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Cancer Epidemiology Biomarkers & Prevention 17, 966, April 1, 2008. doi: 10.1158/1055-9965.EPI-07-2775
© 2008 American Association for Cancer Research
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Line Region Hypomethylation Is Associated with Lifestyle and Differs by Human Papillomavirus Status in Head and Neck Squamous Cell Carcinomas

C. Sloane Furniss1, Carmen J. Marsit2, E. Andres Houseman3, Karen Eddy1 and Karl T. Kelsey1

1 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts; 2 Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island; and 3 Department of Work Environment and Health, University of Massachusetts-Lowell, Lowell, Massachusetts

Requests for reprints: Karl T. Kelsey, Brown University, 70 Ship Street, Providence, RI 02912. Phone: 401-863-6420; Fax: 401-863-9008. E-mail: karl_kelsey{at}brown.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Genomic hypomethylation is a hallmark of essentially all cancers, but the degree of this hypomethylation differs among individual tumors. Little work has explored what leads to these differences and or asked whether they are clinically meaningful. In this study of head and neck squamous cell carcinoma, we assessed hypomethylation in tumors using a semiquantitative fragment analysis approach to determine the relative methylation status of the line retroviral element LRE1 (Line-1.2). Because this is an established marker of genomic methylation status, we examined the relationship between the relative methylation, patient demographics, and other risk factors for head and neck squamous cell carcinoma. We determined relative methylation status for 303 patients, 193 of which had complete data for all variables of interest. Using a generalized linear model, we found that patient body mass index was significantly positively associated with tumor LRE1 methylation level. Smoking duration, particularly in tumors lacking human papillomavirus (HPV) DNA, was significantly negatively associated with relative methylation level. Having previously assessed relative methylation in blood-derived DNA, we compared tumor with the blood DNA methylation level and observed these to be independent. Finally, the lower LRE1 methylation in patients whose tumors were HPV DNA negative was associated with poorer patient survival (hazard ratio, 1.6; 95% confidence interval, 1.0-2.6). These findings suggest that HPV-associated tumors differ molecularly from those arising after heavy tobacco use and that this epigenetic alteration may affect survival in HPV-negative patients already exhibiting a more aggressive disease. (Cancer Epidemiol Biomarkers Prev 2008;17(4):966–71)


   Introduction
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 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Each year, ~40,000 new cases of head and neck squamous cell carcinoma are diagnosed in the United States, with ~31,000 cases in the oral cavity and pharynx and ~10,000 new cases in the larynx (1). Known risk factors for head and neck squamous cell carcinoma in the United States include tobacco smoke, chewing tobacco, and alcohol with a well-demonstrated synergistic interaction between smoking and drinking such that an attributable fraction of close to 90% has been estimated for these two exposures (2, 3). Human papillomavirus (HPV), specifically high-risk type 16, is also a risk factor for head and neck squamous cell carcinoma, particularly among younger patients and women, and is detected in ~25% of cases (4-14). Nutritional factors have also been linked to risk of head and neck squamous cell carcinoma, with a particular risk associated with lower body mass index (BMI; refs. 10, 15-20). It remains unclear if the risk associated with leanness is causal to the disease or is a consequence of the developing tumor or the heavy alcohol and tobacco use associated with the disease.

Global DNA hypomethylation is one hallmark of cancers, and it has been hypothesized that this loss of DNA methylation may lead to alterations in the expression of proto-oncogenes, as well as in facilitating chromosomal instability (21, 22). Vogelstein's model of multistep carcinogenesis places a genome-wide demethylation step (23) that mostly affects transposon regions (24) as an early event in carcinogenesis. Investigators have used the methylation status of repeat elements as a biomarker for global methylation status including the Line-1 retrotransposon (LRE1). Various types of cancer, including urothelial bladder carcinoma (25), malignant testicular tumors (26), hepatocellular carcinoma (27), chronic lymphocytic leukemia (28), prostate carcinomas (29), and head and neck squamous cell carcinoma (25), show hypomethylation of LRE1. Head and neck cancers studied to date have been observed to have significantly greater degree of LRE1 hypomethylation compared with their normal tissue counterparts (30), and a lower level of LRE1 methylation in peripheral blood–derived DNA has been associated with risk for head and neck squamous cell carcinoma (31).

Although it is clear that genomic hypomethylation is a hallmark of cancer, little work has addressed the etiology of this epigenetic alteration or the variability in the extent of genomic methylation across tumors. Therefore, we sought to determine the possible association of head and neck squamous cell carcinoma risk factors, specifically alcohol, tobacco, HPV, and BMI, with the methylation status of LRE1.


   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study Population
LRE1 methylation status was determined in 303 incident cases of head and neck squamous cell carcinoma, drawn from an ongoing population-based case-control study of head and neck squamous cell carcinoma. One hundred ninety-three of these 303 cases had complete data for all variables of interest. The details of the parent study have previously been described in detail (32) and all patients provided informed consent under protocols approved by the appropriate institutional review board. Information on risk factors including age, sex, ethnicity, tobacco and alcohol use, occupation, and diet was collected through patient questionnaires that were explained by study technicians, and clinical and pathologic information on the tumor was collected by chart review. Patient survival was ascertained by interrogating a national vital statistics database.

DNA Extraction and Sodium Bisulfite Modification
Three 20-µm sections were cut from each fixed, paraffin-embedded head and neck squamous cell carcinoma tumor sample and transferred into microcentrifuge tubes. The paraffin was dissolved with Histochoice Clearing Agent (Sigma-Aldrich) followed by two washes with 100% ethanol and one wash with PBS. The samples were then incubated in SDS lysis solution [50 mmol/L Tris-HCl (pH 8.1), 10 mmol/L EDTA, 1% SDS] with proteinase K (Qiagen) overnight at 55°C. De-crosslinking was done by adding NaCl (final concentration, 0.7 mol/L) and incubating at 65°C for 4 h. DNA was recovered using the Wizard DNA clean-up kit (Promega) according to the manufacturer's protocols. Sodium bisulfite modification of the DNA was done using the EZ DNA Methylation Kit (Zymo Research) following the manufacturer's protocol, with the addition of a 5-min initial incubation at 95°C before addition of the denaturation reagent. The de-crosslinking incubation as well as the 95°C incubation ensures complete melting of the DNA and thus complete sodium bisulfite conversion, particularly for the formalin-fixed specimens.

LRE1 Relative Methylation Assay
We used a modified version of the combined bisulfite restriction assay of Line-1.2 described by Chalitchagorn et al. (30) as we have used in a previous examination of LRE1 relative methylation level (31).

Tumor HPV Determination
A 65-bp region of the L1 gene of HPV16 was amplified using primers previously published (33). SPF1A (forward) and SPF2B (reverse), primers specific to HPV16, were used in the assay. PCR conditions were followed as previously described (14).

Statistical Methods
All statistical analyses were done with SAS software, version 9.1. The relative methylation index was calculated by taking the ratio of Taq1 positive amplicons to the sum of the Tas1 and Taq1 amplicons and fitting this value to a standard curve based on dilutions of methylase treated DNA and LRE1 PCR product. To estimate predictors of LRE1 methylation, we modeled LRE1 relative methylation as a linear function of covariates, using generalized linear models. Because LRE1 methylation values fall between zero and one inclusive, we used settings appropriate to a beta response: an identity link function, the binomial variance Var (µ) = µ (1 – µ), and a scale parameter estimated with Pearson residuals. We assessed goodness-of-fit using the cumulative residual method of Lin et al. (34), with 10,000 resampled residual processes. Our model examined the effect of tobacco smoking (examined first as pack-years smoking, but then specifically as years smoking because this variable better captured the response), alcohol consumption as decade-specific lifetime average drinks per week, patient BMI 5 years before diagnosis, tumor HPV DNA status, tumor stage, age, and gender. Alcohol consumption and tumor stage were removed from the model because they showed no significant association with LRE1 methylation level and their removal did not effect the estimates of the other predictors. Age, gender, and tumor HPV status, although insignificant, were retained in the model to control for residual confounding. To examine the modification of the effect of smoking duration by tumor HPV DNA status, a multiplicative interaction term was added to the model to determine if the interaction was statistically significant.

To examine the effect of LRE1 methylation status on patient survival, LRE1 methylation level was dichotomized at its median (0.45) and a Kaplan-Meier survival probability curve was constructed for the groups using the log-rank test to examine the difference in survival by LRE1 methylation level. To control for additional confounders of patient survival, including tumor stage and patient age and gender, Cox proportional hazards models were used. Survival examinations were done overall and in models stratified by tumor HPV DNA status. All statistical tests were two sided, with P < 0.05 considered statistically significant.


   Results
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 Abstract
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 Subjects and Methods
 Results
Discussion
 References
 
The demographics of the study population are shown in Table 1 , which compares the full case population to the subjects included in the final model of LRE1 methylation (those subjects with complete data for all variables of interest, n = 193). The subjects included in the model did not differ significantly from the complete case population in terms of demographics or in the distribution of the risk factors of the disease. The LRE1 methylation level ranged from 0.01 to 0.95, with a median value of 0.45.


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  		Table 1. Demographic comparison of head and neck squamous cell carcinoma cases from full population and samples included in analysis

 
We observed in a model of LRE1 methylation level that the patient's BMI 5 years before diagnosis was significantly associated with an increased LRE1 methylation level (P < 0.003; Table 2 ). At the same time, lifetime smoking duration was significantly associated with a reduced LRE1 methylation level (P < 0.02; Table 2) or a greater degree of tumor hypomethylation. Tumor HPV16 DNA status was not significantly associated with LRE1 relative methylation level or with patient age or gender. Further, because we have previously measured LRE1 relative methylation level in the blood from these patients (31), we compared the levels in blood to those in the tumors and found no association (data not shown). As it has been hypothesized that distinct molecular phenotypes may exist in tumor arising from HPV infection (4, 35), we examined how tumor HPV DNA status may modify the effect of exposure on LRE1 relative methylation level. Although the interaction between smoking duration and tumor HPV status was of borderline significance [P < 0.1 (not significant)], the model suggests that smoking duration in HPV-negative individuals has an enhanced negative effect on LRE1 methylation status (estimate of effect, –0.0025; P < 0.006), and that HPV status may also negatively effect LRE1 methylation level in nonsmoking individuals [estimate of effect, –0.11; P < 0.06 (not significant)], in models controlled for age, gender, and BMI.


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  		Table 2. Main effect of demographics and exposure on relative LRE1 methylation in head and neck squamous cell carcinoma tumors

 
LRE1 relative methylation level also had a borderline statistically significant effect on overall patient survival, with patients who have LRE1 relative methylation below the median (<0.45) showing reduced survival time [P < 0.06 (not significant); Fig. 1A ]. In proportional hazards models, the low LRE1 methylation level showed a statistically significant (P < 0.04) 70% increased instantaneous risk of death (Table 3 ) in a model controlled for age, gender, tumor stage, and tumor HPV DNA status, which was previously shown to be associated with better survival (4-6). We also explored whether this association of hypomethylation with survival differed by HPV status. Stratified survival analyses suggested that among HPV16-negative individuals, low LRE1 relative methylation level is associated with poorer survival [log rank P < 0.07 (not significant); Fig. 1B; Table 3], whereas there was no effect on survival by LRE1 methylation level in patients whose tumors are positive for HPV16 DNA (Fig. 1C; Table 3).


Figure 1
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  		Figure 1. Kaplan-Meier survival probability curves comparing patients whose tumors exhibit LRE1 methylation level less than the median of 0.45 versus those whose tumor LRE1 methylation level is ≥0.45, in the overall patient population (A), among patients with HPV-negative tumors (B) and among patients with HPV positive tumors (C). Censored values are represented by hatch marks, and difference in the survival was examined using the log-rank test.

 

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  		Table 3. Cox proportional hazards model of survival, overall and stratified by tumor HPV DNA status

 

   Discussion
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Consistent with previous examinations of tumor-specific genomic methylation extent (30), we observed a wide range of LRE1 relative methylation level in our population of head and neck squamous cell carcinoma tumors. This could suggest that even a relatively small loss of methylation in normally highly methylated DNA among susceptible individuals may be a precipitating factor in carcinogenesis. Of the cancers studied, head and neck squamous cell carcinoma displayed one of the greatest increases in hypomethylation compared with normal tissue (30). The wide range is also strikingly different than what was observed in peripheral blood–derived DNA from this same population, where the range of LRE1 level was more narrowed and differed only slightly, but significantly, between cases and controls (31).

We observed an association between smoking duration and tumor genomic hypomethylation, particularly in HPV16-negative tumors, where smoking is the major etiologic contributor to the disease. We did not observe, however, any association with alcohol consumption, the other major risk factor in non-HPV–associated disease. Previous studies have observed a significant association between global DNA methylation in buccal mucosal cells and malignant tissues of the lung, but not between methylation in peripheral leukocytes and lung tissues (36), consistent with our data. However, this does suggest a possible role for tobacco smoke in the induction of tissue-specific global hypomethylation. The mechanism for tobacco smoke leading to global hypomethylation is unknown, but studies have shown that hydrocarbons such as those present in tobacco smoke are capable of inactivating folic acid and vitamin B12 (37), and thus may alter the availability and enzymatic pathways responsible for methylating DNA appropriately. A study supporting this hypothesis showed that oral mucosal folate levels were much lower in the buccal mucosal cells of smokers than in nonsmokers (38).

We hypothesized that global hypomethylation would be differential based on HPV DNA presence, but found that HPV DNA presence was not associated with global hypomethylation. Although global hypomethylation has not been shown to be associated with HPV presence in tumors, HPV16 itself is targeted by methylation, with hypomethylation correlating with carcinogenic progression (39). The fact that HPV becomes methylated in the host and the fact that it integrates into repeat regions, including transposons (40), led us to hypothesize that global hypomethylation status in the tumor would differ by HPV DNA presence. The lack of association between HPV and global hypomethylation in our study may reflect the fact that HPV is known to integrate randomly throughout the genome (41) and may not induce or alter methylation levels to perform this integration.

Our observation that smoking duration had an association with hypomethylation only in HPV-negative tumors suggests that distinct molecular phenotypes may characterize tumors arising from HPV compared with those arising from exposure to tobacco smoke and alcohol use. These molecular distinctions may also better explain the striking difference in patient response by tumor HPV status, which has been hypothesized to be related to better radiation response of HPV-positive tumors (42-44).

There was, however, a strong positive association between patient BMI 5 years before diagnosis and LRE1 relative methylation level. The ascertainment of BMI before diagnosis is to prevent potential confounding of patient weight by the development of disease. This finding is of interest, as greater BMI has been linked to protection from risk for head and neck squamous cell carcinoma (15-19), although the mechanism underlying this protective association remains elusive. These results suggest that a higher BMI may play a role in maintaining the DNA methylation status of target tissue, and through this effect a protection from the disease. This is most likely metabolic, perhaps related to better or greater nutrient intake by individuals with higher BMI. It would be of interest to examine if similar trends can be observed in nondiseased tissues based on BMI, or if specific somatic tissues are more susceptible to the effect of nutrition on the epigenetic state.

We observed that tumors with lower LRE1 relative methylation predict poorer survival, and this is particularly the case in HPV-negative individuals. Those HPV-negative individuals already have a poorer overall survival compared with those individuals whose tumors were HPV16 DNA positive (14, 42-44). This suggests that tumors with reduced LRE1 methylation have a more aggressive phenotype or have greater resistance to the therapeutic modalities aimed at the disease. These differences may be due to the genomic instability linked to reduced methyl-cytosine content (45-47). This finding could be clinically useful, as patients with reduced methylation levels may benefit from more aggressive therapies.

We have shown an association between smoking and hypomethylation in tumors from head and neck squamous cell carcinoma patients, suggesting an additional mechanism for tobacco smoke in initiating genomic instability. Additionally, we observed that alterations in the extent of genomic methylation may have clinical relevance, a finding that should be further explored.


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

Note: Current address for K.T. Kelsey: Department of Community Health and Department of Pathology and Laboratory Medicine, Center for Environmental Health and Technology, Brown University, Providence, RI 02912.

Received 11/ 7/07; revised 1/ 3/08; accepted 1/15/08.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43–66.[Abstract/Free Full Text]
  2. Herrero R. Chapter 7: Human papillomavirus and cancer of the upper aerodigestive tract. J Natl Cancer Inst Monogr 2003;2003:47–51.[Abstract/Free Full Text]
  3. Zeka A, Gore R, Kriebel D. Effects of alcohol and tobacco on aerodigestive cancer risks: a meta-regression analysis. Cancer Causes Control 2003;14:897–906.[CrossRef][Medline]
  4. Koch WM, Lango M, Sewell D, Zahurak M, Sidransky D. Head and neck cancer in nonsmokers: a distinct clinical and molecular entity. Laryngoscope 1999;109:1544–51.[CrossRef][Medline]
  5. Myers JN, Elkins T, Roberts D, Byers RM. Squamous cell carcinoma of the tongue in young adults: increasing incidence and factors that predict treatment outcomes. Otolaryngol Head Neck Surg 2000;122:44–51.[CrossRef][Medline]
  6. Mendez P, Jr., Maves MD, Panje WR. Squamous cell carcinoma of the head and neck in patients under 40 years of age. Arch Otolaryngol 1985;111:762–4.[Abstract/Free Full Text]
  7. Loning T, Lango M, Becker J, Zahurak M, Sidransky D. Head and neck cancer in nonsmokers: a distinct clinical and molecular entity. Laryngoscope 1985;84:417–20.
  8. Smith EM, Summersgill KF, Allen J, et al. Human papillomavirus and risk of laryngeal cancer. Ann Otol Rhinol Laryngol 2000;109:1069–76.[Medline]
  9. Mendez P, Jr., Maves M, Panje WR. Squamous cell carcinoma of the head and neck. Clin Cancer Res 2002;8:762–4.
  10. Tuyns AJ. Aetiology of head and neck cancer: tobacco, alcohol and diet. Adv Otorhinolaryngol 1991;46:98–106.[Medline]
  11. Gillison ML, Shah KV. Human papillomavirus-associated head and neck squamous cell carcinoma: mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol 2001;13:183–8.[CrossRef][Medline]
  12. Ringstrom E, Peters E, Hasegawa M, Posner M, Liu M, Kelsey KT. Human papillomavirus type 16 and squamous cell carcinoma of the head and neck. Clin Cancer Res 2002;8:3187–92.[Abstract/Free Full Text]
  13. Schwartz SM, Daling JR, Doody DR, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998;90:1626–36.[Abstract/Free Full Text]
  14. Furniss CS, McClean MD, Smith JF, et al. Human papillomavirus 16 and head and neck squamous cell carcinoma. Int J Cancer 2007;120:2386–92.[CrossRef][Medline]
  15. Franceschi S, Dal Maso L, Levi F, Conti E, Talamini R, La Vecchia C. Leanness as early marker of cancer of the oral cavity and pharynx. Ann Oncol 2001;12:331–6.[Abstract/Free Full Text]
  16. Nieto A, Sanchez MJ, Quintana MJ, et al. BMI throughout life, intake of vitamin supplements and oral cancer in Spain. IARC Sci Publ 2002;156:259–61.[Medline]
  17. Rajkumar T, Sridhar H, Balaram P, et al. Oral cancer in Southern India: the influence of body size, diet, infections and sexual practices. Eur J Cancer Prev 2003;12:135–43.[CrossRef][Medline]
  18. Nieto A, Sanchez MJ, Martinez C, et al. Lifetime body mass index and risk of oral cavity and oropharyngeal cancer by smoking and drinking habits. Br J Cancer 2003;89:1667–71.[CrossRef][Medline]
  19. Kreimer AR, Randi G, Herrero R, Castellsague X, La Vecchia C, Franceschi S. Diet and body mass, and oral and oropharyngeal squamous cell carcinomas: analysis from the IARC multinational case-control study. Int J Cancer 2006;118:2293–7.[CrossRef][Medline]
  20. Sturgis EM, Wei Q, Spitz MR. Descriptive epidemiology and risk factors for head and neck cancer. Semin Oncol 2004;31:726–33.[CrossRef][Medline]
  21. Dunn BK. Hypomethylation: one side of a larger picture. Ann N Y Acad Sci 2003;983:28–42.[Medline]
  22. Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science 2003;300:489–92.[Abstract/Free Full Text]
  23. Lengauer C, Kinzler KW, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells. Proc Natl Acad Sci U S A 1997;94:2545–50.[Abstract/Free Full Text]
  24. Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997;13:335–40.[CrossRef][Medline]
  25. Florl AR, Franke KH, Niederacher D, Gerharz CD, Seifert HH, Schulz WA. DNA methylation and the mechanisms of CDKN2A inactivation in transitional cell carcinoma of the urinary bladder. Lab Invest 2000;80:1513–22.[Medline]
  26. Bratthauer GL, Fanning TG. Active LINE-1 retrotransposons in human testicular cancer. Oncogene 1992;7:507–10.[Medline]
  27. Lin CH, Hsieh SY, Sheen IS, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res 2001;61:4238–43.[Abstract/Free Full Text]
  28. Dante R, Dante-Paire J, Rigal D, Roizes G. Methylation patterns of long interspersed repeated DNA and alphoid repetitive DNA from human cell lines and tumors. Anticancer Res 1992;12:559–63.[Medline]
  29. Santourlidis S, Florl A, Ackermann R, Wirtz HC, Schulz WA. High frequency of alterations in DNA methylation in adenocarcinoma of the prostate. Prostate 1999;39:166–74.[CrossRef][Medline]
  30. Chalitchagorn K, Shuangshoti S, Hourpai N, et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 2004;23:8841–6.[CrossRef][Medline]
  31. Ting Hsiung D, Marsit CJ, Houseman EA, et al. Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev 2007;16:108–14.[Abstract/Free Full Text]
  32. Peters ES, McClean MD, Liu M, Eisen EA, Mueller N, Kelsey KT. The ADH1C polymorphism modifies the risk of squamous cell carcinoma of the head and neck associated with alcohol and tobacco use. Cancer Epidemiol Biomarkers Prev 2005;14:476–82.[Abstract/Free Full Text]
  33. Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 1998;153:1731–9.[Abstract/Free Full Text]
  34. Lin DY, Wei LJ, Ying Z. Model-checking techniques based on cumulative residuals. Biometrics 2002;58:1–12.[CrossRef][Medline]
  35. Marsit CJ, McClean MD, Furniss CS, Kelsey KT. Epigenetic inactivation of the SFRP genes is associated with drinking, smoking and HPV in head and neck squamous cell carcinoma. Int J Cancer 2006;119:1761–6.[CrossRef][Medline]
  36. Johanning GL, Heimburger DC, Piyathilake CJ. DNA methylation and diet in cancer. J Nutr 2002;132:3814–8S.
  37. Francis KT, Thompson RW, Krumdieck CL. Reaction of tetrahydrofolic acid with cyanate from urea solutions: formation of an inactive folate derivative. Am J Clin Nutr 1977;30:2028–32.[Abstract/Free Full Text]
  38. Piyathilake CJ, Hine RJ, Dasanayake AP, et al. Effect of smoking on folate levels in buccal mucosal cells. Int J Cancer 1992;52:566–9.[Medline]
  39. Badal V, Chuang LS, Tan EH, et al. CpG methylation of human papillomavirus type 16 DNA in cervical cancer cell lines and in clinical specimens: genomic hypomethylation correlates with carcinogenic progression. J Virol 2003;77:6227–34.[Abstract/Free Full Text]
  40. Kahn T, Turazza E, Ojeda R, et al. Integration of human papillomavirus type 6a DNA in a tonsillar carcinoma: chromosomal localization and nucleotide sequence of the genomic target region. Cancer Res 1994;54:1305–12.[Abstract/Free Full Text]
  41. Luft F, Klaes R, Nees M, et al. Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells. Int J Cancer 2001;92:9–17.[CrossRef][Medline]
  42. Hoffmann M, Gorogh T, Gottschlich S, et al. Human papillomaviruses in head and neck cancer: 8 year-survival-analysis of 73 patients. Cancer Lett 2005;218:199–206.[CrossRef][Medline]
  43. Lindel K, Beer KT, Laissue J, Greiner RH, Aebersold DM. Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma. Cancer 2001;92:805–13.[CrossRef][Medline]
  44. Schwartz SR, Yueh B, McDougall JK, Daling JR, Schwartz SM. Human papillomavirus infection and survival in oral squamous cell cancer: a population-based study. Otolaryngol Head Neck Surg 2001;125:1–9.[CrossRef][Medline]
  45. Pufulete M, Al-Ghnaniem R, Leather AJ, et al. Folate status, genomic DNA hypomethylation, and risk of colorectal adenoma and cancer: a case control study. Gastroenterology 2003;124:1240–8.[CrossRef][Medline]
  46. Weisenberger DJ, Campan M, Long TI, et al. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 2005;33:6823–36.[Abstract/Free Full Text]
  47. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004;32:e38.[Abstract/Free Full Text]



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