The online version of this article (https://doi.org/10.1186/s12885-018-4787-6) contains supplementary material, which is available to authorized users.
Hongwei Liu, Zhaojun Liu and Xue-wei Liu contributed equally to this work.
Total P16 methylation (P16M), including P16 hydroxymethylation (P16H) and true-P16M, correlates with malignant transformation of oral epithelial dysplasia (OED). Both true-P16M and P16H are early events in carcinogenesis. The aim of this study is to prospectively determine if discrimination of true-P16M from P16H is necessary for prediction of cancer development from OEDs.
Patients (n = 265) with mild or moderate OED were recruited into the double blind two-center cohort. Total-P16M and P16H were analyzed using the 115-bp MethyLight, TET-assisted bisulfite (TAB) methylation-specific PCR (MSP), and TAB-sequencing. Total-P16M-positive and P16H-negative samples were defined as true-P16M-positive. Progression of OEDs was monitored for a minimum 24 months follow-up period.
P16H was detected in 23 of 73 (31.5%) total-P16M-positive OEDs. Follow-up information was obtained from 247 patients with an ultimate compliance rate of 93.2%. OED-derived squamous cell carcinomas were observed in 13.0% (32/247) patients during follow-up (median, 41.0 months). The cancer progression rate for total-P16M-positive patients was significantly increased when compared to total-P16M-negative patients [23.3% vs 8.6%; adjusted odds ratio = 2.67 (95% CI: 1.19–5.99)]. However, the cancer progression rates were similar between P16H- and true-P16M-positive OEDs [26.1% (6/23) vs 22.0% (11/50); odds ratio = 0.80 (95% CI: 0.22–2.92)]. The cancer-free survival was also similar for these patients.
P16H and true-P16M are similar biomarkers for determining malignant potential of OEDs. Discrimination of P16H from true-P16M, at least in OED, may be not necessary in clinical applications.
This study is registered prospectively in the U.S. National Institutes of Health Clinical Trials Protocol Registration System (trial number NCT02967120, available at https://ClinicalTrials.gov/ct2/show/NCT02967120).
Additional file 1: Figure S1. Characterization of the true methylation and hydroxymethylation states of CpG sites in the M.sssI-methylated and 5hmC-containing λ-DNA controls (5mC-Ctrl and 5hmC-Ctrl). Bisulfite-modified DNA templates were used to discriminate 5mC or 5hmC from unmethylated cytosine. TAB-modified DNA templates were used to discriminate 5hmC from 5mC or unmethylated cytosine. The CpG sites within the consensus sequences were listed above the corresponding clone sequences. The number of 5hmC or 5mC sites within each clone was also listed on the left side. These control DNA was added into test samples to monitor the conversion status of 5mC, 5hmC, and unmethylated-cytosine in genomic DNA by bisulfite and TAB treatments. (TIF 197 kb)12885_2018_4787_MOESM1_ESM.tif
Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930–5. CrossRef
Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009;324:929–30. CrossRef
He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011;333:1303–7. CrossRef
Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333:1300–3. CrossRef
Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, Xie ZG, Shi L, He X, Jin SG, Iqbal K, Shi YG, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2011;477:606–10. CrossRef
Williams K, Christensen J, Pedersen MT, Johansen JV, Cloos PA, Rappsilber J, Helin K. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature. 2011;473:343–8. CrossRef
Hackett JA, Sengupta R, Zylicz JJ, Murakami K, Lee C, Down TA, Surani MA. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine. Science. 2013;339:448–52. CrossRef
Pastor WA, Pape UJ, Huang Y, Henderson HR, Lister R, Ko M, McLoughlin EM, Brudno Y, Mahapatra S, Kapranov P, Tahiliani M, Daley GQ, et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature. 2011;473:394–7. CrossRef
Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature. 2011;473:398–402. CrossRef
Song CX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen CH, Zhang W, Jian X, Wang J, Zhang L, et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol. 2011;29:68–72. CrossRef
Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q, Shen Y, Park B, Min JH, Jin P, et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 2012;149:1368–80. CrossRef
Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D. 5' CPG island methylation is associated with transcriptional silencing of the tumor-suppressor P16/CDKN2/MTS1 in human cancers. Nat Med. 1995;1:686–92. CrossRef
Herman JG, Merlo A, Mao L, Lapidus RG, Issa JPJ, Davidson NE, Sidransky D, Baylin SB. Inactivation of the Cdkn2/P16/Mts1 gene is frequently associated with aberrant Dna methylation in all common human cancers. Cancer Res. 1995;55:4525–30. PubMed
Sun Y, Deng DJ, You WC, Bai H, Zhang L, Zhou J, Shen L, Ma JL, Xie YQ, Li JY. Methylation of p16 CpG islands associated with malignant transformation of gastric dysplasia in a population-based study. Clin Cancer Res. 2004;10:5087–93. CrossRef
Belinsky SA, Liechty KC, Gentry FD, Wolf HJ, Rogers J, Vu K, Haney J, Kennedy TC, Hirsch FR, Miller Y, Franklin WA, Herman JG, et al. Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res. 2006;66:3338–44. CrossRef
Hall GL, Shaw RJ, Field EA, Rogers SN, Sutton DN, Woolgar JA, Lowe D, Liloglou T, Field JK, Risk JM. p16 promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomark Prev. 2008;17:2174–9. CrossRef
Cao J, Zhou J, Gao Y, Gu L, Meng H, Liu H, Deng D. Methylation of p16 CpG Island associated with malignant progression of oral epithelial dysplasia: a prospective cohort study. Clin Cancer Res. 2009;15:5178–83. CrossRef
Jin Z, Cheng Y, Gu W, Zheng Y, Sato F, Mori Y, Olaru A, Paun B, Yang J, Kan T, Ito T, Hamilton J, et al. A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett’s esophagus. Cancer Res. 2009;69:4112–5. CrossRef
Liu HW, Liu XW, Dong GY, Zhou J, Liu Y, Gao Y, Liu XY, Gu LK, Sun Z, Deng DJ. P16 methylation as an early predictor for Cancer development from oral epithelial dysplasia: a double-blind multicentre prospective study. EBioMedicine. 2015;2:6.
Gao H, Zhang Y, Zhou J, Li Z, Ma JL, Liu WD, Deng DJ, You WC, Pan KF. Association between p16 methylation and malignant transformation of gastric dysplasia. Chin J Cancer Prev Treat. 2017;24:6.
Cui C, Gan Y, Gu L, Wilson J, Liu Z, Zhang B, Deng D. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis. Genome Biol. 2015;16:252. CrossRef
Gan Y, Ma W, Wang X, Qiao J, Zhang B, Cui C, Liu Z, Deng D. Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation. Chin J Cancer Res. 2018;30:93–103. CrossRef
Qin SS, Li Q, Zhou J, Liu ZJ, Su N, Wilson J, Lu ZM, Deng DJ. Homeostatic maintenance of allele-specific p16 methylation in Cancer cells accompanied by dynamic focal methylation and Hydroxymethylation. PLoS One. 2014;9:E97785. CrossRef
Zhou J, Cao J, Lu Z, Liu H, Deng D. A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues. Bmc Med Genet. 2011;12:67. CrossRef
Herman JG, Graff JR, Myöhänen 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;93:9821–6. CrossRef
Deng DJ, Deng GR, Smith MF, Zhou J, Xin HJ, Powell SM, Lu YY. Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography. Nucleic Acids Res. 2002;30:13E. CrossRef
Luo DY, Zhang BZ, Lv LB, Xiang SY, Liu YH, Ji JF, Deng DJ. Methylation of CpG islands of p16 associated with progression of primary gastric carcinomas. Lab Investig. 2006;86:591–8. CrossRef
Deng D, Liu Z, Du Y. Epigenetic alterations as Cancer diagnostic, prognostic, and predictive biomarkers. In: Herceg Z, Ushijima T Epigenetics and Cancer, Pt Bed, vol. 71, 2010: 125–76. CrossRef
Deng DJ, Lu ZM. Differentiation and Adaptation epigenetic networks: translational research in gastric carcinogenesis. Chin Sci Bull. 2013;58:1–6. CrossRef
Guo S, Diep D, Plongthongkum N, Fung HL, Zhang K. Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA. Nat Genet. 2017;49:635–42. CrossRef
Moran S, Martinez-Cardús A, Boussios S, Esteller M. Precision medicine based on epigenomics: the paradigm of carcinoma of unknown primary. Nat Rev Clin Oncol. 2017;14:682–94. CrossRef
Liu L, Lassam NJ, Slingerland JM, Bailey D, Cole D, Jenkins R, Hogg D. Germline p16INK4A mutation and protein dysfunction in a family with inherited melanoma. Oncogene. 1995;11:405–12. PubMed
Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, Sheahan MD, Clark WH Jr, Tucker MA, Dracopoli NC. Germline p16 mutations in familial melanoma. Nat Genet. 1994;8:15–21. CrossRef
Kannengiesser C, Brookes S, del Arroyo AG, Pham D, Bombled J, Barrois M, Mauffret O, Avril MF, Chompret A, Lenoir GM, Sarasin A, French hereditary melanoma study group, Peters G, Bressac-de Paillerets B. Functional, structural, and genetic evaluation of 20 CDKN2A germ line mutations identified in melanoma-prone families or patients. Hum Mutat. 2009;30:564–74. CrossRef
- A similar effect of P16 hydroxymethylation and true-methylation on the prediction of malignant transformation of oral epithelial dysplasia: observation from a prospective study
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II