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Epigenome-Wide DNA Methylation Profiling of Normal Mucosa Reveals HLA-F Hypermethylation as a Biomarker Candidate for Serrated Polyposis Syndrome

2022; Elsevier BV; Volume: 24; Issue: 6 Linguagem: Inglês

10.1016/j.jmoldx.2022.03.010

1943-7811

Gerhard Jung, Eva Hernández‐Illán, Juan José Lozano, Julia Sidorova, Jenifer Muñoz, Yasuyuki Okada, Enrique Quintero, Goretti Hernández, Rodrigo Jover, Sabela Carballal, Míriam Cuatrecasas, Lorena Moreno, Mireia Díaz, Teresa Ocaña, Ariadna Sánchez, Liseth Rivero, Oswaldo Ortíz, Joan Llach, Antoni Castells, María Pellisé, Ajay Goel, Eduard Batlle, Francesc Balaguer,

Colorectal Cancer Screening and Detection

Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 (Δβ = 10%; P < 0.05). Expression levels of HLA-F, SLFN12, and HLA-DMA were significantly different between SPS patients and healthy individuals and correlated well with the methylation status of the corresponding differentially methylated region (fold change, >20%; r > 0.55; P < 0.001). Significant hypermethylation of CpGs in the gene body of HLA-F was also found in serrated lesions (Δβ = 23%; false discovery rate = 0.01). Epigenome-wide methylation profiling has revealed numerous differentially methylated CpGs in normal mucosa from SPS patients. Significant hypermethylation of HLA-F is a novel biomarker candidate for SPS. Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 (Δβ = 10%; P < 0.05). Expression levels of HLA-F, SLFN12, and HLA-DMA were significantly different between SPS patients and healthy individuals and correlated well with the methylation status of the corresponding differentially methylated region (fold change, >20%; r > 0.55; P < 0.001). Significant hypermethylation of CpGs in the gene body of HLA-F was also found in serrated lesions (Δβ = 23%; false discovery rate = 0.01). Epigenome-wide methylation profiling has revealed numerous differentially methylated CpGs in normal mucosa from SPS patients. Significant hypermethylation of HLA-F is a novel biomarker candidate for SPS. Serrated polyposis syndrome (SPS) is characterized by the presence of multiple and/or large serrated polyps along the colorectum. According to the 2019 updated World Health Organization definition, at least one of the following two criteria need to be fulfilled to establish the diagnosis of SPS: at least five serrated polyps proximal to the rectum, of which at least two are ≥10 mm; and >20 serrated polyps of any size along the colon, of which at least five are located proximal to the rectum.1Rosty C. Brosens L.A.A. Dekker E. Nagtegaal I.D. Serrated Polyposis.ed 5. International Agency for Research on Cancer, World Health Organization, Lyon, France2019Google Scholar Serrated polyposis syndrome is currently perceived as a highly relevant disease because of three reasons: first, with increasingly implemented colorectal cancer (CRC) screening programs, the prevalence of SPS seems to be much higher than previously assumed (approximately 1 in 200 screening colonoscopies in fecal occult blood test screening programs)2IJspeert J.E.G. Bevan R. Senore C. Kaminski M.F. Kuipers E.J. Mroz A. Bessa X. Cassoni P. Hassan C. Repici A. Balaguer F. Rees C.J. Dekker E. Detection rate of serrated polyps and serrated polyposis syndrome in colorectal cancer screening cohorts: a European overview.Gut. 2017; 66: 1225-1232Crossref PubMed Scopus (82) Google Scholar; second, given their morphologic characteristics (pale, small, flat, and often covered by mucus), serrated lesions are often missed and the prevalence of SPS is likely even higher than the actual reported3Rivero-Sanchez L. Lopez-Ceron M. Carballal S. Moreira L. Bessa X. Serradesanferm A. Pozo A. Augé J.M. Ocaña T. Sánchez A. Leoz M.L. Cuatrecasas M. Grau J. Llach J. Castells A. Balaguer F. Pellisé M. Reassessment colonoscopy to diagnose serrated polyposis syndrome in a colorectal cancer screening population.Endoscopy. 2017; 49: 44-53PubMed Google Scholar; and, third, SPS has been associated with a higher risk for developing CRC and therefore surveillance programs need to be implemented.4Boparai K.S. Mathus-Vliegen E.M.H. Koornstra J.J. Nagengast F.M. van Leerdam M. van Noesel C.J.M. Houben M. Cats A. van Hest L.P. Fockens P. Dekker E. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study.Gut. 2010; 59: 1094-1100Crossref PubMed Scopus (198) Google Scholar, 5Bleijenberg A.G.G.C. Ijspeert J.E.E.G. van Herwaarden Y.J. Carballal S. Pellisé M. Jung G. Bisseling T.M. Nagetaal I.D. Van Leerdam M.E. van Lelyveld N. Bessa X. Rodríguez-Moranta F. Bastiaansen B. de Klaver W. Rivero L. Spaander M.C.W.C. Koornstra J.J. Bujanda L. Balaguer F. Dekker E. Personalised surveillance for serrated polyposis syndrome: results from a prospective 5-year international cohort study.Gut. 2020; 69: 112-121Crossref PubMed Scopus (31) Google Scholar, 6Bleijenberg A.G.C. IJspeert J.E.G. Hazewinkel Y. Boparai K.S. Oppeneer S.C. Bastiaansen B.A.J. Dekker E. The long-term outcomes and natural disease course of serrated polyposis syndrome: over 10 years of prospective follow-up in a specialized center.Gastrointest Endosc. 2020; 92: 1098-1107.e1Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar In addition to the long-known adenoma-carcinoma sequence, around 10% to 15% of all sporadic CRCs are believed to arise from the serrated pathway, with the serrated lesion as their precursor.7García-Solano J. Pérez-Guillermo M. Conesa-Zamora P. Acosta-Ortega J. Trujillo-Santos J. Cerezuela-Fuentes P. Mäkinen M.J. Clinicopathologic study of 85 colorectal serrated adenocarcinomas: further insights into the full recognition of a new subset of colorectal carcinoma.Hum Pathol. 2010; 41: 1359-1368Crossref PubMed Scopus (77) Google Scholar,8García-Solano J. García-Solano M.E. Torres-Moreno D. Carbonell P. Trujillo-Santos J. Pérez-Guillermo M. Conesa-Zamora P. Biomarkers for the identification of precursor polyps of colorectal serrated adenocarcinomas.Cell Oncol. 2016; 39: 243-252Crossref Scopus (6) Google Scholar However, the enormous phenotypic, genomic, and epigenomic heterogeneity of serrated lesions is posing a challenge in sorting out the underlying molecular mechanisms, which are still widely unknown. In this sense, the serrated pathway can arise from hyperplastic polyps (HPs), sessile serrated lesions (SSLs), and traditional serrated adenomas, and all show different morphologic characteristics and molecular features. In addition, some SSLs might progress to serrated adenocarcinoma, whereas others develop as conventional CRC with histologic and molecular features of microsatellite instability high cancers.9De Palma F.D.E. D'Argenio V. Pol J. Kroemer G. Maiuri M.C. Salvatore F. The molecular hallmarks of the serrated pathway in colorectal cancer.Cancers (Basel). 2019; 11: 1017Crossref PubMed Scopus (76) Google Scholar Nonetheless, one common histologic feature of all the subtypes is the saw-tooth shape of the crypt base, which is believed to arise from altered apoptotic pathways.10Tateyama H. Li W. Takahashi E. Miura Y. Sugiura H. Eimoto T. Apoptosis index and apoptosis-related antigen expression in serrated adenoma of the colorectum.Am J Surg Pathol. 2002; 26: 249-256Crossref PubMed Scopus (96) Google Scholar,11O'Brien M.J. Yang S. Mack C. Xu H. Huang C.S. Mulcahy E. Amorosino M. Farraye F.A. 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. 2006; 30: 1491-1501Crossref PubMed Scopus (433) Google Scholar Serrated polyposis syndrome has been associated with hypermethylation of multiple gene promoters. In a study published in 2006, Minoo et al12Minoo P. Baker K. Goswami R. Chong G. Foulkes W.D. Ruszkiewicz A.R. Barker M. Buchanan D. Young J. Jass J.R. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.Gut. 2006; 55: 1467-1474Crossref PubMed Scopus (129) Google Scholar analyzed the DNA methylation at promoters of 14 markers and found a higher methylation level in patients with SPS compared with those with serrated lesions but without fulfilling criteria for SPS. More important, these differences were even more evident when normal mucosa was analyzed.12Minoo P. Baker K. Goswami R. Chong G. Foulkes W.D. Ruszkiewicz A.R. Barker M. Buchanan D. Young J. Jass J.R. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.Gut. 2006; 55: 1467-1474Crossref PubMed Scopus (129) Google Scholar More recent studies have shown that, compared with conventional adenomas, SSLs show more hypermethylation, particularly in CpG islands and shores.13Parker H.R. Orjuela S. Martinho Oliveira A. Cereatti F. Sauter M. Heinrich H. Tanzi G. Weber A. Komminoth P. Vavricka S. Albanese L. Buffoli F. Robinson M.D. Marra G. The proto CpG island methylator phenotype of sessile serrated adenomas/polyps.Epigenetics. 2018; 13: 1088-1105Crossref PubMed Scopus (12) Google Scholar In addition, an important fraction of serrated CRCs present the so-called CpG island methylator phenotype (CIMP), and this is associated to various molecular features and risk factors, including BRAF mutation, microsatellite instability, proximal tumor site, female sex, older age, and smoking.14Weisenberger D.J. Levine A.J. Long T.I. Buchanan D.D. Walters R. Clendenning M. Rosty C. Joshi A.D. Stern M.C. Le Marchand L. Lindor N.M. Daftary D. Gallinger S. Selander T. Bapat B. Newcomb P.A. Campbell P.T. Casey G. Ahnen D.J. Baron J.A. Haile R.W. Hopper J.L. Young J.P. Laird P.W. Siegmund K.D. Colon Cancer Family RegistryAssociation of the colorectal CpG island methylator phenotype with molecular features, risk factors, and family history.Cancer Epidemiol Biomarkers Prev. 2015; 24: 512-519Crossref PubMed Scopus (69) Google Scholar Another frequent finding of sporadic CRCs, overlapping with the serrated pathway, is the presence of microsatellite instability secondary to MLH1 promoter hypermethylation.12Minoo P. Baker K. Goswami R. Chong G. Foulkes W.D. Ruszkiewicz A.R. Barker M. Buchanan D. Young J. Jass J.R. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.Gut. 2006; 55: 1467-1474Crossref PubMed Scopus (129) Google Scholar A third molecular mechanism hypothesized to initiate the serrated pathway is based on activating mutations in the mitogen-activated protein kinase pathway. Particularly, BRAF mutations have been associated with a high level of promoter methylation and MLH1 silencing.15Rad R. Cadiñanos J. Rad L. Varela I. Strong A. Kriegl L. Constantino-Casas F. Eser S. Hieber M. Seidler B. Price S. Fraga M.F. Calvanese V. Hoffman G. Ponstingl H. Schneider G. Yusa K. Grove C. Schmid R.M. Wang W. Vassiliou G. Kirchner T. McDermott U. Liu P. Saur D. Bradley A. A genetic progression model of Braf V600E-induced intestinal tumorigenesis reveals targets for therapeutic intervention.Cancer Cell. 2013; 24: 15-29Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar,16Nagasaka T. Sasamoto H. Notohara K. Cullings H.M. Takeda M. Kimura K. Kambara T. MacPhee D.G. Young J. Leggett B.A. Jass J.R. Tanaka N. Matsubara N. Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation.J Clin Oncol. 2004; 22: 4584-4594Crossref PubMed Scopus (197) Google Scholar The observation that patients with a history of colorectal polyps or cancer are more likely to develop metachronous lesions than those without such previous lesions has led to the field cancerization (or field defect) hypothesis.17Bernstein C. Bernstein H. Payne C.M. Dvorak K. Garewal H. Field defects in progression to gastrointestinal tract cancers.Cancer Lett. 2008; 260: 1-10Crossref PubMed Scopus (52) Google Scholar,18Luo Y. Yu M. Grady W.M. Field cancerization in the colon: a role for aberrant DNA methylation?.Gastroenterol Rep. 2014; 2: 16-20Crossref PubMed Scopus (36) Google Scholar Thus far, some genetic and epigenetic alterations have been identified that arise in the colonic epithelium in a patchy way (fields) and predispose to the formation of polyps and eventually cancer, yet the mucosa appears macroscopically and microscopically normal.19Belshaw N.J. Elliott G.O. Foxall R.J. Dainty J.R. Pal N. Coupe A. Garg D. Bradburn D.M. Mathers J.C. Johnson I.T. Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa.Br J Cancer. 2008; 99: 136-142Crossref PubMed Scopus (75) Google Scholar This has become particularly evident for proximal serrated lesions where aberrant DNA methylation of a specific gene panel (CIMP panel) detected in normal mucosa was associated with the presence of advanced proximal serrated lesions.20Worthley D.L. Whitehall V.L.J. Buttenshaw R.L. Irahara N. Greco S.A. Ramsnes I. Mallitt K.A. Le Leu R.K. Winter J. Hu Y. Ogino S. Young G.P. Leggett B.A. DNA methylation within the normal colorectal mucosa is associated with pathway-specific predisposition to cancer.Oncogene. 2010; 29: 1653-1662Crossref PubMed Scopus (88) Google Scholar From a clinical viewpoint, it has been proposed that these aberrations in normal mucosa could be leveraged as a tool to identify patients early at risk for harboring or developing lesions.12Minoo P. Baker K. Goswami R. Chong G. Foulkes W.D. Ruszkiewicz A.R. Barker M. Buchanan D. Young J. Jass J.R. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.Gut. 2006; 55: 1467-1474Crossref PubMed Scopus (129) Google Scholar However, the clinical implications of this hypothesis remain largely unexplored. Intense colonoscopy surveillance for SPS is costly and uncomfortable for patients. Besides, recent studies suggest that the risk of developing cancer under surveillance is low and that lower-risk patients could benefit from relaxation of the intervals.5Bleijenberg A.G.G.C. Ijspeert J.E.E.G. van Herwaarden Y.J. Carballal S. Pellisé M. Jung G. Bisseling T.M. Nagetaal I.D. Van Leerdam M.E. van Lelyveld N. Bessa X. Rodríguez-Moranta F. Bastiaansen B. de Klaver W. Rivero L. Spaander M.C.W.C. Koornstra J.J. Bujanda L. Balaguer F. Dekker E. Personalised surveillance for serrated polyposis syndrome: results from a prospective 5-year international cohort study.Gut. 2020; 69: 112-121Crossref PubMed Scopus (31) Google Scholar To optimize patient management, it is clear that we first need to better understand the molecular fundamentals of the serrated pathway to then dispose of molecular biomarkers for a better cancer risk prediction. This study describes the methylation profile of normal mucosa in a large cohort of SPS and identified biomarker candidates for SPS. We also aimed to verify the field defect hypothesis by comparing the methylation profiles found in normal mucosa and in serrated polyp tissue.18Luo Y. Yu M. Grady W.M. Field cancerization in the colon: a role for aberrant DNA methylation?.Gastroenterol Rep. 2014; 2: 16-20Crossref PubMed Scopus (36) Google Scholar This study consisted of four phases (Figure 1). In the exploratory phase, a genome-wide methylation analysis was performed using the 850K Epic BeadChip Array (Infinium) in fresh-frozen tissue samples of normal colon mucosa from 50 patients diagnosed with SPS and 19 individuals without colorectal neoplasia. Because aberrant methylation has been shown to differ along the colorectum,21Ogino S. Nosho K. Kirkner G.J. Kawasaki T. Meyerhardt J.A. Loda M. Giovannucci E.L. Fuchs C.S. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer.Gut. 2009; 58: 90-96Crossref PubMed Scopus (641) Google Scholar samples from the proximal and distal colon were obtained. Proximal colon was defined as proximal to the splenic flexure. The gene expression was then analyzed in the same cohort and samples and the expression levels were correlated with the methylation status of the corresponding differentially methylated regions (DMRs). In the next step, the results in formalin-fixed, paraffin-embedded (FFPE) tissue samples of the normal mucosa of an independent cohort of 17 SPS patients and 10 individuals without colorectal neoplasia were validated. As a further validation step, genome-wide methylation analysis was performed using the same technique to interrogate differentially methylated CpGs directly in FFPE tissue of serrated lesions derived from another independent cohort: 6 HPs and 18 SSLs. Patients with SPS for the exploratory phase were prospectively recruited between 2016 and 2018 at the High-Risk CRC Clinic from the Hospital Clínic of Barcelona (Barcelona, Spain). For the validation cohort, patients were recruited retrospectively from the same clinic attended between 2004 and 2016. Patients included in this study fulfilled criterion I and/or III, according to the 2010 World Health Organization criteria: criterion I, five or more serrated polyps proximal to the sigmoid and at least two of them measure ≥10 mm; and criterion III, ≥20 serrated polyps of any size at any localization. Healthy controls with a normal colonoscopy (ie, without any adenoma, serrated lesion, or any other lesion) in the exploratory and validation phase derived from the CRC screening programs at the General Hospital of Alicante and the Hospital Clinic of Barcelona, respectively. In both cases, they were matched for age and sex with the study cohorts. Briefly, in these average risk, population-based screening programs, asymptomatic residents, aged 50 to 69 years, are identified by the community health registry and invited for CRC screening based on fecal immunochemical testing.22Castells A. Quintero E. Programmatic screening for colorectal cancer: the COLONPREV study.Dig Dis Sci. 2015; 60: 672-680Crossref PubMed Scopus (21) Google Scholar In those with a positive result (>155 ng Hb/mL; OC-SENSOR; Eiken Chemical Co., Taito-ku, Tokyo, Japan), a colonoscopy is performed. In this program, patients with a history of CRC, adenoma, inflammatory bowel disease, family history of hereditary or familial CRC, severe comorbidity, or previous colectomy are excluded, because these patients are considered high risk and may be under different programs of surveillance or screening. Those patients with a normal colonoscopy (ie, without any pathology) were eligible to serve as healthy controls for the present study, and biopsies from the proximal and distal colon were taken. The study was conducted according to the guidelines of the Declaration of Helsinki. Informed consent was retrieved from all patients, and the study was approved by our local ethics committee by February 29, 2016 (registered HCB/2016/0099). Fresh-frozen normal mucosa tissue samples (from the proximal and distal colon) from patients with SPS were obtained during the surveillance colonoscopies.1Rosty C. Brosens L.A.A. Dekker E. Nagtegaal I.D. Serrated Polyposis.ed 5. International Agency for Research on Cancer, World Health Organization, Lyon, France2019Google Scholar,23van Leerdam M.E. Roos V.H. van Hooft J.E. Dekker E. Jover R. Kaminski M.F. Latchford A. Neumann H. Pellisé M. Saurin J.-C. Tanis P.J. Wagner A. Balaguer F. Ricciardiello L. Endoscopic management of polyposis syndromes: European Society of Gastrointestinal Endoscopy (ESGE) guideline.Endoscopy. 2019; 51: 877-895Crossref PubMed Scopus (102) Google Scholar After the biopsy, samples were stored in RNAlater solution (reference AM7021; Invitrogen, Waltham, MA) at −80°C in the freezer at the laboratories of Institut d'Investigacions Biomèdiques August Pi i Sunyer (Barcelona, Spain). FFPE tissue samples of normal mucosa from patients who had previously received partial or total colectomy because of SPS with (N = 7) or without (N = 10) CRC were obtained. Samples were collected from the proximal and distal colon. An expert pathologist (M.C.) confirmed histologically that the sample only contained normal mucosa and not tumor or serrated lesions. The epithelial compartment comprised about 80% to 90% of each sample and therefore microdissection was not performed. Serrated polyp samples that were enrolled as a second validation step were obtained by endoscopic mucosal resection between 2014 and 2018. Each serrated polyp was classified as HP and SSL to the World Health Organization classification. Fresh-frozen samples were disaggregated by using UFO beads (Stainless Steel UFO Beads 3.5 mm RNAse free; reference F7SSUFO35-RNA; Cultek, Madrid, Spain) and the TissueLyser LT (reference 85600; Qiagen, Hilden, Germany). Genomic DNA from the tissue samples was extracted using the PureLink Genomic DNA MiniKit following the manufacturer's instructions (reference K1820-02; Invitrogen, ThermoFisher Scientific, Waltham, MA). The double-stranded DNA concentration was measured by a fluorometric method (Qubit 3.0; reference Q33216; Invitrogen). For gene expression analysis, RNA was extracted using the Rneasy miniKit following the manufacturer's instructions (reference 74104; Qiagen). Quality and quantity of RNA samples were measured on an automated electrophoresis TapeStation System Device (RNA ScreenTape Analysis; Agilent Technologies, Santa Clara, CA). The extracted DNA and RNA were then stored at −20°C and −80°C, respectively, which allowed adequate preservation of the quality of DNA and RNA for subsequent analysis of methylation and gene expression, respectively. Global methylome analysis was performed by using an 850K Epic BeadChip Array (reference WG-317-1003; Illumina, San Diego, CA). This kit targets >850,000 CpG sites on a single-nucleotide level and covers CpGs located in CpG islands and outside of islands (ie, in shores and opensea areas). A CpG island is defined as a region of 500 to 2000 nucleotides, of which >55% are CpGs. The shores extend around 2000 bp upstream and downstream from the islands, whereas opensea represents areas with CpGs not related to CpG islands and that have a low CpG content. On the other hand, the 850K Epic BeadChip Array (Infinium) covers CpGs located in promoter regions but also in gene bodies and intergenic regions. Promoter regions of miRNAs were also covered. In the case of the FFPE samples, the Infinium HD FFPE DNA Restore Kit (reference WG-321-1002; Illumina) was used previously to allow DNA recovery for the subsequent analysis using the Infinium platform.24De Ruijter T.C. De Hoon J.P. Slaats J. De Vries B. Janssen M.J. Van Wezel T. Aarts M.J. Van Engeland M. Tjan-Heijnen V.C. Van Neste L. Veeck J. Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays.Lab Invest. 2015; 95: 833-842Crossref PubMed Scopus (34) Google Scholar Briefly, 1 μg of DNA was treated with sodium bisulfite (EZ DNA Methylation-Gold kit; reference D5005; Zymo Research, Tustin, CA). Subsequently, an isothermal amplification was performed at 37°C, followed by enzymatic fragmentation, purification, and hybridization (all according to the manufacturer's instructions). The sets of methylation data were extracted using the GenomeStudio software v2011.1 (Illumina). Methylation values for individual CpG sites were obtained as β-values, calculated as the ratio of the methylated signal intensity/the sum of both methylated and unmethylated signals after background subtraction. The β-values were reported as a DNA methylation score, ranging from 0 (completely unmethylated) to 1 (completely methylated). To correct for the bias introduced by different bead types in the methylation array, a β-mixture quantile normalization method, described by Teschendorff et al25Teschendorff A.E. 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: 189-196Crossref PubMed Scopus (951) Google Scholar (2013), and which is included in the R package Chip Analysis Methylation Pipeline (ChAMP) for Infinium Illumina EPIC chips was used.26Morris T.J. Butcher L.M. Feber A. Teschendorff A.E. Chakravarthy A.R. Wojdacz T.K. Beck S. ChAMP: 450k Chip analysis methylation pipeline.Bioinformatics. 2014; 30: 428-430Crossref PubMed Scopus (546) Google Scholar The batch effect was corrected by using the open-source Bioconductor package ChAMP version 2.8.9 with default parameters.27Tian Y. Morris T.J. Webster A.P. Yang Z. Beck S. Feber A. Teschendorff A.E. ChAMP: updated methylation analysis pipeline for Illumina BeadChips.Bioinformatics. 2017; 33: 3982-3984Crossref PubMed Scopus (356) Google Scholar This R package also generates the gene set enrichment analysis and DMR analysis (ie, differentially methylated regions). A differentially methylated region is a set of multiple CpGs, usually adjacent to each other, which display a predicted strong functional and regional correlation alongside the genome and possibly form a functional unit for the transcriptional regulation.28Rakyan V.K. Down T.A. Balding D.J. Beck S. Rakyan V.K. Down T.A. Balding D.J. Beck S. Epigenome-wide association studies for common human diseases.Nat Rev Genet. 2011; 12: 529-541Crossref PubMed Scopus (914) Google Scholar To detect DMRs between two populations on normalized β-values, the Bioconductor package bump hunter was used with default parameters,29Jaffe A.E. Murakami P. Lee H. Leek J.T. Fallin M.D. Feinberg A.P. Irizarry R.A. Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies.Int J Epidemiol. 2012; 41: 200-209Crossref PubMed Scopus (433) Google Scholar included in the ChAMP. DMR function was assessed, to estimate regions for which a genomic profile deviates from its baseline value. Differentially methylated cytosines and regions between groups (SPS and healthy) were assessed by linear models,30Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. Smyth G.K. Limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (16497) Google Scholar including clinical covariates: age, sex, colon location (proximal and distal), and tobacco use after a logit transforming of β-values. Because differences in normal mucosa were predicted to be subtle, a difference in the relative methylation level (Δβ value) of >0.1 (ie, an absolute 10% increase or decrease) was defined as clinically relevant and an adjusted P value (Benjamini-Hochberg method) of 0.2 (ie, an absolute 20% increase or decrease) was defined as clinically relevant and an adjusted P value of 20,000 genes in a transcriptome-wide manner (Human Clariom S Assay; reference 902927; ThermoFisher Scientific). The raw gene expression was processed using bioconductor tools.31Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. Hornik K. Hothorn T. Huber W. Iacus S. Irizarry R. Leisch F. Li C. Maechler M. Rossini A.J. Sawitzki G. Smith C. Smyth G. Tierney L. Yang J.Y.H. Zhang J. Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar Briefly, the entrez-based probe definition,32Dai M. Wang P. Boyd A.D. Kostov G. Athey B. Jones E.G. Bunney W.E. Myers R.M. Speed T.P. Akil H. Watson S.J. Meng F. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data.Nucleic Acids Res. 2005; 33: e175Crossref PubMed Scopus (1404) Google Scholar normalization using rma, and differential expressed ranking using moderated t-statistics were used.30Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. Smyth G.K. Limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (16497) Google Scholar Correlation between genes and differentially methylated regions was computed using Spearman correlation. A fold change of at least ±1.1 (ie, a 10% increase or decrease in expression level) with a P value of <0.05 was considered as relevant and statistically significant. Clinical characteristics of the study cohorts are summarized in Table 1. Mean age in the discovery phase was 59 and 62 years for patients with SPS and healthy controls, respectively. Of the 50 SPS cases, four patients had CRC (8%) in the discovery phase. In the validation cohort, mean age was 60 and 62 years for patients with SPS and healthy individuals, respectively. There were seven cases of CRC in the SPS group (41%).Table 1Study PopulationCharacteristicSPS gro