Clinical Cancer Research Versailles No Abst Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Widschwendter, A.
Right arrow Articles by Widschwendter, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Widschwendter, A.
Right arrow Articles by Widschwendter, M.
Clinical Cancer Research Vol. 10, 3396-3400, May 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Analysis of Aberrant DNA Methylation and Human Papillomavirus DNA in Cervicovaginal Specimens to Detect Invasive Cervical Cancer and Its Precursors

Andreas Widschwendter1, Conny Gattringer1, Lennart Ivarsson1, Heidi Fiegl1, Alois Schneitter1, Angela Ramoni1, Hannes M. Müller1, Annemarie Wiedemair1, Susanne Jerabek1, Elisabeth Müller-Holzner1, Georg Goebel2, Christian Marth1 and Martin Widschwendter1

1 Department of Obstetrics and Gynecology, Innsbruck University Hospital, Innsbruck, and 2 Department of Biostatistics and Documentation, University of Innsbruck, Innsbruck, Austria


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status.

Experimental Design: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCR-based methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions.

Results: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs.

Conclusions: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer of the uterine cervix is an important cause of death in women worldwide (1) . Since the introduction of Pap smears in screening programs, the incidence and mortality of cervical cancer have decreased dramatically (2) . However, successful screening strongly depends on the coverage rate of the population and the sensitivity and specificity of the screening test. A meta-analysis of studies investigating the Pap test for the detection of cervical cancer and its precursors revealed a sensitivity ranging from 30 to 87% and a specificity ranging from 86 to 100% (3) . Converging evidence from epidemiological and molecular studies suggests that infection with genital human papillomavirus (HPV) is causally linked to the development of cervical cancer (4) . Therefore, testing for HPV DNA has been evaluated to improve cervical cancer screening. Numerous studies showed a high sensitivity for the HPV test in detecting cervical cancer and its precursors, whereas specificity was usually lower in comparison to cytology (5) . To reduce the inconvenience and cost of repeated clinical visits, it has been proposed that women collect cervicovaginal specimens themselves, hopefully increasing the coverage of screening programs.

Several studies investigated HPV DNA detection rates between self-collected and physician-collected samples with varying concordance between the two different collection methods (6, 7, 8) .

In addition to HPV infection, it is clear that other factors are also involved in cervical carcinogenesis because the majority of patients with HPV-associated lesions do not progress to invasive cancer. Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasias (9) . Recently, an aberrant methylation pattern was found during the multistage pathogenesis of cervical cancer with a trend to increasing methylation with increasing pathological changes (10) . This study analyzed the methylation status of a panel of genes that are frequently methylated in cervical cancer or other malignancies.

The aim of this proof of principle study was to examine whether HPV DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens, collected on a tampon, are able to detect invasive cervical cancer and whether these changes are already present in the precursor lesions.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
A total of 34 patients with cervical intraepithelial neoplasia (cervical intraepithelial neoplasia 1: 3 cases, cervical intraepithelial neoplasia 2: 18 cases, and cervical intraepithelial neoplasia 3: 13 cases) and 11 patients with invasive cervical cancer were included in this study. Patients were referred to our hospital for additional treatment because of abnormal Pap smear or obvious cervical lesion. All Pap smears were taken within 3 months before tampon collection. Pap smears were classified according to the Munich Cytological Classification. The correlation between the Munich Cytological Classification and the Bethesda System are shown in Supplemental Data. Patients underwent cervical conization or, in the case of obvious carcinomatous lesion, cervical biopsy to obtain a histological diagnosis. The level of dysplasia was assigned based on the highest grade of dysplasia seen in the cone specimens. All patients with invasive cervical cancer were staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Five patients had a cervical cancer classified as FIGO stage I, 3 had FIGO stage II, 2 had FIGO stage III, and 1 had FIGO stage IV. Additionally, 10 patients with normal cervical cytology and without cervical dysplasia in the histological examination who underwent hysterectomy because of fibroids were investigated. All patients, except 6 patients with invasive cervical cancer, were taken from a prior study performed to determine whether it is possible to detect endometrial cancer by analyzing methylated DNA in cervicovaginal specimens collected by tampon (11) . Aberrant DNA methylation of 5 genes, namely CDH13, HSPA2, MLH1, RASSF1A, and SOCS2 was investigated in that study.

Sample Collection.
Samples and clinical data were collected after informed consent was obtained. To ensure standardized sample collection, a tampon (o.b. comfort mini tampons; Johnson & Johnson GmbH, Hallein, Austria) was inserted in the vagina by a physician after speculum examination before surgery (the day before) and retained intravaginal for 30 min. Preparation of the samples was described previously (11) . Briefly, the tampon was transferred to a 50-ml tube after removal, and 1.2 ml of PBS buffer were added to the tampon. Centrifugation at 1000 x g for 10 min produced supernatant and a pellet. Aliquots (0.2 ml) of the supernatant were each mixed with 0.2 ml from the working solution of the High Pure Viral Nucleic Acid kit (Roche Diagnostics, Mannheim, Germany) and stored at –30°C until DNA isolation.

DNA Isolation and Methylation Analysis.
Genomic DNA from samples was isolated using the High Pure Viral Nucleic Acid kit (Roche Diagnostics) according to the manufacturer’s protocol with some modifications for multiple loading of the DNA extraction columns to gain a sufficient amount of DNA. Eleven genes known to be methylated in cervical cancer were selected for methylation analysis (10 , 12, 13, 14) . After sodium bisulfite conversion, the 11 genes (SOCS1, CDH1, TIMP3, GSTP1, DAPK, hTERT, CDH13, HSPA2, MLH1, RASSF1A, and SOCS2) underwent methylation analysis by means of the fluorescence-based, real-time PCR MethyLight assay as described previously (15 , 16) . Briefly, two sets of primers and probes, designed specifically for bisulfite-converted DNA, were used: a methylated set for the gene of interest and a reference set, ß-actin (ACTB) to normalize for input DNA. Specificity of the reactions for methylated DNA was confirmed separately using SssI (New England Biolabs)-treated human WBC DNA (heavily methylated). 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 SssI-treated DNA and multiplying by 100. The result is given as percentage of fully methylated reference. A gene was deemed methylated if the percentage of fully methylated reference value was >0. Primers and probes used for MethyLight reactions are listed in Table 1Citation .


View this table:
[in this window]
[in a new window]

  		Table 1 Primers and probes used for MethyLight reactions

 
HPV DNA Analysis.
HPV PCR enzyme immunoassay was done as described previously (17) . Briefly, 10 µl of purified total cellular DNA were used for PCR using consensus primers GP5+/bioGP6+. The reaction mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3.5 mM MgCl2, 200 µM of each deoxynucleoside triphosphate, 1 unit of thermostable DNA polymerase (TaqDNA Polymerase; Roche, Vienna, Austria), and 50 pmol each of the GP5+ (5-TTTGTTACTGTGGTAGATATACTAC-3) and bioGP6+ (5-GAAAAATAAACTGTAAATCATATT-3) primers (MWG-Biotech, Ebersberg, Germany). A 4-min denaturation step at 94°C was followed by 40 cycles of amplification in a PCR cycler (Gene Amp PCR System 9600; Perkin-Elmer, Norwalk, CT). To determine the HPV type, the PCR product was subjected to an enzyme immunoassay recognizing 14 different high-risk HPV types as described by Jacobs et al. (17) .

Statistical Analysis.
Associations between categorical variables were tested with Pearson’s {chi}2 test (or Fisher’s exact test) and the Mantel-Haenzel Test. Correlations between ordinal variables were evaluated using the Spearman Rank Correlation Coefficient. For unsupervised hierarchical clustering of clinical cases and genes, we used the complete linkage aggregation method and the Manhattan distance function.

All statistical calculations were performed using SPSS, version 11.0, for Windows and Gene Expression Similarity Suite.3


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
This proof of principle study investigated cervicovaginal specimens sampled by intravaginal tampon application in patients without cervical dysplasia, patients with low-grade and high-grade squamous intraepithelial lesion (SIL), and patients with invasive cervical cancer for HPV DNA and aberrant methylation of 11 genes. Pap smear revealed normal cytology (Pap II) for all patients without dysplasia. The three cases with low-grade SIL had Pap III, Pap IIID, and Pap IV, and all patients with high-grad SIL had abnormal cytology (Fig. 1)Citation . From patients with invasive cervical cancer, Pap smear was available in 4 of 11 cases, and all 4 cases had abnormal cytology (Fig. 1)Citation . In 7 cases, a carcinomatous cervical lesion was obvious, and a cervical biopsy was taken instead of a Pap smear. High-risk HPV DNA was detected in 2 of 3, 21 of 31, and 9 of 11 in low-grade SIL, high-grade SIL, and invasive cervical cancer, respectively (Table 2)Citation . Patients without cervical dysplasia showed no HPV infection. HPV-positive cervical cancers were large cell squamous cancers and one adeno cancer, whereas both HPV-negative cases were small cell cervical cancers. Differences in methylation of the investigated genes between the nondysplasia group (n = 10) and the low-grade SIL group (n = 3) were not statistically significant. Therefore, the results from these two groups were combined and compared with the high-grade SIL and invasive cancer groups. An overview of the frequency of methylated genes is given in Table 2Citation . All investigated genes, except GSTP1 and SOCS2, were significantly more frequently methylated in high-grade SIL and/or invasive cancer in comparison to the nondysplasia/low-grade SIL group. CDH1 and SOCS2 were found to be methylated in nearly a quarter of the nondysplasia/low-grade SIL patients, whereas hTERT was methylated exclusively in specimens obtained from cervical cancer patients. No methylated genes were found in 61 and 42% of the cervicovaginal specimens from patients with nondysplasia/low-grade SIL and high-grade SIL, respectively, whereas cervical cancer patients revealed three or more methylated genes in each investigated sample. The percentage of methylation-positive samples showed a significantly increasing trend from nondysplasia/low-grade SIL to invasive cervical cancer, as shown in Fig. 2Citation . No significant correlation between HPV positivity and aberrant hypermethylation was observed (P = 0.561, Fisher’s exact test). Also, comparison between abnormal Pap smear and aberrant hypermethylation revealed no significant correlation (P = 0.21, Fisher’s exact test)



View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. DNA methylation status of 11 genes in 55 cervicovaginal specimens was analyzed using the MethyLight technique. A gene was deemed methylated if the percentage of fully methylated reference value was >0 ("Materials and Methods"; white and pink indicate unmethylated and methylated, respectively; gray indicates not done). Groups of patients were determined using unsupervised agglomerative hierarchical cluster analysis (average linkage, Manhattan distance) to group specimens and CpG regions. Gene names are given at the top of the figure. We observed two clusters: 1 (red) and 2 (blue). Nine of 11 patients with cervical cancer are grouped together in one cluster (blue). Pap smear result is reported in accordance with the Second Munich Cytological Classification. In 7 cases, a carcinomatous cervical lesion was obvious. In these cases, a cervical biopsy was taken instead of a Pap smear (no Pap). SIL, squamous intraepithelial lesion.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Methylated genes and high-risk human papillomavirus DNA in patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer

 


View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Trend to increasing methylation from low-grade squamous intraepithelial lesion (SIL) to invasive cervical cancer. A statistically significant correlation between type of histology and number of methylated genes ({rho} = 0. 6, P < 0.001) was found.

 
Two clusters were formed by unsupervised hierarchical cluster analysis using solely information on the DNA methylation of the 11 genes tested. One of the two clusters contained 9 of 11 invasive cancers as well as two high-grade SILs (Fig. 2)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Up to 60% of women diagnosed as having invasive cervical cancer were not screened despite health maintenance organization enrollment (18) . It has been shown that HPV is causally linked to the development of cervical cancer (4) , and numerous studies have revealed a high sensitivity for the HPV test in detecting cervical cancer and its precursors, whereas specificity was usually lower in comparison to cytology (5) . To reduce the inconvenience and cost of repeated clinical visits, it has been proposed that women collect cervicovaginal specimens themselves, thus hopefully increasing the coverage of screening programs. Previous studies investigating HPV DNA detection rates between self-collected and physician-collected samples described varying concordance between the two different collection methods (6, 7, 8) . Our study detected high-risk HPV DNA obtained in 68% of high-grade SIL and in 82% of invasive cervical cancer patients. Recently, it was shown that clinician-directed swabs detect up to 28% more HPV-positive women in comparison to tampon-collected specimens (7) . In light of the fact that numerous studies have revealed a ~100% sensitivity in detecting SIL and cervical cancer by HPV DNA testing of physician-collected samples (5) , specimen collection by tampon seems not to be a feasible method for HPV DNA detection.

It has been proposed that in addition to HPV infection, genetic or epigenetic alterations may be required to maintain a malignant phenotype. Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasias (9) . Recently, it was suggested that aberrant methylation may play a role in cervical carcinogenesis (10) . Therefore, we additionally investigated the methylation status of genes known to be methylated in cervical cancer (10 , 12, 13, 14 , 19) and in various malignancies (20, 21, 22, 23, 24, 25) . hTERT was never found to be methylated in cervical intraepithelial neoplasias, whereas 82% of the specimens obtained from cervical cancer patients were methylated. This finding suggests that methylation of hTERT is a late event in cervical carcinogenesis. SOCS2 and CDH1 were methylated in nearly a quarter of patients from the nondysplasia/low-grade SIL groups, indicating that methylation of these genes is an early event in cervical carcinogenesis. Our results clearly show that an increasing percentage of methylation-positive samples is associated with increasing pathological changes of the cervix uteri (Fig. 2)Citation , suggesting that methylation, in addition to HPV infection, is an important factor in cervical carcinogenesis. Comparison between aberrant hypermethylation and abnormal Pap smear revealed no significant correlation. This comparison is of limited value because Pap smear was not available in 7 of 11 cervical cancer patients. Additionally, no significant correlation between HPV positivity and aberrant hypermethylation was observed. It can be speculated that aberrant methylation in women with or without HPV infection may help identify subgroups at increased risk for histological progression or cancer development.

Our proof of principle study shows that it is possible to detect HPV and aberrant hypermethylation of various genes in DNA from tampon-collected samples. We were able to identify 9 of 11 invasive cervical cancers by unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes. Both methods combined detect a high percentage of high-grade cervical lesions, although sensitivity and specificity of this test for high-grade SIL are too low to be applicable as a screening test. Because the selection of the 11 genes used for analysis was based mainly on the fact that they have been described as methylated in invasive cancers, additional studies need to define genes that become methylated at an earlier time in cervical carcinogenesis. Once that is the case, this simple approach may become useful for the early detection of SIL and invasive cervical cancers by means of DNA methylation analysis in cervicovaginal specimens isolated from tampons inserted by women and sent to a laboratory for testing.


   FOOTNOTES
 
Grant support: "Fonds zur Förderung der wissenschaftlichen Forschung" Grants P15995-B05 and P16159-B05 and "Jubiläumsfonds der Österreichischen Nationalbank" Grant 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.

Note: A. Widschwendter and C. Gattringer contributed equally to this work.

Requests for reprints: Martin Widschwendter, Department of 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{at}uibk.ac.at

3 Internet address: http://genome.tugraz.at/Software/Genesis/Genesis.html. Back

Received 9/ 2/03; revised 2/ 2/04; accepted 2/10/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pisani P, Parkin DM, Bray F, Ferlay J. Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer, 83: 18-29, 1999.[Medline]
  2. Quinn M, Babb P, Jones J, Allen E. Effect of screening on incidence of and mortality from cancer of cervix in England: evaluation based on routinely collected statistics. BMJ, 318: 904-8, 1999.[Abstract/Free Full Text]
  3. Nanda K, McCrory DC, Myers ER, et al Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Ann Intern Med, 132: 810-9, 2000.[Abstract/Free Full Text]
  4. zur Hausen H. Papillomavirus infections: a major cause of human cancers. Biochim Biophys Acta, 1288: F55-78, 1996.[Medline]
  5. Lorincz AT, Richart RM. Human papillomavirus DNA testing as an adjunct to cytology in cervical screening programs. Arch Pathol Lab Med, 127: 959-68, 2003.[Medline]
  6. Gravitt PE, Lacey JV, Jr, Brinton LA, et al Evaluation of self-collected cervicovaginal cell samples for human papillomavirus testing by polymerase chain reaction. Cancer Epidemiol Biomark Prev, 10: 95-100, 2001.[Abstract/Free Full Text]
  7. Harper DM, Noll WW, Belloni DR, Cole BF. Randomized clinical trial of PCR-determined human papillomavirus detection methods: self-sampling versus clinician-directed biologic concordance and women’s preferences. Am J Obstet Gynecol, 186: 365-73, 2002.[CrossRef][Medline]
  8. Lorenzato FR, Singer A, Ho L, et al Human papillomavirus detection for cervical cancer prevention with polymerase chain reaction in self-collected samples. Am J Obstet Gynecol, 186: 962-8, 2002.[CrossRef][Medline]
  9. Jones PA. DNA methylation errors and cancer. Cancer Res, 56: 2463-7, 1996.[Free Full Text]
  10. Virmani AK, Muller C, Rathi A, Zoechbauer-Mueller S, Mathis M, Gazdar AF. Aberrant methylation during cervical carcinogenesis. Clin Cancer Res, 7: 584-9, 2001.[Abstract/Free Full Text]
  11. Fiegl H, Gattringer C, Widschwendter A, et al. Methylated DNA collected by tampons: a new tool to detect endometrial cancer. Cancer Epidemiol Biomark Prev 2004. In press.
  12. Dong SM, Kim HS, Rha SH, Sidransky D. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res, 7: 1982-6, 2001.[Abstract/Free Full Text]
  13. Muller HM, Widschwendter A, Fiegl H, et al. A DNA methylation pattern similar to normal tissue is associated with better prognosis in human cervical cancer. Cancer Lett 2003. In press.
  14. Widschwendter A, Muller HM, Fiegl H, et al DNA methylation in serum and tumors of cervical cancer patients. Clin Cancer Res, 10: 565-71, 2003.
  15. Eads CA, Danenberg KD, Kawakami K, et al MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res, 28: E32 2000.
  16. Eads CA, Lord RV, Wickramasinghe K, et al Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res, 61: 3410-8, 2001.[Abstract/Free Full Text]
  17. Jacobs MV, Snijders PJ, van den Brule AJ, Helmerhorst TJ, Meijer CJ, Walboomers JM. A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings. J Clin Microbiol, 35: 791-5, 1997.[Abstract]
  18. Kinney W, Sung HY, Kearney KA, Miller M, Sawaya G, Hiatt RA. Missed opportunities for cervical cancer screening of HMO members developing invasive cervical cancer (ICC). Gynecol Oncol, 71: 428-30, 1998.[CrossRef][Medline]
  19. Kuzmin I, Liu L, Dammann R, et al Inactivation of RAS association domain family 1A gene in cervical carcinomas and the role of human papillomavirus infection. Cancer Res, 63: 1888-93, 2003.[Abstract/Free Full Text]
  20. Dessain SK, Yu H, Reddel RR, Beijersbergen RL, Weinberg RA. Methylation of the human telomerase gene CpG island. Cancer Res, 60: 537-41, 2000.[Abstract/Free Full Text]
  21. Grady WM, Willis J, Guilford PJ, et al Methylation of the CDH1 promoter as the second genetic hit in hereditary diffuse gastric cancer. Nat Genet, 26: 16-7, 2000.[CrossRef][Medline]
  22. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A. The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet, 103: 96-101, 1998.[CrossRef][Medline]
  23. Yoshikawa H, Matsubara K, Qian GS, et al SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet, 28: 29-35, 2001.[CrossRef][Medline]
  24. Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res, 61: 249-55, 2001.[Abstract/Free Full Text]
  25. Muller HM, Widschwendter M. Methylated DNA as a possible screening marker for neoplastic disease in several body fluids. Expert Rev Mol Diagn, 3: 443-58, 2003.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
S. S. Wang, D. J. Smiraglia, Y.-Z. Wu, S. Ghosh, J. S. Rader, K. R. Cho, T. A. Bonfiglio, R. Nayar, C. Plass, and M. E. Sherman
Identification of Novel Methylation Markers in Cervical Cancer Using Restriction Landmark Genomic Scanning
Cancer Res., April 1, 2008; 68(7): 2489 - 2497.
[Abstract] [Full Text] [PDF]

Home page
 ADRHome page
J. Palefsky
Biology of HPV in HIV Infection
Advances in Dental Research, April 1, 2006; 19(1): 99 - 105.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
J. Zhang, C. R. Martins, Z. B. Fansler, K. L. Roemer, E. A. Kincaid, K. S. Gustafson, D. F. Heitjan, and D. P. Clark
DNA Methylation in Anal Intraepithelial Lesions and Anal Squamous Cell Carcinoma
Clin. Cancer Res., September 15, 2005; 11(18): 6544 - 6549.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
P. W. Laird
Cancer epigenetics
Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R65 - R76.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Widschwendter, A.
Right arrow Articles by Widschwendter, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Widschwendter, A.
Right arrow Articles by Widschwendter, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online