DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients and Samples
2.2. Study of Methylation by MLH1 and MGMT Pyrosequencing
2.3. Methylation Quantification
2.4. Variables and Collected Data
2.5. TCGA Cohort Sample Selection and Data Downloading
2.6. Statistical Analysis
3. Results
3.1. Sample Description
3.2. Methylation State
3.3. Follow-Up
3.4. Validation in External Dataset
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Warnakulasuriya, S. Global Epidemiology of Oral and Oropharyngeal Cancer. Oral Oncol. 2009, 45, 309–316. [Google Scholar] [CrossRef] [PubMed]
- Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef] [PubMed]
- Chi, A.C.; Day, T.A.; Neville, B.W. Oral Cavity and Oropharyngeal Squamous Cell Carcinoma–an Update. CA Cancer J. Clin. 2015, 65, 401–421. [Google Scholar] [CrossRef] [PubMed]
- Perez-Sayans, M.; Somoza-Martin, J.M.; Barros-Angueira, F.; Reboiras-Lopez, M.D.; Gandara Rey, J.M.; Garcia-Garcia, A. Genetic and Molecular Alterations Associated with Oral Squamous Cell Cancer (Review). Oncol. Rep. 2009, 22, 1277–1282. [Google Scholar] [CrossRef] [Green Version]
- Petti, S. Lifestyle Risk Factors for Oral Cancer. Oral Oncol. 2009, 45, 340–350. [Google Scholar] [CrossRef]
- Johnson, N.W.; Warnakulasuriya, S.; Gupta, P.C.; Dimba, E.; Chindia, M.; Otoh, E.C.; Sankaranarayanan, R.; Califano, J.; Kowalski, L. Global Oral Health Inequalities in Incidence and Outcomes for Oral Cancer: Causes and Solutions. Adv. Dent. Res. 2011, 23, 237–246. [Google Scholar] [CrossRef] [Green Version]
- Gomez, I.; Warnakulasuriya, S.; Varela-Centelles, P.I.; Lopez-Jornet, P.; Suarez-Cunqueiro, M.; Diz-Dios, P.; Seoane, J. Is Early Diagnosis of Oral Cancer a Feasible Objective? Who is to Blame for Diagnostic Delay? Oral Dis. 2010, 16, 333–342. [Google Scholar] [CrossRef] [PubMed]
- Speight, P.M.; Khurram, S.A.; Kujan, O. Oral Potentially Malignant Disorders: Risk of Progression to Malignancy. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2018, 125, 612–627. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kadyrov, F.A.; Dzantiev, L.; Constantin, N.; Modrich, P. Endonucleolytic Function of MutLalpha in Human Mismatch Repair. Cell 2006, 126, 297–308. [Google Scholar] [CrossRef] [Green Version]
- Gao, D.; Herman, J.G.; Guo, M. The Clinical Value of Aberrant Epigenetic Changes of DNA Damage Repair Genes in Human Cancer. Oncotarget 2016, 7, 37331–37346. [Google Scholar] [CrossRef]
- Ushijima, T. Detection, and Interpretation of Altered Methylation Patterns in Cancer Cells. Nat. Rev. Cancer 2005, 5, 223–231. [Google Scholar] [CrossRef]
- Scully, C. Challenges in Predicting which Oral Mucosal Potentially Malignant Disease Will Progress to Neoplasia. Oral Dis. 2014, 20, 1–5. [Google Scholar] [CrossRef]
- Gleber-Netto, F.O.; Braakhuis, B.J.; Triantafyllou, A.; Takes, R.P.; Kelner, N.; Rodrigo, J.P.; Strojan, P.; Vander Poorten, V.; Rapidis, A.D.; Rinaldo, A.; et al. Molecular Events in Relapsed Oral Squamous Cell Carcinoma: Recurrence Vs. Secondary Primary Tumor. Oral Oncol. 2015, 51, 738–744. [Google Scholar] [CrossRef]
- Shia, J. Evolving Approach and Clinical Significance of Detecting DNA Mismatch Repair Deficiency in Colorectal Carcinoma. In Seminars in Diagnostic Pathology; WB Saunders: Philadelphia, PA, USA, 2015; Volume 32, pp. 352–361. [Google Scholar]
- González-Ramírez, I.; Ramírez-Amador, V.; Irigoyen-Camacho, M.E.; Sánchez-Pérez, Y.; Anaya-Saavedra, G.; Granados-García, M.; García-Vázquez, F.; García-Cuellar, C.M. hMLH1 Promoter Methylation is an Early Event in Oral Cancer. Oral Oncol. 2011, 47, 22–26. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- De Oliveira, S.R.; Da Silva, I.C.; Mariz, B.A.; Pereira, A.M.; De Oliveira, N.F. DNA Methylation Analysis of Cancer-Related Genes in Oral Epithelial Cells of Healthy Smokers. Arch. Oral Biol. 2015, 60, 825–833. [Google Scholar] [CrossRef] [PubMed]
- Gerson, S.L. MGMT: Its Role in Cancer Aetiology and Cancer Therapeutics. Nat. Rev. Cancer 2004, 4, 296–307. [Google Scholar] [CrossRef]
- Von Elm, E.; Altman, D.G.; Egger, M.; Pocock, S.J.; Gøtzsche, P.C.; Vandenbroucke, J.P.; STROBE Initiative. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: Guidelines for Reporting Observational Studies. J. Clin. Epidemiol. 2008, 61, 344–349. [Google Scholar] [CrossRef] [Green Version]
- Cancer Genome Atlas Network. Comprehensive Genomic Characterization of Head and Neck Squamous Cell Carcinomas. Nature 2015, 517, 576–582. [Google Scholar] [CrossRef] [Green Version]
- Flausino, C.S.; Daniel, F.I.; Modolo, F. DNA Methylation in Oral Squamous Cell Carcinoma: From its Role in Carcinogenesis to Potential Inhibitor Drugs. Crit. Rev. Oncol. Hematol. 2021, 164, 103399. [Google Scholar] [CrossRef]
- Choi, S.; Myers, J.N. Molecular Pathogenesis of Oral Squamous Cell Carcinoma: Implications for Therapy. J. Dent. Res. 2008, 87, 14–32. [Google Scholar] [CrossRef] [PubMed]
- Jayaprakash, C.; Radhakrishnan, R.; Ray, S.; Satyamoorthy, K. Promoter Methylation of MGMT in Oral Carcinoma: A Population-Based Study and Meta-Analysis. Arch. Oral Biol. 2017, 80, 197–208. [Google Scholar] [CrossRef] [PubMed]
- Li, Q.; Hong, J.; Shen, Z.; Deng, H.; Shen, Y.; Wu, Z.; Zhou, C. A Systematic Review and Meta-Analysis Approach on Diagnostic Value of MLH1 Promoter Methylation for Head and Neck Squamous Cell Carcinoma. Medicine 2019, 98, e17651. [Google Scholar] [CrossRef] [PubMed]
- Murakami, J.; Asaumi, J.; Maki, Y.; Tsujigiwa, H.; Nagatsuka, H.; Kokeguchi, S.; Inoue, T.; Kawasaki, S.; Tanaka, N.; MacPhee, D.; et al. Influence of CpG Island Methylation Status in O6-Methylguanine-DNA Methyltransferase Expression of Oral Cancer Cell Lines. Oncol. Rep. 2004, 12, 339–345. [Google Scholar] [CrossRef] [Green Version]
- Ferlazzo, N.; Currò, M.; Zinellu, A.; Caccamo, D.; Isola, G.; Ventura, V.; Carru, C.; Matarese, G.; Ientile, R. Influence of MTHFR Genetic Background on p16 and MGMT Methylation in Oral Squamous Cell Cancer. Int. J. Mol. Sci. 2017, 18, 724. [Google Scholar] [CrossRef] [Green Version]
- Kordi-Tamandani, D.M.; Moazeni-Roodi, A.K.; Rigi-Ladiz, M.A.; 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. [Google Scholar] [CrossRef]
- Taioli, E.; Ragin, C.; Wang, X.H.; Chen, J.; Langevin, S.M.; Brown, A.R.; Gollin, S.M.; Garte, S.; Sobol, R.W. Recurrence in Oral and Pharyngeal Cancer is Associated with Quantitative MGMT Promoter Methylation. BMC Cancer 2009, 9, 354. [Google Scholar] [CrossRef] [Green Version]
- 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. [Google Scholar] [CrossRef]
- Donís, S.P.; González, A.P.; Alves, M.G.O.; do Carmo Carvalho, B.F.; Ferreira, C.C.P.; Almeida, J.D.; Iruegas, E.P.; Petronacci, C.M.C.; Peñaranda, J.M.S.; Sayáns, M.P. MLH1, MSH2, MRE11, and XRCC1 in Oral Leukoplakia and Oral Squamous Cell Carcinoma. Appl. Immunohistochem. Mol. Morphol. 2021, 29, 613–618. [Google Scholar] [CrossRef]
Qualitative Variables | N | % | |||
---|---|---|---|---|---|
Study group | OSCC | 16 | 18.8 | ||
Transformation | 47 | 55.3 | |||
Simultaneous | 22 | 25.9 | |||
Gender | Woman | 48 | 56.5 | ||
Man | 37 | 43.5 | |||
Location | Gingiva | 13 | 15.3 | ||
Tongue | 27 | 31.8 | |||
Buccal mucosa | 24 | 28.2 | |||
Hard palate | 2 | 2.4 | |||
Floor mouth | 2 | 2.4 | |||
Retromolar trigone | 8 | 9.4 | |||
Lip | 3 | 3.5 | |||
Soft palate | 3 | 3,5 | |||
Tonsil | 3 | 3.5 | |||
Exitus | No | 68 | 80.0 | ||
Yes | 17 | 20.0 | |||
Relapse | No | 54 | 63.5 | ||
yes | 31 | 36.5 | |||
Treatment | Only surgery | 56 | 65.9 | ||
Radiotherapy | 22 | 25.9 | |||
Radiotherapy and chemotherapy | 7 | 8.2 | |||
Quantitative variables | N | Minimum | Maximum | Average | SD |
Percentage MLH1 | 75 | 0.4 | 63.8 | 8.6 | 11.5 |
Percentage MGMT | 82 | 1.0 | 41.0 | 8.0 | 9.2 |
Time until malignancy | 58 | 0.4 | 116.6 | 33.4 | 39.4 |
Lesion | Study Group | MLH1 Methylation | p-Value | MGMT Methylation | p-Value | |||
---|---|---|---|---|---|---|---|---|
No | Yes | No | Yes | |||||
Initial lesion | Leukoplakia without dysplasia | Transformation | 10 (55.6%) | 3 (75.0%) | 0.474 | 11 (64.7%) | 3 (60.0%) | 0.848 |
Simultaneous | 8 (44.4%) | 1 (25.0%) | 6 (35.3%) | 2 (40.0%) | ||||
Oral lichen planus | Transformation | 2 (40.0%) | 0 (0.0%) | 0.439 | 2 (50.0%) | 0 (0.0%) | 0.221 | |
Simultaneous | 3 (60.0%) | 1 (100.0%) | 2 (50.0%) | 2 (100.0%) | ||||
Papilloma | Transformation | 2 (100.0%) | 2 (100.0%) | |||||
Simultaneous | ||||||||
Verrucous carcinoma | Transformation | 1 (100.0%) | 1 (100.0%) | |||||
Simultaneous | ||||||||
Erythroplasia | Transformation | 1 (100.0%) | ||||||
Simultaneous | ||||||||
Nonspecific ulcer | Transformation | 2 (100.0%) | 2 (100.0%) | 2 (100.0%) | 2 (100.0%) | |||
Simultaneous | ||||||||
Final lesion | Leukoplakia without dysplasia | Transformation | 2 (50.0%) | 1 (100.0%) | 0.361 | 3 (60.0%) | 1 (100.0%) | 0.439 |
Simultaneous | 2 (50.0%) | 0 (0.0%) | 2 (40.0%) | 0 (0.0%) | ||||
Leukoplakia low grade | Transformation | 6 (85.7%) | 1 (50.0%) | 0.284 | 6 (60.0%) | 1 (100.0%) | 0.428 | |
Simultaneous | 1 (14.3%) | 1 (50.0%) | 4 (40.0%) | 0 (0.0%) | ||||
Leukoplakia high grade | Transformation | 2 (100.0%) | 1 (100.0%) | 5 (100.00%) | ||||
Simultaneous | ||||||||
Oral lichen planus | Transformation | 1 (33.3%) | 0 (0.0%) | 0.505 | 1 (50.0%) | |||
Simultaneous | 2 (66.7%) | 1 (100.0%) | 1 (50.0%) | |||||
Papilloma | Transformation | 2 (100.0%) | 1 (100.0%) | 1 (100.0%) | ||||
Simultaneous | ||||||||
Verrucous carcinoma | Transformation | 3 (100.0%) | 3 (100.0%) | |||||
Simultaneous | ||||||||
Nonspecific ulcer | Transformation | 2 (100.0%) | 1 (100.0%) | 2 (100.0%) | 2 (100.0%) | |||
Simultaneous | ||||||||
OSCC | OSCC | 8 (25.0%) | 6 (42.9%) | 12 (33.3%) | 4 (30.8%) | 0.825 | ||
Transformation | 13 (40.6%) | 6 (42.9%) | 16 (44.4%) | 5 (38.5%) | ||||
Simultaneous | 11 (34.4%) | 2 (14.3%) | 8 (22.2%) | 4 (30.8%) |
Methylation | Average | Standard Deviation | Confidential Interval 95% | p-Value | |||
---|---|---|---|---|---|---|---|
Lower Limit | Upper Limit | ||||||
MLH1 methylation | Study group | OSCC | 9.3 | 7.3 | 5.1 | 13.6 | 0.934 |
Transformation | 8.7 | 13.8 | 4.3 | 13.0 | |||
Simultaneous | 7.9 | 8.8 | 3.8 | 11.9 | |||
Sex | Woman | 6.8 | 9.3 | 3.9 | 9.7 | 0.120 | |
Man | 11.0 | 13.8 | 5.9 | 16.1 | |||
Location | Gingiva | 10.6 | 18.0 | −1.5 | 22.7 | 0.780 | |
Tongue | 11.5 | 13.4 | 5.9 | 17.0 | |||
Buccal mucosa | 6.9 | 8.2 | 3.0 | 10.7 | |||
Hard palate | 2.4 | − | − | − | |||
Floor mouth | 3.2 | 0 | 3.2 | 3.2 | |||
Retromolar trigone | 4.8 | 3.9 | 1.2 | 8.4 | |||
Lip | 3.5 | 1.3 | 0.3 | 6.7 | |||
Soft palate | 11.3 | 10.0 | −13.6 | 36.3 | |||
Tonsil | 4.8 | 4.7 | −6.8 | 16.4 | |||
MGMT methylation | Study group | OSCC | 6.6 | 6.4 | 3.2 | 10.0 | 0.515 |
Transformation | 7.7 | 7.7 | 5.4 | 10.0 | |||
Simultaneous | 10.0 | 13.5 | 3.5 | 16.5 | |||
Sex | Woman | 7.3 | 9.1 | 4.6 | 10.0 | 0.444 | |
Man | 8.9 | 9.4 | 5.7 | 12.1 | |||
Location | Gum | 9.4 | 11.4 | 2.1 | 16.6 | 0.506 | |
Tongue | 5.8 | 5.9 | 3.5 | 8.2 | |||
Buccal mucosa | 10.9 | 12.0 | 5.6 | 16.2 | |||
Hard palate | 8.5 | 6.4 | -48.7 | 65.7 | |||
Floor mouth | 2.1 | 0.5 | −2.6 | 6.9 | |||
Retromolar trigone | 5.1 | 5.1 | 0.8 | 9.4 | |||
Lip | 4.3 | 1.9 | −0.3 | 8.9 | |||
Soft palate | 12 | 10.3 | −13.5 | 37.5 | |||
Tonsil | 12.8 | 13.9 | −21.9 | 47.4 |
Kaplan Meier | Months (Mean) | Confidential Interval 95% | Log Rank Test p Value | |||
---|---|---|---|---|---|---|
Lower Limit | Upper Limit | |||||
Survival | MLH1 methylation | No | 9.1 | 5.2 | 13.1 | 0.827 |
Yes | 9.3 | 1.3 | 17.3 | |||
MGMT methylation | No | 9.4 | 6.0 | 12.8 | 0.134 | |
Yes | 19.1 | 19.1 | 19.1 | |||
Recurrence | MLH1 methylation | No | 19.7 | 12.0 | 27.3 | 0.137 |
Yes | 42.4 | 9.9 | 74.9 | |||
MGMT methylation | No | 30.9 | 19.2 | 42.6 | 0.267 | |
Yes | 18.2 | 9.4 | 26.9 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Padin-Iruegas, E.; Chamorro-Petronacci, C.M.; Sines-Cajade, I.; Lorenzo-Pouso, A.I.; Blanco-Carrión, A.; Pérez-Jardón, A.; Gándara-Vila, P.; Pérez-Sayans, M. DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization. Medicina 2022, 58, 878. https://doi.org/10.3390/medicina58070878
Padin-Iruegas E, Chamorro-Petronacci CM, Sines-Cajade I, Lorenzo-Pouso AI, Blanco-Carrión A, Pérez-Jardón A, Gándara-Vila P, Pérez-Sayans M. DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization. Medicina. 2022; 58(7):878. https://doi.org/10.3390/medicina58070878
Chicago/Turabian StylePadin-Iruegas, Elena, Cintia M. Chamorro-Petronacci, Iria Sines-Cajade, Alejandro I. Lorenzo-Pouso, Andrés Blanco-Carrión, Alba Pérez-Jardón, Pilar Gándara-Vila, and Mario Pérez-Sayans. 2022. "DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization" Medicina 58, no. 7: 878. https://doi.org/10.3390/medicina58070878