A similar effect of P16 hydroxymethylation and true-methylation on the prediction of malignant transformation of oral epithelial dysplasia: observation from a prospective study

Hongwei Liu, Zhaojun Liu, Xue-Wei Liu, Si Xu, Lei Wang, Yang Liu, Jing Zhou, Liankun Gu, Yan Gao, Xiao-Yong Liu, Huidong Shi, Zheng Sun, Dajun Deng, Hongwei Liu, Zhaojun Liu, Xue-Wei Liu, Si Xu, Lei Wang, Yang Liu, Jing Zhou, Liankun Gu, Yan Gao, Xiao-Yong Liu, Huidong Shi, Zheng Sun, Dajun Deng

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Trial registration: 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 ).

Keywords: Hydroxymethylation; Malignant transformation; Oral epithelial dysplasia; P16; Prospective cohort.

Conflict of interest statement

Ethics approval and consent to participate

The Institutional Review Boards of the Peking University Cancer Hospital approved this study. All patients gave written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Participant flow diagram. P16H, P16 hydroxymethylation-positive; True-P16M, true P16 methylation-positive; P16U, total P16 methylation-negative
Fig. 2
Fig. 2
Comparison of the cancer rates of oral epithelial dysplasia (OED) patient groups based on the P16 alleles with various methylation and hydroxymethylation states. a OED-derived cancer rate (%); b Estimated cancer-free survival curves in Kaplan-Meier analysis (P < 0.001)
Fig. 3
Fig. 3
Characterization of the methylation and hydroxymethylation states of CpG sites in a 392-bp fragment of P16 exon-1 in representative OED samples. a DHPLC chromatogram of total-P16M (red arrow-pointed) and total-P16U PCR products amplified from regular bisulfite-templates (Bis); or P16H (green arrow-pointed) and non-hydroxymethylated-P16 (P16N) PCR products amplified from TAB-templates (TAB). P16 hemi-methylated cell line HCT116 was used as standard control; Sample-C was total-P16M-negative and P16H-negative; b Results of TAB-sequencing and bisulfite-sequencing for two representative total-P16M-positive and P16H-positive samples, respectively; each line represents one clone; blue-dot, methylated or hydroxymethylated CpG site; #1–35, CpG ID; location of the 115-bp MethyLight amplicon and the 150-bp TAB-hMSP amplicon were also indicated
Fig. 4
Fig. 4
Illustration of both P16 methylation and hydroxymethylation increase cancer risk of OED

References

    1. 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;324:930–935. doi: 10.1126/science.1170116.
    1. Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009;324:929–930. doi: 10.1126/science.1169786.
    1. 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;333:1303–1307. doi: 10.1126/science.1210944.
    1. 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;333:1300–1303. doi: 10.1126/science.1210597.
    1. 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;477:606–610. doi: 10.1038/nature10443.
    1. 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;473:343–348. doi: 10.1038/nature10066.
    1. 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;339:448–452. doi: 10.1126/science.1229277.
    1. 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;473:394–397. doi: 10.1038/nature10102.
    1. 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. doi: 10.1038/nature10008.
    1. 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. doi: 10.1038/nbt.1732.
    1. 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;149:1368–1380. doi: 10.1016/j.cell.2012.04.027.
    1. 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;1:686–692. doi: 10.1038/nm0795-686.
    1. 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;55:4525–4530.
    1. 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;10:5087–5093. doi: 10.1158/1078-0432.CCR-03-0622.
    1. 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;66:3338–3344. doi: 10.1158/0008-5472.CAN-05-3408.
    1. 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;17:2174–2179. doi: 10.1158/1055-9965.EPI-07-2867.
    1. 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;15:5178–5183. doi: 10.1158/1078-0432.CCR-09-0580.
    1. 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;69:4112–4115. doi: 10.1158/0008-5472.CAN-09-0028.
    1. 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.
    1. 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.
    1. 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. doi: 10.1186/s13059-015-0819-6.
    1. 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. doi: 10.21147/j.issn.1000-9604.2018.01.10.
    1. 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. doi: 10.1371/journal.pone.0097785.
    1. Qin S, Zhang B, Tian W, Gu L, Lu Z, Deng D. Kaiso mainly locates in the nucleus in vivo and binds to methylated, but not hydroxymethylated DNA. Chin J Cancer Res. 2015;27:148–155.
    1. 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. doi: 10.1186/1471-2350-12-67.
    1. Liu Z, Zhou J, Gu L, Deng D. Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure. Oncotarget. 2016;7:56447–56455.
    1. 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;93:9821–9826. doi: 10.1073/pnas.93.18.9821.
    1. 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. doi: 10.1093/nar/30.3.e13.
    1. 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;86:591–598. doi: 10.1038/labinvest.3700415.
    1. Deng Dajun, Liu Zhaojun, Du Yantao. Epigenetics and Cancer, Part B. 2010. Epigenetic Alterations as Cancer Diagnostic, Prognostic, and Predictive Biomarkers; pp. 125–176.
    1. Deng DJ, Lu ZM. Differentiation and Adaptation epigenetic networks: translational research in gastric carcinogenesis. Chin Sci Bull. 2013;58:1–6. doi: 10.1007/s11434-012-5578-0.
    1. 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;49:635–642. doi: 10.1038/ng.3805.
    1. Moran Sebastián, Martinez-Cardús Anna, Boussios Stergios, Esteller Manel. Precision medicine based on epigenomics: the paradigm of carcinoma of unknown primary. Nature Reviews Clinical Oncology. 2017;14(11):682–694. doi: 10.1038/nrclinonc.2017.97.
    1. 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;11:405–412.
    1. 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. doi: 10.1038/ng0994-15.
    1. 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;30:564–574. doi: 10.1002/humu.20845.

Source: PubMed

스폰서 및 공동 작업자

건강 상태

약물 개입

3
구독하다