MYOD1, PGR, and TIMP3, as being methylated significantly more frequently in cervical cancer tissue than in normal cervical tissue.3 In our study, all of the patients with methylated serum DNA revealed the same methylation pattern in the corresponding cervical cancer tissue, except one patient who revealed TIMP3 methylated serum DNA but no methylation of the corresponding tissue sample. These results are in accordance with previous studies (17, 18, 19) and strengthen the evidence that methylated serum DNA is tumor derived. Correlation of PMR values of the investigated genes between tissue and serum showed no significant results. The lack of correlation can be due to several reasons, e.g., different grade of neoangiogenesis, different biological behavior, different development of tumor necrosis, and genetic heterogeneity of the tumors (20). Numerous studies have analyzed the methylation status of cancer-related genes in plasma or serum. The correlation between detection of methylated genes in serum samples and clinical or histopathological features is conflicting in these studies. Whereas several studies report an association with prognosis (19), stage of the disease (21), or occurrence of metastases (18), others found no significant correlation with clinical or histopathological characteristics (22, 23, 24).

When investigating methylation patterns of serum DNA and their association with clinical and histopathological parameters, several factors seem to influence the outcome. On the one hand, the choice of appropriate target genes is essential to attain prognostic significance. Although Usadel et al. (19) found that methylation of the APC (adenomatous polyposis coli) gene in serum samples of lung cancer patients is an independent prognostic factor, Esteller et al. (22), analyzing p16 INK4a, DAPK, GSTP1 (glutathione _S_-transferase), and MGMT genes in serum samples of lung cancer patients, observed no correlation between methylation status and prognosis. On the other hand, sample size is a crucial factor to obtain statistically significant results. Wong et al. (25) described detection of aberrant p16 methylation in the plasma and serum of 22 liver cancer patients without observing clinical associations. Incorporating 23 additional patients into the same study, they detected a significant association between the presence of p16 methylation and the development of tumor recurrence or metastasis (26).

In our study, MYOD1 and TIMP3 in serum were methylated only in squamous cell carcinomas, whereas methylation of PGR was more frequent in adeno- and adenosquamous carcinomas, implying a specific methylation pattern according to histology. A different pattern of promoter hypermethylation in cervical cancer tissue between squamous cell carcinomas and adenocarcinomas was described previously for DAPK, APC, and the HIC-1 (hypermethylated in cancer-1) genes (6). Furthermore, an increase in methylation frequency in serum was observed with a decrease in differentiation of the tumor. In all of the patients with cervical cancer grade 3, at least one of the investigated genes was methylated, whereas only 77% of patients with well-differentiated tumors revealed aberrant methylation. Additionally, aberrant promoter hypermethylation was detected in all of the patients with distant metastases and in 11 of the 13 patients who experienced recurrence with distant metastasis. These results suggest that multiple methylation is associated with less differentiated and, therefore, more aggressive tumor cells.

Aberrant methylation of MYOD1 was significantly associated with tumor stage in our study. This is in accordance with a recently published study reporting an association between hypermethylated APC DNA in sera of patients with esophageal adenocarcinoma and advanced disease stage (21). Additionally, a higher methylation level of several genes in stage II cervical cancer patients has been described in comparison with patients with a stage I tumor (7). These results suggest that hypermethylation of several genes is associated with advanced-stage and less-differentiated tumor cells and, therefore, that methylation of serum DNA might be a useful marker to identify patients with more aggressive disease.

In a prior study, we analyzed the methylation status of 25 genes in 14 normal cervical tissue specimens and in 65 tissue specimens of cervical cancer patients.3 Surgically treated lymph node positive patients from this cohort showed statistically significantly higher MYOD1 PMR values in comparison with lymph node-negative patients.4 In the present study, patients with unmethylated MYOD1 serum DNA revealed both better disease-free survival (P = 0.04) and better overall survival (P = 0.02). These results strengthen the evidence that aberrant MYOD1 methylation is associated with a more aggressive tumor.

Additionally, an in vitro study revealed an increase in the methylation status of MYOD1 CpG islands during oncogenic transformation (27). Hypermethylation of MYOD1 has also been described in tissue samples of various malignancies, e.g., breast cancer, colorectal cancers, and malignant lymphoproliferative disorders (28, 29, 30). In these studies, aberrant promoter hypermethylation of MYOD1 was associated with poorly differentiated and more invasive tumors, whereas hypermethylation was not detected in normal tissue

From our findings, we hypothesize that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse, who could benefit from radiochemotherapy, versus cervical cancer patients at lower risk for disseminated disease. Additional studies, involving a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Grant support: Grants from “Fonds zur Förderung der wissenschaftlichen Forschung,” P15995-B05 and P16159-B05, and from “Jubiläumsfonds der Österreichischen Nationalbank,” Project 9856.

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.

Notes: Drs. Andreas Widschwendter and Hannes M. Müller contributed equally to this work. Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).

Requests for reprints: Martin Widschwendter, Department Obstetrics and Gynecology, Innsbruck University Hospital, Anichstrasse 35, A-6020 Innsbruck, Austria. Phone: 0043-512-504-4155; Fax: 0043-512-504-3112; E-mail: martin.widschwendter@uibk.ac.at

H. M. Müller, A. Widschwendter, G. Goebel, H. Fiegl, E. Müller-Holzner, C. Marth, and M. Widschwendter. A DNA methylation pattern similar to normal tissue is associated with better prognosis in human cervical cancer, submitted for publication.

See Supplemental Data at http://cancerres.aacrjournals.org.

Fig. 1.

Fig. 1. Methylation [percentage of fully methylated reference (PMR) values] in normal and cervical cancer tissues of CALCA, hTERT, MYOD1, PGR, and TIMP3. Differences in PMR values between normal and cancer tissue were tested with the Mann-Whitney U test.

Methylation [percentage of fully methylated reference (PMR) values] in normal and cervical cancer tissues of CALCA, hTERT, MYOD1, PGR, and TIMP3. Differences in PMR values between normal and cancer tissue were tested with the Mann-Whitney U test.

Fig. 1.

Close modal

Fig. 2.

Fig. 2. Comparison of cervical cancer tissue methylation [percentage of fully methylated reference (PMR) values] between unmethylated (negative) and methylated (positive) serum samples for each investigated gene. Box blots for hTERT are not shown because no serum sample revealed hTERT methylation. Differences were examined with the Mann-Whitney U test.

Comparison of cervical cancer tissue methylation [percentage of fully methylated reference (PMR) values] between unmethylated (negative) and methylated (positive) serum samples for each investigated gene. Box blots for hTERT are not shown because no serum sample revealed hTERT methylation. Differences were examined with the Mann-Whitney U test.

Fig. 2.

Close modal

Fig. 3.

Fig. 3. Disease-free and overall survival according to MYOD1 methylation status in serum samples.

Disease-free and overall survival according to MYOD1 methylation status in serum samples.

Fig. 3.

Fig. 3. Disease-free and overall survival according to MYOD1 methylation status in serum samples.

Disease-free and overall survival according to MYOD1 methylation status in serum samples.

Close modal

Table 1

Methylation of multiple genes in serum samples of cervical cancer patients

Characteristics	n a	CALCA	hTERT	MYOD1	PGR	TIMP3	At least one gene
Stage
FIGO_b_ I	23	57%	0%	4%	74%	4%	78%
FIGO II	24	58%	0%	21%	75%	0%	88%
FIGO III	33	70%	0%	36%	82%	9%	88%
FIGO IV	13	62%	0%	39%c	85%	0%	100%
Tumor grade_d_
1	22	55%	0%	14%	68%	5%	77%
2	50	62%	0%	28%	84%	4%	90%
3	16	81%	0%	38%	81%	6%	100%
Histology
Squamous	84	63%	0%	27%	77%	5%	87%
Adenocarcinoma	6	67%	0%	0%	83%	0%	83%
Adenosquamous	3	33%	0%	0%	100%	0%	100%
Age
<50	38	66%	0%	21%	82%	5%	87%
≥50	55	60%	0%	27%	76%	4%	87%
Total	93	62%	0%	25%	79%	4%	87%

Characteristics	n a	CALCA	hTERT	MYOD1	PGR	TIMP3	At least one gene
Stage
FIGO_b_ I	23	57%	0%	4%	74%	4%	78%
FIGO II	24	58%	0%	21%	75%	0%	88%
FIGO III	33	70%	0%	36%	82%	9%	88%
FIGO IV	13	62%	0%	39%c	85%	0%	100%
Tumor grade_d_
1	22	55%	0%	14%	68%	5%	77%
2	50	62%	0%	28%	84%	4%	90%
3	16	81%	0%	38%	81%	6%	100%
Histology
Squamous	84	63%	0%	27%	77%	5%	87%
Adenocarcinoma	6	67%	0%	0%	83%	0%	83%
Adenosquamous	3	33%	0%	0%	100%	0%	100%
Age
<50	38	66%	0%	21%	82%	5%	87%
≥50	55	60%	0%	27%	76%	4%	87%
Total	93	62%	0%	25%	79%	4%	87%

n, number of cases examined.

FIGO, International Federation of Gynecology and Obstetrics.

Tumor grade was unknown in five cases.

References

Pisani P., Parkin D. M., Bray F., Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990.

Int. J. Cancer

-29,

1999

Graflund M., Sorbe B., Hussein A., Bryne M., Karlsson M. The prognostic value of histopathologic grading parameters and microvessel density in patients with early squamous cell carcinoma of the uterine cervix.

Int. J. Gynecol. Cancer

-41,

2002

Takeda N., Sakuragi N., Takeda M., Okamoto K., Kuwabara M., Negishi H., Oikawa M., Yamamoto R., Yamada H., Fujimoto S. Multivariate analysis of histopathologic prognostic factors for invasive cervical cancer treated with radical hysterectomy and systematic retroperitoneal lymphadenectomy.

Acta Obstet. Gynecol. Scand.

1144

-1151,

2002

Jones P. A. DNA methylation errors and cancer.

Cancer Res.

2463

-2467,

1996

Jones P. A., Laird P. W. Cancer epigenetics comes of age.

Nat. Genet.

163

-167,

1999

Dong S. M., Kim H. S., Rha S. H., Sidransky D. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix.

Clin. Cancer Res.

1982

-1986,

2001

Virmani A. K., Muller C., Rathi A., Zoechbauer-Mueller S., Mathis M., Gazdar A. F. Aberrant methylation during cervical carcinogenesis.

Clin. Cancer Res.

584

-589,

2001

Anker P., Mulcahy H., Chen X. Q., Stroun M. Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.

Cancer Metastasis Rev.

-73,

1999

Leon S. A., Shapiro B., Sklaroff D. M., Yaros M. J. Free DNA in the serum of cancer patients and the effect of therapy.

Cancer Res.

646

-650,

1977

Ziegler A., Zangemeister-Wittke U., Stahel R. A. Circulating DNA: a new diagnostic gold mine?.

Cancer Treat. Rev.

255

-271,

2002

Muller H. M., Widschwendter M. Methylated DNA as a possible screening marker for neoplastic disease in several body fluids.

Expert Rev. Mol. Diagn.

443

-458,

2003

Widschwendter A., Blassnig A., Wiedemair A., Muller-Holzner E., Muller H. M., Marth C. Human papillomavirus DNA in sera of cervical cancer patients as tumor marker.

Cancer Lett.

202

231

-239,

2003

Eads C. A., Danenberg K. D., Kawakami K., Saltz L. B., Blake C., Shibata D., Danenberg P. V., Laird P. W. MethyLight: a high-throughput assay to measure DNA methylation.

Nucleic Acids Res.

E32

2000

Eads C. A., Lord R. V., Wickramasinghe K., Long T. I., Kurumboor S. K., Bernstein L., Peters J. H., DeMeester S. R., DeMeester T. R., Skinner K. A., Laird P. W. Epigenetic patterns in the progression of esophageal adenocarcinoma.

Cancer Res.

3410

-3418,

2001

Wong Y. F., Cheung T. H., Lo K. W. K., Chan L. K. Y., Heung M. S., Cheung A. Y. K., Herman J. G., Chung T. K. H. Multiple gene promoter hypermethylation in tumor and serum of cervical squamous cell carcinoma patients.

Proc. Am. Assoc. Cancer Res.

2003

Wong I. H., Lo Y. M., Johnson P. J. Epigenetic tumor markers in plasma and serum: biology and applications to molecular diagnosis and disease monitoring.

Ann. N. Y. Acad. Sci.

945

-50,

2001

Lee T. L., Leung W. K., Chan M. W., Ng E. K., Tong J. H., Lo K. W., Chung S. C., Sung J. J., To K. F. Detection of gene promoter hypermethylation in the tumor and serum of patients with gastric carcinoma.

Clin. Cancer Res.

1761

-1766,

2002

Sanchez-Cespedes M., Esteller M., Wu L., Nawroz-Danish H., Yoo G. H., Koch W. M., Jen J., Herman J. G., Sidransky D. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients.

Cancer Res.

892

-895,

2000

Usadel H., Brabender J., Danenberg K. D., Jeronimo C., Harden S., Engles J., Danenberg P. V., Yang S., Sidransky D. Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer.

Cancer Res.

371

-375,

2002

Klein C. A., Blankenstein T. J., Schmidt-Kittler O., Petronio M., Polzer B., Stoecklein N. H., Riethmuller G. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer.

Lancet

360

683

-689,

2002

Kawakami K., Brabender J., Lord R. V., Groshen S., Greenwald B. D., Krasna M. J., Yin J., Fleisher A. S., Abraham J. M., Beer D. G., Sidransky D., Huss H. T., DeMeester T. R., Eads C., Laird P. W., Ilson D. H., Kelsen D. P., Harpole D., Moore M. B., Danenberg K. D., Danenberg P. V., Meltzer S. J. Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma.

J. Natl. Cancer Inst. (Bethesda)

1805

-1811,

2000

Esteller M., Sanchez-Cespedes M., Rosell R., Sidransky D., Baylin S. B., Herman J. G. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients.

Cancer Res.

-70,

1999

Hibi K., Taguchi M., Nakayama H., Takase T., Kasai Y., Ito K., Akiyama S., Nakao A. Molecular detection of p16 promoter methylation in the serum of patients with esophageal squamous cell carcinoma.

Clin. Cancer Res.

3135

-3138,

2001

Wong T. S., Chang H. W., Tang K. C., Wei W. I., Kwong D. L., Sham J. S., Yuen A. P., Kwong Y. L. High frequency of promoter hypermethylation of the death-associated protein-kinase gene in nasopharyngeal carcinoma and its detection in the peripheral blood of patients.

Clin. Cancer Res.

433

-437,

2002

Wong I. H., Lo Y. M., Zhang J., Liew C. T., Ng M. H., Wong N., Lai P. B., Lau W. Y., Hjelm N. M., Johnson P. J. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients.

Cancer Res.

-73,

1999

Wong I. H., Johnson P. J., Lai P. B., Lau W. Y., Lo Y. M. Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring.

Ann. N. Y. Acad. Sci.

906

102

-105,

2000

Rideout W. M., III, Eversole-Cire P., Spruck C. H., III, Hustad C. M., Coetzee G. A., Gonzales F. A., Jones P. A. Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Mol. Cell. Biol.

6143

-6152,

1994

Hahnel R., Harvey J., Kay P. Hypermethylation of the myogenic gene Myf-3 in human breast carcinomas.

Anticancer Res.

2111

-2115,

1996

Shannon B., Kay P., House A., Iacopetta B. Hypermethylation of the MYF-3 gene in colorectal cancers: associations with pathological features and with microsatellite instability.

Int. J. Cancer

109

-113,

1999

Taylor J. M., Kay P. H., Spagnolo D. V. The diagnostic significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders.

Leukemia (Baltimore)

583

-589,

2001