Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

Clinical Epigenetics

Alexander Dobrovic³  Benjamin Solomon⁹  Danny Rischin¹  Bernard Lyons^1,10  Elizabeth Sigston⁷  Stephen Kleid⁴  David Wiesenfeld⁵  June Corry²  Richard J Young⁹  Christopher Angel³  Elena A Takano³  Hongdo Do³  Marnie Collins⁸  Nicholas Wong⁶  Ida LM Candiloro^1,11  Annette M Lim⁹ 

[1] The University of Melbourne, Parkville, Victoria 3010, Australia;Department of Radiation Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia;Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia;Department of Surgical Oncology, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia;Department of Surgery, Royal Melbourne Hospital, 300 Grattan St, Parkville, Victoria 3050, Australia;Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Health, 145-163 Studley Road, Heidelberg, Victoria 3084, Australia;Department of Surgery, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia;Department of Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia;Research Division, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia;Department of Surgery, St Vincent’s Hospital, PO Box 2900, Fitzroy, Victoria 3065, Australia;Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3010, Australia

关键词: Tongue;    RUNX3;    Head and neck cancer;    Methylation;    Quantitative;   

Others  :  1092760
DOI  :  10.1186/1868-7083-6-22

 received in 2014-08-12, accepted in 2014-10-17,  发布年份 2014

【 摘 要 】

Background

DNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC).

Results

The methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing.

In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated.

Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76).

Conclusions

We demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.

【 授权许可】

   
2014 Lim et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Sandoval J, Esteller M: Cancer epigenomics: beyond genomics. Curr Opin Genet Dev 2012, 22:50-55.
• [2]Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JE, Hau P, Mirimanoff RO, Cairncross JG, Janzer RC, Stupp R: MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005, 352:997-1003.
• [3]Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A, Schoch R, Gattermann N, Sanz G, List A, Gore SD, Seymour JF, Bennett JM, Byrd J, Backstrom J, Zimmerman L, McKenzie D, Beach CL, Silverman LR: Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. The Lancet Oncology 2009, 10:223-232.
• [4]Baylin SB, Jones PA: A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer 2011, 11:726-734.
• [5]Reed AL, Califano J, Cairns P, Westra WH, Jones RM, Koch W, Ahrendt S, Eby Y, Sewell D, Nawroz H, Bartek J, Sidransky D: High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res 1996, 56:3630-3633.
• [6]Carvalho AL, Henrique R, Jeronimo C, Nayak CS, Reddy AN, Hoque MO, Chang S, Brait M, Jiang WW, Kim MM, Claybourne Q, Goldenberg D, Khan Z, Khan T, Westra WH, Sidranksy D, Koch W, Califano JA: Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance. Clin Cancer Res 2011, 17:4782-4789.
• [7]Sinha P, Bahadur S, Thakar A, Matta A, Macha M, Ralhan R, Gupta SD: Significance of promoter hypermethylation of p16 gene for margin assessment in carcinoma tongue. Head Neck 2009, 31:1423-1430.
• [8]Huang KH, Huang SF, Chen IH, Liao CT, Wang HM, Hsieh LL: Methylation of RASSF1A, RASSF2A, and HIN-1 is associated with poor outcome after radiotherapy, but not surgery, in oral squamous cell carcinoma. Clin Cancer Res 2009, 15:4174-4180.
• [9]Dikshit RP, Gillio-Tos A, Brennan P, De Marco L, Fiano V, Martinez-Penuela JM, Boffetta P, Merletti F: Hypermethylation, risk factors, clinical characteristics, and survival in 235 patients with laryngeal and hypopharyngeal cancers. Cancer 2007, 110:1745-1751.
• [10]Rosas SL, Koch W, da Costa Carvalho MG, Wu L, Califano J, Westra W, Jen J, Sidransky D: Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res 2001, 61:939-942.
• [11]Kristensen LS, Hansen LL: PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 2009, 55:1471-1483.
• [12]Brandes JC, Carraway H, Herman JG: Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. Oncogene 2007, 26:6229-6237.
• [13]Lee ES, Issa JP, Roberts DB, Williams MD, Weber RS, Kies MS, El-Naggar AK: Quantitative promoter hypermethylation analysis of cancer-related genes in salivary gland carcinomas: comparison with methylation-specific PCR technique and clinical significance. Clin Cancer Res 2008, 14:2664-2672.
• [14]Mikeska T, Candiloro IL, Dobrovic A: The implications of heterogeneous DNA methylation for the accurate quantification of methylation. Epigenomics 2010, 2:561-573.
• [15]Wojdacz TK: The limitations of locus specific methylation qualification and quantification in clinical material. Front Genet 2012, 3:21.
• [16]Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996, 93:9821-9826.
• [17]Tawfik HM, El-Maqsoud NM, Hak BH, El-Sherbiny YM: Head and neck squamous cell carcinoma: mismatch repair immunohistochemistry and promoter hypermethylation of hMLH1 gene. Am J Otolaryngol 2011, 32:528-536.
• [18]Ai L, Vo QN, Zuo C, Li L, Ling W, Suen JY, Hanna E, Brown KD, Fan CY: Ataxia-telangiectasia-mutated (ATM) gene in head and neck squamous cell carcinoma: promoter hypermethylation with clinical correlation in 100 cases. Cancer Epidemiol Biomarkers Prev 2004, 13:150-156.
• [19]Lima LM, de Souza LR, da Silva TF, Pereira CS, Guimaraes AL, de Paula AM, de Andrade CH: DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors. Histopathology 2012, 60:489-496.
• [20]Szaumkessel M, Richter J, Giefing M, Jarmuz M, Kiwerska K, Tonnies H, Grenman R, Heidemann S, Szyfter K, Siebert R: Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma. Int J Oncol 2011, 39:505-514.
• [21]Supic G, Kozomara R, Jovic N, Zeljic K, Magic Z: Hypermethylation of RUNX3 but not WIF1 gene and its association with stage and nodal status of tongue cancers. Oral Dis 2011, 17:794-800.
• [22]Gao S, Eiberg H, Krogdahl A, Liu CJ, Sorensen JA: Cytoplasmic expression of E-cadherin and beta-Catenin correlated with LOH and hypermethylation of the APC gene in oral squamous cell carcinomas. J Oral Pathol Med 2005, 34:116-119.
• [23]Tsunematsu T, Kudo Y, Iizuka S, Ogawa I, Fujita T, Kurihara H, Abiko Y, Takata T: RUNX3 has an oncogenic role in head and neck cancer. PLoS One 2009, 4:e5892.
• [24]Worsham MJ, Chen KM, Meduri V, Nygren AO, Errami A, Schouten JP, Benninger MS: Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2006, 132:668-677.
• [25]Chang HW, Chow V, Lam KY, Wei WI, Yuen A: Loss of E-cadherin expression resulting from promoter hypermethylation in oral tongue carcinoma and its prognostic significance. Cancer 2002, 94:386-392.
• [26]Gao S, Worm J, Guldberg P, Eiberg H, Krogdahl A, Liu CJ, Reibel J, Dabelsteen E: Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma. Int J Cancer 2004, 109:230-237.
• [27]Supic G, Kozomara R, Brankovic-Magic M, Jovic N, Magic Z: Gene hypermethylation in tumor tissue of advanced oral squamous cell carcinoma patients. Oral Oncol 2009, 45:1051-1057.
• [28]Gao S, Bennett EP, Reibel J, Chen X, Christensen ME, Krogdahl A, Dabelsteen E: Histo-blood group ABO antigen in oral potentially malignant lesions and squamous cell carcinoma–genotypic and phenotypic characterization. APMIS 2004, 112:11-20.
• [29]Supic G, Kozomara R, Jovic N, Zeljic K, Magic Z: Prognostic significance of tumor-related genes hypermethylation detected in cancer-free surgical margins of oral squamous cell carcinomas. Oral Oncol 2011, 47:702-708.
• [30]Stephen JK, Chen KM, Shah V, Havard S, Kapke A, Lu M, Benninger MS, Worsham MJ: DNA hypermethylation markers of poor outcome in laryngeal cancer. Clin Epigenetics 2010, 1:61-69.
• [31]Rigi-Ladiz MA, Kordi-Tamandani DM, Torkamanzehi A: Analysis of hypermethylation and expression profiles of APC and ATM genes in patients with oral squamous cell carcinoma. Clin Epigenetics 2011, 3:6. BioMed Central Full Text
• [32]Yalniz Z, Demokan S, Suoglu Y, Ulusan M, Dalay N: Simultaneous methylation profiling of tumor suppressor genes in head and neck cancer. DNA Cell Biol 2011, 30:17-24.
• [33]Bolt J, Vo QN, Kim WJ, McWhorter AJ, Thomson J, Hagensee ME, Friedlander P, Brown KD, Gilbert J: The ATM/p53 pathway is commonly targeted for inactivation in squamous cell carcinoma of the head and neck (SCCHN) by multiple molecular mechanisms. Oral Oncol 2005, 41:1013-1020.
• [34]Shaw RJ, Hall GL, Lowe D, Liloglou T, Field JK, Sloan P, Risk JM: The role of pyrosequencing in head and neck cancer epigenetics: correlation of quantitative methylation data with gene expression. Arch Otolaryngol Head Neck Surg 2008, 134:251-256.
• [35]Marsit CJ, Liu M, Nelson HH, Posner M, Suzuki M, Kelsey KT: Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival. Oncogene 2004, 23:1000-1004.
• [36]Maruya S, Issa JP, Weber RS, Rosenthal DI, Haviland JC, Lotan R, El-Naggar AK: Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications. Clin Cancer Res 2004, 10:3825-3830.
• [37]Shaw R, Hobkirk A, Nikolaidis G, Woolgar JA, Triantafyllou A, Brown JS, Liloglou T, Risk JM: Molecular staging of surgical margins in oral squamous cell carcinoma using promoter methylation of p16(INK4A), cytoglobin, E-cadherin, and TMEFF2. Ann Surg Oncol 2013, 20:2796-2802.
• [38]Calmon MF, Colombo J, Carvalho F, Souza FP, Filho JF, Fukuyama EE, Camargo AA, Caballero OL, Tajara EH, Cordeiro JA, Rahal P: Methylation profile of genes CDKN2A (p14 and p16), DAPK1, CDH1, and ADAM23 in head and neck cancer. Cancer Genet Cytogenet 2007, 173:31-37.
• [39]Marsit CJ, Posner MR, McClean MD, Kelsey KT: Hypermethylation of E-cadherin is an independent predictor of improved survival in head and neck squamous cell carcinoma. Cancer 2008, 113:1566-1571.
• [40]De Schutter H, Geeraerts H, Verbeken E, Nuyts S: Promoter methylation of TIMP3 and CDH1 predicts better outcome in head and neck squamous cell carcinoma treated by radiotherapy only. Oncol Rep 2009, 21:507-513.
• [41]Steinmann K, Sandner A, Schagdarsurengin U, Dammann RH: Frequent promoter hypermethylation of tumor-related genes in head and neck squamous cell carcinoma. Oncol Rep 2009, 22:1519-1526.
• [42]Kordi-Tamandani DM, Moazeni-Roodi AK, Rigi-Ladiz MA, Hashemi M, Birjandian E, Torkamanzehi A: Promoter hypermethylation and expression profile of MGMT and CDH1 genes in oral cavity cancer. Arch Oral Biol 2010, 55:809-814.
• [43]Nakayama S, Sasaki A, Mese H, Alcalde RE, Tsuji T, Matsumura T: The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas. Int J Cancer 2001, 93:667-673.
• [44]Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, Field JK, Risk JM: Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 2006, 94:561-568.
• [45]Hasegawa M, Nelson HH, Peters E, Ringstrom E, Posner M, Kelsey KT: Patterns of gene promoter methylation in squamous cell cancer of the head and neck. Oncogene 2002, 21:4231-4236.
• [46]Su PF, Huang WL, Wu HT, Wu CH, Liu TY, Kao SY: p16(INK4A) promoter hypermethylation is associated with invasiveness and prognosis of oral squamous cell carcinoma in an age-dependent manner. Oral Oncol 2010, 46:734-739.
• [47]Viswanathan M, Tsuchida N, Shanmugam G: Promoter hypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cell carcinoma. Int J Cancer 2003, 105:41-46.
• [48]Weiss D, Basel T, Sachse F, Braeuninger A, Rudack C: Promoter methylation of cyclin A1 is associated with human papillomavirus 16 induced head and neck squamous cell carcinoma independently of p53 mutation. Mol Carcinog 2011, 50:680-688.
• [49]Sasiadek MM, Stembalska-Kozlowska A, Smigiel R, Ramsey D, Kayademir T, Blin N: Impairment of MLH1 and CDKN2A in oncogenesis of laryngeal cancer. Br J Cancer 2004, 90:1594-1599.
• [50]Wong TS, Man MWL, Lam AKY, Wei WI, Kwong YL, Yuen APW: The study of p16 and p15 gene methylation in head and neck squamous cell carcinoma and their quantitative evaluation in plasma by real-time PCR. Eur J Cancer 2003, 39:1881-1887.
• [51]Puri SK, Si L, Fan CY, Hanna E: Aberrant promoter hypermethylation of multiple genes in head and neck squamous cell carcinoma. Am J Otolaryngol 2005, 26:12-17.
• [52]Kato K, Hara A, Kuno T, Mori H, Yamashita T, Toida M, Shibata T: Aberrant promoter hypermethylation of p16 and MGMT genes in oral squamous cell carcinomas and the surrounding normal mucosa. J Cancer Res Clin Oncol 2006, 132:735-743.
• [53]Taioli E, Ragin C, Wang XH, Chen J, Langevin SM, Brown AR, Gollin SM, Garte S, Sobol RW: Recurrence in oral and pharyngeal cancer is associated with quantitative MGMT promoter methylation. BMC Cancer 2009, 9:354. BioMed Central Full Text
• [54]Kaur J, Demokan S, Tripathi SC, Macha MA, Begum S, Califano JA, Ralhan R: Promoter hypermethylation in Indian primary oral squamous cell carcinoma. Int J Cancer 2010, 127:2367-2373.
• [55]Khor GH, Froemming GR, Zain RB, Abraham MT, Omar E, Tan SK, Tan AC, Vincent-Chong VK, Thong KL: DNA methylation profiling revealed promoter hypermethylation-induced silencing of p16, DDAH2 and DUSP1 in primary oral squamous cell carcinoma. Int J Med Sci 2013, 10:1727-1739.
• [56]O’Regan EM, Toner ME, Finn SP, Fan CY, Ring M, Hagmar B, Timon C, Smyth P, Cahill S, Flavin R, Sheils OM, O’Leary JJ: p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas. Hum Pathol 2008, 39:452-458.
• [57]Lin SC, Chang KW, Chang CS, Liu TY, Tzeng YS, Yang FS, Wong YK: Alterations of p16/MTS1 gene in oral squamous cell carcinomas from Taiwanese. J Oral Pathol Med 2000, 29:159-166.
• [58]Farias LC, Fraga CA, De Oliveira MV, Silva TF, Marques-Silva L, Moreira PR, De-Paula AM, Gomez RS, Guimaraes AL: Effect of age on the association between p16CDKN2A methylation and DNMT3B polymorphism in head and neck carcinoma and patient survival. Int J Oncol 2010, 37:167-176.
• [59]Martone T, Gillio-Tos A, De Marco L, Fiano V, Maule M, Cavalot A, Garzaro M, Merletti F, Cortesina G: Association between hypermethylated tumor and paired surgical margins in head and neck squamous cell carcinomas. Clin Cancer Res 2007, 13:5089-5094.
• [60]Laytragoon-Lewin N, Chen F, Castro J, Elmberger G, Rutqvist LE, Lewin F, Turesson I, Lundgren J: DNA content and methylation of p16, DAPK and RASSF1A gene in tumour and distant, normal mucosal tissue of head and neck squamous cell carcinoma patients. Anticancer Res 2010, 30:4643-4648.
• [61]de Freitas C-SM, Stur E, Agostini LP, de Podesta JR, de Oliveira JC, Soares MS, Mendonca EF, Gouvea SA, Von Zeidler SV, Louro ID: Promoter hypermethylation in primary squamous cell carcinoma of the oral cavity and oropharynx: a study of a Brazilian cohort. Mol Biol Rep 2012, 39:10111-10119.
• [62]Gonzalez MV, Pello MF, Lopez-Larrea C, Suarez C, Menendez MJ, Coto E: Deletion and methylation of the tumour suppressor gene p16/CDKN2 in primary head and neck squamous cell carcinoma. J Clin Pathol 1997, 50:509-512.
• [63]Ogi K, Toyota M, Ohe-Toyota M, Tanaka N, Noguchi M, Sonoda T, Kohama G, Tokino T: Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma. Clin Cancer Res 2002, 8:3164-3171.
• [64]Sailasree R, Abhilash A, Sathyan KM, Nalinakumari KR, Thomas S, Kannan S: Differential roles of p16INK4A and p14ARF genes in prognosis of oral carcinoma. Cancer Epidemiol Biomarkers Prev 2008, 17:414-420.
• [65]Sanchez-Cespedes M, Esteller M, Wu L, Nawroz-Danish H, Yoo GH, Koch WM, Jen J, Herman JG, Sidransky D: Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000, 60:892-895.
• [66]Shiga K, Ogawa T, Katagiri K, Yoshida F, Tateda M, Matsuura K, Kobayashi T: Differences between oral cancer and cancers of the pharynx and larynx on a molecular level. Oncol Lett 2012, 3:238-243.
• [67]Wu CL, Roz L, McKown S, Sloan P, Read AP, Holland S, Porter S, Scully C, Paterson I, Tavassoli M, Thakker N: DNA studies underestimate the major role of CDKN2A inactivation in oral and oropharyngeal squamous cell carcinomas. Genes Chromosomes Cancer 1999, 25:16-25.
• [68]Paluszczak J, Misiak P, Wierzbicka M, Wozniak A, Baer-Dubowska W: Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in laryngeal squamous cell carcinomas and adjacent normal mucosa. Oral Oncol 2011, 47:104-107.
• [69]Huang S, Lee H, Mar K, Ji D, Huang M, Hsia K: Loss expression of O6-methylguanine DNA methyltransferase by promoter hypermethylation and its relationship to betel quid chewing in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010, 109:883-889.
• [70]Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG: Inactivation of the DNA Repair Gene O6-Methylguanine-DNA Methyltransferase by Promoter Hypermethylation is a Common Event in Primary Human Neoplasia. Cancer Res 1999, 59:793-797.
• [71]Zuo C, Ai L, Ratliff P, Suen JY, Hanna E, Brent TP, Fan CY: O6-methylguanine-DNA methyltransferase gene: epigenetic silencing and prognostic value in head and neck squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev 2004, 13:967-975.
• [72]Czerninski R, Krichevsky S, Ashhab Y, Gazit D, Patel V, Ben-Yehuda D: Promoter hypermethylation of mismatch repair genes, hMLH1 and hMSH2 in oral squamous cell carcinoma. Oral Dis 2009, 15:206-213.
• [73]Wang Y, Irish J, MacMillan C, Brown D, Xuan Y, Boyington C, Gullane P, Kamel-Reid S: High frequency of microsatellite instability in young patients with head-and-neck squamous-cell carcinoma: lack of involvement of the mismatch repair genes hMLH1 AND hMSH2. Int J Cancer 2001, 93:353-360.
• [74]Demokan S, Suoglu Y, Demir D, Gozeler M, Dalay N: Microsatellite instability and methylation of the DNA mismatch repair genes in head and neck cancer. Ann Oncol 2006, 17:995-999.
• [75]Sengupta S, Chakrabarti S, Roy A, Panda CK, Roychoudhury S: Inactivation of human mutL homolog 1 and mutS homolog 2 genes in head and neck squamous cell carcinoma tumors and leukoplakia samples by promoter hypermethylation and its relation with microsatellite instability phenotype. Cancer 2007, 109:703-712.
• [76]Liu K, Zuo C, Luo QK, Suen JY, Hanna E, Fan CY: Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma. Diagn Mol Pathol 2003, 12:50-56.
• [77]Gonzalez-Ramirez I, Ramirez-Amador V, Irigoyen-Camacho ME, Sanchez-Perez Y, Anaya-Saavedra G, Granados-Garcia M, Garcia-Vazquez F, Garcia-Cuellar CM: hMLH1 promoter methylation is an early event in oral cancer. Oral Oncol 2011, 47:22-26.
• [78]Hogg RP, Honorio S, Martinez A, Agathanggelou A, Dallol A, Fullwood P, Weichselbaum R, Kuo MJ, Maher ER, Latif F: Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma. Eur J Cancer 2002, 38:1585-1592.
• [79]Gao F, Huang C, Lin M, Wang Z, Shen J, Zhang H, Jiang L, Chen Q: Frequent inactivation of RUNX3 by promoter hypermethylation and protein mislocalization in oral squamous cell carcinomas. J Cancer Res Clin Oncol 2009, 135:739-747.
• [80]Lee HW, Hwang YH, Han JH, Choi JH, Kang SY, Jeong SH, Ann MS, Oh YT, Kim JH, Kim CH, Sheen SS: High expression of excision repair cross-complementation group 1 protein predicts poor outcome in patients with nasopharyngeal cancer. Oral Oncol 2010, 46:209-213.
• [81]Handra-Luca A, Hernandez J, Mountzios G, Taranchon E, Lacau-St-Guily J, Soria JC, Fouret P: Excision repair cross complementation group 1 immunohistochemical expression predicts objective response and cancer-specific survival in patients treated by Cisplatin-based induction chemotherapy for locally advanced head and neck squamous cell carcinoma. Clin Cancer Res 2007, 13:3855-3859.
• [82]Jun HJ, Ahn MJ, Kim HS, Yi SY, Han J, Lee SK, Ahn YC, Jeong HS, Son YI, Baek JH, Park K: ERCC1 expression as a predictive marker of squamous cell carcinoma of the head and neck treated with cisplatin-based concurrent chemoradiation. Br J Cancer 2008, 99:167-172.
• [83]Olaussen KA, Dunant A, Fouret P, Brambilla E, Andre F, Haddad V, Taranchon E, Filipits M, Pirker R, Popper HH, Stahel R, Sabatier L, Pignon JP, Tursz T, Le Chevalier T, Soria JC, Ialt Bio Investigators: DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 2006, 355:983-991.
• [84]Do H, Wong NC, Murone C, John T, Solomon B, Mitchell PL, Dobrovic A: A critical re-assessment of DNA repair gene promoter methylation in non-small cell lung carcinoma. Sci Rep 2014, 4:4186.
• [85]Cervigne NK, Machado J, Goswami RS, Sadikovic B, Bradley G, Perez-Ordonez B, Galloni NN, Gilbert R, Gullane P, Irish JC, Jurisica I, Reis PP, Kamel-Reid S: Recurrent genomic alterations in sequential progressive leukoplakia and oral cancer: drivers of oral tumorigenesis? Hum Mol Genet 2014, 23:2618-2628.
• [86]Wolff E, Girod S, Liehr T, Vorderwülbecke U, Ries J, Steininger H, Gebhart E: Oral squamous cell carcinomas are characterized by a rather uniform pattern of genomic imbalances detected by comparative genomic hybridisation. Oral Oncol 1998, 34:186-190.
• [87]Hecht F, Hecht BK: Cancer in ataxia-telangiectasia patients. Cancer Genet Cytogenet 1990, 46:9-19.
• [88]Lazar AD, Winter MR, Nogueira CP, Larson PS, Finnemore EM, Dolan RW, Fuleihan N, Chakravarti A, Zietman A, Rosenberg CL: Loss of heterozygosity at 11q23 in squamous cell carcinoma of the head and neck is associated with recurrent disease. Clin Cancer Res 1998, 4:2787-2793.
• [89]Lavin MF: The Mre11 complex and ATM: a two-way functional interaction in recognising and signaling DNA double strand breaks. DNA Repair (Amst) 2004, 3:1515-1520.
• [90]Lim AM, Young RJ, Collins M, Fox SB, McArthur GA, Corry J, Peters L, Rischin D, Solomon B: Correlation of Ataxia-Telangiectasia-Mutated (ATM) gene loss with outcome in head and neck squamous cell carcinoma. Oral Oncol 2012, 48:698-702.
• [91]Joenje H, Arwert F: Connecting Fanconi anemia to BRCA1. Nat Med 2001, 7:406-407.
• [92]Timmers C, Taniguchi T, Hejna J, Reifsteck C, Lucas L, Bruun D, Thayer M, Cox B, Olson S, D’Andrea AD, Moses R, Grompe M: Positional cloning of a novel Fanconi anemia gene, FANCD2. Mol Cell 2001, 7:241-248.
• [93]Oksuzoglu B, Yalcin S: Squamous cell carcinoma of the tongue in a patient with Fanconi’s anemia: a case report and review of the literature. Ann Hematol 2002, 81:294-298.
• [94]Sun D, Zhang Z, Van do N, Huang G, Ernberg I, Hu L: Aberrant methylation of CDH13 gene in nasopharyngeal carcinoma could serve as a potential diagnostic biomarker. Oral Oncol 2007, 43:82-87.
• [95]Brock MV, Hooker CM, Ota-Machida E, Han Y, Guo M, Ames S, Glockner S, Piantadosi S, Gabrielson E, Pridham G, Pelosky K, Belinsky SA, Yang SC, Baylin SB, Herman JG: DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med 2008, 358:1118-1128.
• [96]Lim AM, Do H, Young RJ, Wong SQ, Angel C, Collins M, Takano EA, Corry J, Wiesenfeld D, Kleid S, Sigston E, Lyons B, Fox SB, Rischin D, Dobrovic A, Solomon B: Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome. Int J Cancer 2014, 135:887-895.
• [97]Kristensen LS, Mikeska T, Krypuy M, Dobrovic A: Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res 2008, 36:e42.
• [98]Claus R, Wilop S, Hielscher T, Sonnet M, Dahl E, Galm O, Jost E, Plass C: A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR. Epigenetics 2012, 7:772-780.
• [99]Wojdacz TK, Dobrovic A, Hansen LL: Methylation-sensitive high-resolution melting. Nat Protoc 2008, 3:1903-1908.
• [100]Candiloro IL, Mikeska T, Dobrovic A: Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation. Epigenetics 2011, 6:500-507.
• [101]Liu Y, Ji Y, Qiu P: Identification of thresholds for dichotomizing DNA methylation data. EURASIP J Bioinform Syst Biol 2013, 2013:8. BioMed Central Full Text
• [102]Cameron EE, Baylin SB, Herman JG: p15INK4B CpG Island Methylation in Primary Acute Leukemia Is Heterogeneous and Suggests Density as a Critical Factor for Transcriptional Silencing. Blood 1999, 94:2445-2451.
• [103]Wee EJ, Rauf S, Shiddiky MJ, Dobrovic A, Trau M: DNA ligase-based strategy for quantifying heterogeneous DNA methylation without sequencing. Clin Chem 2014.
• [104]Claus R, Lucas DM, Stilgenbauer S, Ruppert AS, Yu L, Zucknick M, Mertens D, Buhler A, Oakes CC, Larson RA, Kay NE, Jelinek DF, Kipps TJ, Rassenti LZ, Gribben JG, Dohner H, Heerema NA, Marcucci G, Plass C, Byrd JC: Quantitative DNA methylation analysis identifies a single CpG dinucleotide important for ZAP-70 expression and predictive of prognosis in chronic lymphocytic leukemia. J Clin Oncol 2012, 30:2483-2491.
• [105]Pulling LC, Grimes MJ, Damiani LA, Juri DE, Do K, Tellez CS, Belinsky SA: Dual promoter regulation of death-associated protein kinase gene leads to differentially silenced transcripts by methylation in cancer. Carcinogenesis 2009, 30:2023-2030.
• [106]Bibikova M, Barnes B, Tsan C, Ho V, Klotzle B, Le JM, Delano D, Zhang L, Schroth GP, Gunderson KL, Fan JB, Shen R: High density DNA methylation array with single CpG site resolution. Genomics 2011, 98:288-295.
• [107]van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, Garcia-Sanz R, van Krieken JH, Droese J, Gonzalez D, Bastard C, White HE, Spaargaren M, Gonzalez M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA: Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003, 17:2257-2317.
• [108]Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 2007, 35:e41.
• [109]Worm J, Aggerholm A, Guldberg P: In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem 2001, 47:1183-1189.
• [110]Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, Moorman AF: Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 2009, 37:e45.
• [111]Zapparoli GV, Jorissen RN, Hewitt CA, McBean M, Westerman DA, Dobrovic A: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection. BMC Cancer 2013, 13:206. BioMed Central Full Text
• [112]Triche TJ Jr, Weisenberger DJ, Van Den Berg D, Laird PW, Siegmund KD: Low-level processing of Illumina Infinium DNA Methylation BeadArrays. Nucleic Acids Res 2013, 41:e90.
• [113]Maksimovic J, Gordon L, Oshlack A: SWAN: Subset-quantile within array normalization for illumina infinium HumanMethylation450 BeadChips. Genome Biol 2012, 13:R44. BioMed Central Full Text
• [114]Teschendorff AE, Marabita F, Lechner M, Bartlett T, Tegner J, Gomez-Cabrero D, Beck S: A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data. Bioinformatics 2013, 29:189-196.
• [115]Naeem H, Wong NC, Chatterton Z, Hong MK, Pedersen JS, Corcoran NM, Hovens CM, Macintyre G: Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the HumanMethylation450 array. BMC Genomics 2014, 15:51. BioMed Central Full Text
• [116]Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, Shen R, Gunderson KL: Genome-wide DNA methylation profiling using Infinium(R) assay. Epigenomics 2009, 1:177-200.