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Comprehensive DNA Methylation and Mutation Analyses Reveal a Methylation Signature in Colorectal Sessile Serrated Adenomas

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Pathology & Oncology Research

Abstract

Colorectal sessile serrated adenomas (SSA) are hypothesized to be precursor lesions of an alternative, serrated pathway of colorectal cancer, abundant in genes with aberrant promoter DNA hypermethylation. In our present pilot study, we explored DNA methylation profiles and examined selected gene mutations in SSA. Biopsy samples from patients undergoing screening colonoscopy were obtained during endoscopic examination. After DNA isolation and quality analysis, SSAs (n = 4) and healthy controls (n = 5) were chosen for further analysis. DNA methylation status of 96 candidate genes was screened by q(RT)PCR using Methyl-Profiler PCR array system. Amplicons for 12 gene mutations were sequenced by GS Junior Instrument using ligated and barcoded adaptors. Analysis of DNA methylation revealed 9 hypermethylated genes in both normal and SSA samples. 12 genes (CALCA, DKK2, GALR2, OPCML, PCDH10, SFRP1, SFRP2, SLIT3, SST, TAC1, VIM, WIF1) were hypermethylated in all SSAs and 2 additional genes (BNC1 and PDLIM4) were hypermethylated in 3 out of 4 SSAs, but in none of the normal samples. 2 SSAs exhibited BRAF mutation and synchronous MLH1 hypermethylation and were microsatellite instable by immunohistochemical analysis. Our combined mutation and DNA methylation analysis revealed that there is a common DNA methylation signature present in pre-neoplastic SSAs. This study advocates for the use of DNA methylation as a potential biomarker for the detection of SSA; however, further investigation is needed to better characterize the molecular background of these newly recognized colorectal lesions.

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References

  1. East JE, Vieth M, Rex DK (2015) Serrated lesions in colorectal cancer screening: detection, resection, pathology and surveillance. Gut 64:991–1000

    Article  PubMed  Google Scholar 

  2. Patai AV, Molnár B, Tulassay Z, Sipos F (2013) The serrated pathway: the alternative route to colorectal cancer. World J Gastroenterol 19:607–615

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fearon ER, Vogelstein B (1990) A genetic model for colorectal tumorigenesis. Cell 61:759–767

    Article  CAS  PubMed  Google Scholar 

  4. Patai AV, Molnár B, Kalmár A, Schöller A, Tóth K, Tulassay Z (2012) Role of DNA methylation in colorectal carcinogenesis. Dig Dis 30:310–315

    Article  PubMed  Google Scholar 

  5. Tóth K, Sipos F, Kalmár A, Patai AV, Wichmann B, Stoehr R, Golcher H, Schellerer V, Tulassay Z, Molnár B (2012) Detection of methylated SEPT9 in plasma is a reliable screening method for both left- and right-sided colon cancers. PLoS One 7:e46000

    Article  PubMed  PubMed Central  Google Scholar 

  6. Tóth K, Wasserkort R, Sipos F, Kalmár A, Wichmann B, Leiszter K, Valcz G, Juhász M, Miheller P, Patai ÁV, Tulassay Z, Molnár B (2014) Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA. PLoS One 9:e115415

    Article  PubMed  PubMed Central  Google Scholar 

  7. Burgess NG, Tutticci NJ, Pellise M, Bourke MJ (2014) Sessile serrated adenomas/polyps with cytologic dysplasia: a triple threat for interval cancer. Gastrointest Endosc 80:307–310

    Article  PubMed  Google Scholar 

  8. Erichsen R, Baron JA, Hamilton-Dutoit SJ, Snover DC, Torlakovic EE, Pedersen L, Frøslev T, Vyberg M, Hamilton SR, Sørensen HT (2016) Increased risk of colorectal cancer development among patients with serrated polyps. Gastroenterology 150:895–902.e5

  9. Regula J, Rupinski M, Kraszewska E, Polkowski M, Pachlewski J, Orlowska J, Nowacki MP, Butruk E (2006) Colonoscopy in colorectal-cancer screening for detection of advanced neoplasia. N Engl J Med 355:1863–1872

    Article  CAS  PubMed  Google Scholar 

  10. Rothwell PM, Wilson M, Elwin CE, Norrving B, Algra A, Warlow CP, Meade TW (2010) Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 376:1741–1750

    Article  CAS  PubMed  Google Scholar 

  11. Heigh RI, Yab TC, Taylor WR, Hussain FT, Smyrk TC, Mahoney DW, Domanico MJ, Berger BM, Lidgard GP, Ahlquist DA (2014) Detection of colorectal serrated polyps by stool DNA testing: comparison with fecal immunochemical testing for occult blood (FIT). PLoS One 9:e85659

    Article  PubMed  PubMed Central  Google Scholar 

  12. Park SJ, Rashid A, Lee JH, Kim SG, Hamilton SR, Wu TT (2003) Frequent CpG island methylation in serrated adenomas of the colorectum. Am J Pathol 162:815–822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wynter CV, Walsh MD, Higuchi T, Leggett BA, Young J, Jass JR (2004) Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer. Gut 53:573–580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. O'Brien MJ, Yang S, Mack C, Xu H, Huang CS, Mulcahy E, Amorosino M, Farraye FA (2006) Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points. Am J Surg Pathol 30:1491–1501

    Article  PubMed  Google Scholar 

  15. Kim YH, Kakar S, Cun L, Deng G, Kim YS (2008) Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polyps. Int J Cancer 123:2587–2593

    Article  CAS  PubMed  Google Scholar 

  16. Subramaniam MM, Chan JY, Soong R, Ito K, Yeoh KG, Wong R, Guenther T, Will O, Chen CL, Kumarasinghe MP, Ito Y, Salto-Tellez M (2009) RUNX3 inactivation in colorectal polyps arising through different pathways of colonic carcinogenesis. Am J Gastroenterol 104:426–436

    Article  CAS  PubMed  Google Scholar 

  17. Kim KM, Lee EJ, Ha S, Kang SY, Jang KT, Park CK, Kim JY, Kim YH, Chang DK, Odze RD (2011) Molecular features of colorectal hyperplastic polyps and sessile serrated adenoma/polyps from Korea. Am J Surg Pathol 35:1274–1286

    Article  PubMed  Google Scholar 

  18. Kaji E, Uraoka T, Kato J, Hiraoka S, Suzuki H, Akita M, Saito S, Tanaka T, Ohara N, Yamamoto K (2012) Externalization of saw-tooth architecture in small serrated polyps implies the presence of methylation of IGFBP7. Dig Dis Sci 57:1261–1270

    Article  CAS  PubMed  Google Scholar 

  19. Fu X, Li L, Peng Y (2012) Wnt signalling pathway in the serrated neoplastic pathway of the colorectum: possible roles and epigenetic regulatory mechanisms. J Clin Pathol 65:675–679

    Article  CAS  PubMed  Google Scholar 

  20. Beggs AD, Jones A, Shepherd N, Arnaout A, Finlayson C, Abulafi AM, Morton DG, Matthews GM, Hodgson SV, Tomlinson IP (2013) Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation. PLoS Genet 9:e1003488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Muto Y, Maeda T, Suzuki K, Kato T, Watanabe F, Kamiyama H, Saito M, Koizumi K, Miyaki Y, Konishi F, Alonso S, Perucho M, Rikiyama T (2014) DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability. BMC Cancer 14:466

    Article  PubMed  PubMed Central  Google Scholar 

  22. Dhir M, Yachida S, Van Neste L, Glöckner SC, Jeschke J, Pappou EP, Montgomery EA, Herman JG, Baylin SB, Iacobuzio-Donahue C, Ahuja N (2011) Sessile serrated adenomas and classical adenomas: an epigenetic perspective on premalignant neoplastic lesions of the gastrointestinal tract. Int J Cancer 129:1889–1898

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gaiser T, Meinhardt S, Hirsch D, Killian JK, Gaedcke J, Jo P, Ponsa I, Miró R, Rüschoff J, Seitz G, Hu Y, Camps J, Ried T (2013) Molecular patterns in the evolution of serrated lesion of the colorectum. Int J Cancer 132:1800–1810

    Article  CAS  PubMed  Google Scholar 

  24. Patai ÁV, Valcz G, Hollósi P, Kalmár A, Péterfia B, Patai Á, Wichmann B, Spisák S, Barták BK, Leiszter K, Tóth K, Sipos F, Kovalszky I, Péter Z, Miheller P, Tulassay Z, Molnár B (2015) Comprehensive DNA methylation analysis reveals a common ten-gene methylation signature in colorectal adenomas and carcinomas. PLoS One 10:e0133836

    Article  PubMed  PubMed Central  Google Scholar 

  25. Holemon H, Korshunova Y, Ordway JM, Bedell JA, Citek RW, Lakey N, Leon J, Finney M, McPherson JD, Jeddeloh JA (2007) MethylScreen: DNA methylation density monitoring using quantitative PCR. BioTechniques 43:683–693

    Article  CAS  PubMed  Google Scholar 

  26. Péterfia B, Kalmár A, Patai AV, Bodor A, Micsik T, Hollósi P, Egedi K, Wichmann B, Tulassay Z, Molnár B (2016) Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform. J Cancer. doi:10.7150/jca.16037

  27. Valcz G, Patai ÁV, Kalmár A, Péterfia B, Fűri I, Wichmann B, Műzes G, Sipos F, Krenács T, Mihály E, Spisák S, Molnár B, Tulassay Z (2014) Myofibroblast-derived SFRP1 as potential inhibitor of colorectal carcinoma field effect. PLoS One 9:e106143

    Article  PubMed  PubMed Central  Google Scholar 

  28. Lee EJ, Chun SM, Kim MJ, Jang SJ, Kim do S, Lee DH, Youk EG (2016) Reappraisal of hMLH1 promoter methylation and protein expression status in the serrated neoplasia pathway. Histopathology 69:198–210

  29. Hazewinkel Y, de Wijkerslooth TR, Stoop EM, Bossuyt PM, Biermann K, van de Vijver MJ, Fockens P, van Leerdam ME, Kuipers EJ, Dekker E (2014) Prevalence of serrated polyps and association with synchronous advanced neoplasia in screening colonoscopy. Endoscopy 46:219–224

    PubMed  Google Scholar 

  30. Sipos F, Műzes G, Patai AV, Fűri I, Péterfia B, Hollósi P, Molnár B, Tulassay Z (2013) Genome-wide screening for understanding the role of DNA methylation in colorectal cancer. Epigenomics 5:569–581

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Authors are deeply indebted to colleagues at the Endoscopy Unit and also to Gabriella Farkas Kónyáné (2nd Department of Medicine, Semmelweis University) for her skilled technical work and assistance in the immunohistochemical analyses.

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Authors and Affiliations

  1. 2nd Department of Medicine, Semmelweis University, Szentkirályi utca 46, Budapest, 1088, Hungary

    Árpád V. Patai, Barbara Kinga Barták, Réka Horváth, Csaba Sumánszki, Zoltán Péter, Alexandra Kalmár, Kinga Tóth, Zsolt Tulassay & Béla Molnár

  2. Molecular Medicine Research Group, Hungarian Academy of Sciences, Roosevelt tér 9, Budapest, 1051, Hungary

    Bálint Péterfia, Gábor Valcz, Zsolt Tulassay & Béla Molnár

  3. 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, 1085, Hungary

    Tamás Micsik & Tibor Krenács

  4. Department of Gastroenterology and Internal Medicine, Markusovszky University Teaching Hospital, Markusovszky Lajos utca 5, Szombathely, 9700, Hungary

    Árpád Patai

Authors
  1. Árpád V. Patai
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  2. Barbara Kinga Barták
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  3. Bálint Péterfia
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  8. Árpád Patai
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  14. Béla Molnár
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Corresponding author

Correspondence to Árpád V. Patai.

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Financial Support

This study was supported by the National Office for Research and Technology, Hungary, (Grant number: TECH_08-A1/2–2008-0114) and by the Hungarian Scientific Research Fund (OTKA, Grant number: K 111743). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Additional information

Árpád V. Patai, Barbara Kinga Barták and Bálint Péterfia contributed equally to this work.

Electronic supplementary material

Supplementary Fig. 1

Characteristic histopathological image of SSA. SSA: sessile serrated adenoma (JPEG 11 kb)

High resolution image (TIFF 1716 kb)

Supplementary Fig. 2

Principle of Methyl-Profiler DNA methylation analysis (EpiTect Methyl qPCR Array System, figure provided by Qiagen, with permission) (JPEG 4 kb)

High resolution image (TIFF 414 kb)

Supplementary Fig. 3

Principle of methylation-sensitive restriction enzyme analysis. Mock reaction contains no enzymes, double reaction contains both enzymes. Methylation-sensitive restriction enzyme cleaves at unmethylated CpG sites, whereas methylation-dependent restriction enzyme cleaves at methylated CpG sites. Empty circle unmethylated cytosine, full circle: methylated cytosine (JPEG 7 kb)

High resolution image (TIFF 730 kb)

Supplementary Table 1

(DOC 79 kb)

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Patai, Á.V., Barták, B.K., Péterfia, B. et al. Comprehensive DNA Methylation and Mutation Analyses Reveal a Methylation Signature in Colorectal Sessile Serrated Adenomas. Pathol. Oncol. Res. 23, 589–594 (2017). https://doi.org/10.1007/s12253-016-0154-6

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  • DOI: https://doi.org/10.1007/s12253-016-0154-6

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