DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression © 2019 Published by Elsevier B.V. Published version Mäki-Nevala, Satu; Valo, Satu; Ristimäki, Ari; Sarhadi, Virinder; Knuutila, Sakari; Nyström, Minna; Renkonen-Sinisalo, Laura; Lepistö, Anna; Mecklin, Jukka-Pekka; Peltomäki, Päivi Mäki-Nevala, S., Valo, S., Ristimäki, A., Sarhadi, V., Knuutila, S., Nyström, M., Renkonen-Sinisalo, L., Lepistö, A., Mecklin, J.-P., & Peltomäki, P. (2019). DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression. EBioMedicine, 39, 280-291. https://doi.org/10.1016/j.ebiom.2018.12.018 2019 DNA methylation changes and somatic mutations as tumorigenic events in Lynch syndrome-associated adenomas retaining mismatch repair protein expression Satu Mäki-Nevala a, ⁎,SatuValo a , Ari Ristimäki b,c ,VirinderSarhadi b , Sakari Knuutila b , Minna Nyström d , Laura Renkonen-Sinisalo e ,AnnaLepistö e , Jukka-Pekka Mecklin f , Päivi Peltomäki a a Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland b Department of Pathology, HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland c Genome-Scale Biology Research Program, Research Programs Unit, University of Helsinki, Finland d Department of Biosciences, University of Helsinki, Helsinki, Finland e Department of Surgery, Helsinki University Hospital, Helsinki, Finland f Sport and Health Sciences, University of Jyväskylä and Jyväskylä Central Hospital, Jyväskylä, Finland abstractarticle info Article history: Received 16 September 2018 Received in revised form 4 December 2018 Accepted 11 December 2018 Available online 18 December 2018 Background: DNA mismatch repair (MMR) defects are a major factor in colorectal tumorigenesis in Lynch syndrome (LS) and 15% of sporadic cases. Some adenomas from carriers of inherited MMR gene mutations have intact MMR protein expression implying other mechanisms accelerating tumorigenesis. We determined roles of DNA methylation changes and somatic mutations in cancer-associated genes as tumorigenic events in LS-associated colorectal adenomas with intact MMR. Methods: We investigated 122 archival colorectal specimens of normal mucosae, adenomas and carcinomas from 57 LS patients. MMR-deficient (MMR-D, n=49)andMMR-proficient (MMR-P, n= 18) adenomas were of particular interest and were interrogated by methylation-specific multiplex ligation-dependent probe amplification and Ion Torrent sequencing. Findings: Promoter methylation of CpG island methylator phenotype (CIMP)-associated marker genes and selected colorectal cancer (CRC)-associated tumor suppressor genes (TSGs) increased and LINE-1 methylation decreased from normal mucosa to MMR-P adenomas to MMR-D adenomas. Methylation differences were statistically significant when either adenoma group was compared with normal mucosa, but not between MMR-P and MMR-D adenomas. Significantly increased methylation was found in multiple CIMP marker genes (IGF2, NEUROG1,CRABP1, and CDKN2A) and TSGs (SFRP1 and SFRP2) in MMR-P adenomas already. Furthermore, certain CRC-associated somatic mutations, such as KRAS, were prevalent in MMR-P adenomas. Interpretation: We conclude that DNA methylation changes and somatic mutations of cancer-associated genes might serve as an alternative pathway accelerating LS-associated tumorigenesis in the presence of proficient MMR. Fund: Jane and Aatos Erkko Foundation, Academy of Finland, Cancer Foundation Finland, Sigrid Juselius Foundation, and HiLIFE. © 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Lynch syndrome Colorectal adenoma DNA methylation DNA mismatch repair LINE-1 methylation Mutation Tumorigenesis Tumor suppressor 1. Introduction Lynch syndrome (LS) is a hereditary cancer predisposing syndrome caused by germline defects in DNA mismatch repair (MMR) genes MLH1,MSH2,MSH6 and PMS2 [1], or rarely deletions in the 3′end of EPCAM gene that lead to hypermethylation of MSH2 gene promoter [2]. These germline defects cause a reduced level of MMR protein, i.e. haploinsufficiency, which accelerates the occurrence of de novo somatic mutations [3] or compromises other functions of MMR genes, such as apoptosis signaling [4], thereby increasing the risk for early onset malignancies. Those defects together with epigenetic events cause an increased risk of cancer [5], primarily colorectal cancer (CRC) and endometrial cancer [6]. The “second hit”leading to a loss of the remaining functional allele of a MMR gene is typically caused by loss of heterozygosity (LOH) or somatic mutations [7–9]. However, MMR deficiency is thought to appear as a secondary event in LS tumorigenesis, supported by multiple studies observing that 10–46% of adenomas show retained expression of MMR EBioMedicine 39 (2019) 280–291 ⁎Corresponding author at: Department of Medical and Clinical Genetics, P.O. Box 63 (Haartmaninkatu 8), 00014, University of Helsinki, Finland. E-mail address: satu.maki-nevala@helsinki.fi(S. Mäki-Nevala). https://doi.org/10.1016/j.ebiom.2018.12.018 2352-3964/© 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com protein [9–13], and MMR deficiency is associated with larger polyps and higher grade [10,14,15]. The nature, timing and order of other molecular hits driving the malignant transformation are yet to be identified. Most LS CRCs exhibit microsatellite instability (MSI) caused by defects in the MMR system [16]. CpG island methylation phenotype (CIMP) characterized by aberrant CpG island methylation in promoters of various tumor suppressor genes (TSGs) [17], is prevalent in LS CRC, and occurs already at early stages of tumor development, but clearly increases along with dysplasia [9]. The molecular drivers behind CIMP, and its role in hereditary cancer remain obscure. Accumulation of somatic mutations involving the epigenetic regulatory genes may serve as a mechanism for CIMP [8]. Long interspersed elements (LINEs) form a class of retrotransposons that constitute approximately 17% of the human genome, and are normally heavily methylated [18]. In cancer, LINEs can be activated due to DNA hypomethylation. LINE-1 hypomethylation serves as a surrogate marker for global hypomethylation that can lead to chromosomal instability (CIN) [19]. LINE-1 is a 6 kb long retrotransposon that can, once activated, induce transcription of other genes [18]. Moreover, activated LINE-1 can change the epigenetic regulation of adjacent genes [20], and can occur already at early stages in malignant transformation of CRC [21,22]. The aim of this study was to investigate molecular mechanisms, DNA methylation changes and somatic mutations, as events contributing to LS-associated colorectal tumorigenesis. Our focus was on precursors of cancer, adenomas, based on the observation by Valo et al. [9], that many LS-associated adenomas retain the expression of the MMR protein corresponding to the germline mutation. DNA methylation changes were investigated in eight CIMP marker genes, LINE-1, and seven candidate TSGs associated with early colon tumorigenesis [23]. Moreover, adenomas were studied for somatic alterations in mutational hotspots of 22 cancer-associated genes. DNA methylation changes and mutations were compared between MMR-proficient (MMR-P) and MMR-deficient (MMR-D) adenomas to identify possible initiating or promoting molecular changes in adenomas in which the “second hit” leading to the loss of MMR function had not (yet) occurred. 2. Materials and methods 2.1. Patient samples Study material consisted of formalin-fixed, paraffin-embedded (FFPE) specimens of normal mucosae, adenomas and carcinomas from Research in context Evidence before this study Functional DNA mismatch repair is critical for cancer avoidance. DNA mismatch repair genes comply with the Knudson's two-hit paradigm for tumor suppressor genes. Lynch syndrome individuals have inherited a mutant copy of a given DNA mismatch repair gene which makes them susceptible to cancer, but as the product of the remaining normal copy of the same gene is available, tumor development is prevented. Tissues with an intact allele present show normal expression of the respective mismatch repair protein. However, recent evidence reveals that 10–46% of colorectal adenomas from Lynch syndrome patients retain mismatch repair protein expression, yet a tumor has developed, implying that proficient mismatch repair alone is insufficient to prevent tumor initiation. Molecular events needed in addition to or alternative to deficient DNA mismatch repair to initiate tumorigenesis, as well as the chronological sequence of events, is obscure. Deficient mismatch repair is associated with a characteristic mutational signature consisting of instability at repeat sequences and a designated pattern of base substitutions. Utilization of such mutational preferences to determine if a given alteration has occurred before or after mismatch repair deficiency, together with other approaches, has resulted in varying conclusions of early or late appearance of mismatch repair defects in Lynch syndrome tumorigenesis. Based on the above cited and other (recent) literature retrieved from PubMed, together with our previous observation that a mismatch repair-proficient subset exists among adenomas from a nation-wide registry of Lynch syndrome individuals, this study was undertaken to explore the tumorigenic events in Lynch syndrome-associated colorectal adenomas retaining MMR protein expression. Added value of this study Our investigation is one of the very few existing studies focusing on mismatch repair-proficient colorectal adenomas from Lynch syndrome mutation carriers, with the aim to determine the roles of DNA methylation changes and somatic mutations in cancerassociated genes in such neoplastic lesions. We show that tumor initiation may precede the loss of mismatch repair protein and identify molecular alterations present in adenomas with retained mismatch repair protein expression. While many of these changes were more prevalent in mismatch repair deficient adenomas, some were less frequent or even absent in the latter. Specifically, we found that hypermethylation of IGF2, NEUROG1, CDKN2A,CRABP1,SFRP1,andSFRP2, and especially KRAS mutations may accelerate, or possibly initiate, Lynch syndromeassociated tumorigenesis when the wild-type allele of the predisposing mismatch repair gene is still present as judged from retained protein expression. The emerging differences between mismatch repair deficient and proficient adenomas suggest that tumorigenesis in Lynch syndrome may be driven by different pathways, as supported by recent findings from Lynch syndromeassociated colorectal carcinomas. Implications of all the available evidence Recent studies, including this study, have shed light on the neoplastic process associated with colon tumorigenesis in Lynch syndrome. The fact that adenoma development is possible in the absence of biallelic inactivation of the predisposing mismatch repair gene shows that the traditional Knudson's two-hit mechanism is not the only one that applies to mismatch repair gene mutation-associated tumorigenesis. Genetic and epigenetic alterations observed in mismatch repair proficient adenomas may qualify for alternative tumor-initiating or promoting events. The findings have important clinical implications. As 27% of Lynch syndrome adenomas from our investigation and up to 46% of adenomas from published studies express the mismatch repair protein corresponding to the gene mutant in the germline, retained mismatch repair protein expression in a colorectal adenoma cannot be used to rule out Lynch syndrome in diagnostics. Genetic and epigenetic changes detected in mismatch repair proficient adenomas may provide potential biomarkers of increased tumor risk. Genetic heterogeneity in Lynch syndrome adenomas and carcinomas may be relevant for the design of targeted therapies or preventive measures. Larger sample sizes are required to confirm the recent findings described above and the specific contributions of the observed alterations to tumorigenesis. Further investigations are also needed to explore the extent to which the findings from Lynch syndrome may apply to sporadic colorectal tumorigenesis. 281S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 57 LS patients (Table 1). Specimens were collected at the Helsinki University Central Hospital and the Jyväskylä Central Hospital during 2013–2016. All the patients were represented in the nationwide Hereditary Colorectal Cancer Registry of Finland, and the information of verified germline mutations is available in Supplementary Table S1. In this study, we used normal mucosae and adenoma specimens, extending the sample series from our previous study [9] with 23 specimens including 13 adenomas with low-grade dysplasia, seven adenomas with highgrade dysplasia and three carcinomas. Normal mucosae collected for this study were supplemented with 13 older archival specimens of normal mucosae of Lynch syndrome patients to increase a total number of the reference group (n= 29). Tumor material was evaluated for histology and tumor content by the pathologist. The detailed characteristics of adenoma specimens are presented in Supplementary Table S2. Patients whose adenoma specimens were used in this study were between 26 and 74 years old (mean 52·4 ± 2·9 CI 95%) at the time of biopsy. This study was approved by the Institutional Review Board of the Central Finland Health Care District (K\\S shp Dnro4/2011) and the collection of archival specimens by the National Authority for Medicolegal Affairs (Dnro 1272/04/044/07) and the National Supervisory Authority for Welfare and Health (Valvira, Dnro 10,741/06.01.03.01/2015). 2.2. DNA extraction DNA extraction from FFPE samples was performed by the phenylchlorophorm method [24]. DNA concentrations were measured by the Nanodrop spectrophotometer (Thermo Fisher Scientific). Additionally, DNA samples from adenoma specimens were quantified by the Qubit™Fluorometer (Thermo Fisher Scientific), and these values were used in the next generation sequencing (NGS) protocol. 2.3. Immunohistochemical (IHC) staining for MMR protein expression IHC was done according to the standard procedures using the primary antibodies described previously [9,25]. 2.4. Microsatellite instability (MSI) analysis Mononucleotide repeat markers BAT25 and BAT26, which have been shown to sensitive and specific markers of high-degree MSI (MSI-H) [26,27], were used to study MSI status. When either one of the markers was unstable, a tumor was considered MSI, and if both markers were normal a tumor was considered microsatellite-stable (MSS). 2.5. Methylation assay by methylation-specific multiplex ligationdependent probe amplification (MS-MLPA) The MS-MLPA was used to study DNA methylation changes in the promoters of eight CIMP-associated genes, seven selected candidate TSGs, and LINE-1, according to the manufacturer's instructions (MRC Holland, Amsterdam, The Netherlands). The method is described in more detail in Valo et al. [9] Briefly, 150–250 ng of DNA was used for each reaction. MS-MLPA is based on the hybridizing probes including the recognition sequence of GCGC for the methylation-sensitive endonuclease HhaI. Methylation prevents digestion reaction to occur, and after the PCR amplification a signal peak appears if template DNA is methylated [28]. Methylation dosage ratios (D m ) were calculated by the following formula: D m =(P x /P ctrl ) Dig /(P x /P ctrl ) Undig , where D m is a methylation dosage ratio (degree of methylation), P x is a peak area of the given probe, P ctrl is a sum of the peak area of all control probes, Dig stands for sample digested with HhaI, and Undig stands for the undigested sample. The average degree of methylation (D m ) plus one standard deviation or two standard deviations (i.e. stringency level II that was used for the CIMP analysis for the more accurate classification) calculated for normal mucosae samples determined a threshold value for the hypermethylation for each probe. In tumor samples, probes were defined methylated, if the degree of methylation exceeded the threshold value. The results from individual tumor samples were combined to define the relative frequency of hypermethylation. CIMP status was investigated using the SALSA MLPA probemix ME042-B2 (MRC Holland) including 3–6 probes for the promoter of each marker gene: CACNA1G,IGF2,NEUROG1,RUNX3,SOCS1,CDKN2A, MLH1 and CRABP1. The gene was considered methylated when one fourth or more of the probes were methylated [29]. The tumor sample was classified CIMP(+) using the Weisenberger panel: three or more genes out of CACNA1G,IGF2,NEUROG1,RUNX3 and SOCS1 should be methylated [30]. LINE-1 hypomethylation was studied by the custom-made LINE-1MS-MLPA assay described in Pavicic et al. [31]Briefly, the assay included in total of ten probes, of those three were methylation-sensitive probes targeting regions with the HhaI restriction site inside the LINE-1 promoter sequence. The remaining seven probes provided a set of control probes targeting other regions without the restriction site in the LINE1 sequence. LINE-1 hypomethylation was calculated based on L1-M2 probe [31]. Similarly, the custom-made MS-MLPA panel was designed to study promoter methylation of the selected candidate TSGs involved in early colon tumorigenesis in a mouse model for LS: DKK1,SFRP2,CDH1, HOXD1,SFRP5,SLC5A8, and SFRP1 [23]. Detailed design is described in Valo et al. [9] The custom-made probes were used together with the reference probe mix of the SALSA MLPA P300-A2 Human DNA Reference-2 (MRC Holland). 2.6. Amplicon-based next generation sequencing (NGS) In total of 63 DNA samples of FFPE adenoma specimens were eligible for the sequencing study. Libraries were prepared from 4 to 15 ng of input DNA according to the manufacturer's protocol of the Ion AmpliSeq Library Kit 2.0 (Rev. C.0) (Thermo Fisher Scientific) using the Ion AmpliSeq Colon and Lung Cancer Panel (Thermo Fisher Scientific). The panel targets mutational hotspots in 22 genes totaling 14.6 kb: AKT1, ALK,BRAF,CTNNB1,DDR2,EGFR,ERBB2,ERBB4,FBXW7,FGFR1,FGFR2, FGFR3,KRAS,MAP2K1,MET,NOTCH1,NRAS,PIK3CA,PTEN,SMAD4, STK11,andTP53. The libraries were purified using the AMPure XP magnetic beads (Beckman Coulter, Krefeld, Germany), and the quantification was performed with the Qubit Fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Library amplification and enrichment steps were performed according to the manufacturer's protocol of the Ion PGM Hi-Q View OT2 Kit (Rev.C.0) (Thermo Fisher Scientific) on the Ion OneTouch2 and Ion OneTouch ES instruments, respectively (Thermo Fisher Scientific). Sequencing was performed using the Ion PGM Hi-Q View Sequencing Kit (Rev.C.0) on the Ion PGM 316v2 Chip (Thermo Fisher Scientific). The barcoded (Ion Xpress Barcode Adapters, Thermo Fisher Scientific) libraries were pooled in the sets of 8–16 libraries. The sequencing data were analyzed utilizing the Torrent Suite software (v5.2.2). Coverage analysis was performed by using the plug-in Table 1 Lynch syndrome individuals and eligible specimens. No of patients or specimens Patients with germline mutation in MLH1 44 MSH2 8 MSH6 5 Total 57 Colorectal specimens Normal colonic mucosa 29 Low dysplasia adenoma 47 High dysplasia adenoma 21 Carcinoma 25 Total 122 282 S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 of coverageAnalysis (v5.2.1.2), and variant calling was conducted with the variantCaller (v5.2.1.39). Default analysis parameters were applied on signal processing, base calling, read alignment and variant calling. Reads were aligned at the human genome reference assembly of h19. Variant calling files were imported in the VarSeq software (GoldenHelix) and annotated with the databases of COSMIC Cancer Gene Census 71 [32], ExAC Variant Frequencies 0.3, dbSNP 149 (NCBI), and RefSeq Genes 105 Interim v1 (NCBI), and in silico functional prediction tools (SIFT, PolyPhen2 HVAR, MutationTaster, MutationAssessor, FATHMM, and FATHMM MKL). For the final step of variant analysis, only variants with quality score ≥30, read depth ≥50, and VAF ≥0·05 were considered true positives. Moreover, all variants mapped outside the target regions, producing synonymous variant, or variants reported in the 1000 Genomes project (NCBI dbSNP 149) and/or in the ExaC database with minor allele frequency (MAF) of 0·01 or more, were filtered out. 2.7. Statistical analysis Statistical analyses were performed using the SPSS software, version 24.0 (IBM SPSS Inc., Chicago, IL, USA). Group-wise comparisons for categorical variables were calculated by using the Fisher's exact-test. For continuous variables, normal distribution was tested by Shapiro-Wilk test, and a parametric (Student's t-test or one-way ANOVA with Tukey's post hoc test, homogeneity of variances tested by Levene's test) or nonparametric (Mann-Whitney Utest/Kruskal-Wallis one-way ANOVA) test was used, as appropriate. Two-sided pvalues b0·05 were considered significant. The Bonferroni correction method was applied on p values obtained from multiple comparisons. 2.8. Data sharing statement All relevant data supporting this study is published along the paper. Case-specific DNA methylation data is available upon request. 3. Results 3.1. MMR protein expression and MSI status The colorectal specimen collection described in Table 1 was used for IHC investigations to evaluate if the remaining wild-type allele of the germline mutated MMR gene had undergone somatic inactivation. Protein expression of the relevant MMR genes was lost in all carcinomas (Table 2). Among adenomas, 35% (16/46) of low dysplasia cases and 10% (2/21) of high dysplasia cases retained the protein expression of the predisposing MMR gene (Table 2). Normal mucosae (n=43)were tested previously and all showed normal MMR protein expression [9]. Table 2 The proportions of retained MMR protein expression in adenomas and carcinomas by IHC (extended series of the previous study [9]). MLH1 MSH2 MSH6 Total a Adenoma with low-grade dysplasia 11/34 (32%) 3/8 (38%) 2/4 (50%) 16/46 (35%) Adenoma with high-grade dysplasia 1/17 (6%) 0/2 (0%) 1/2 (50%) 2/21 (10%) Carcinoma 0/15 (0%) 0/3 (0%) 0/5 (0%) 0/23 (0%) a MMR IHC could not be performed on two carcinoma and one adenoma with lowgrade dysplasia specimens, and thus, they are missing from this table compared to the numbers presented in Table 1. Normal – MMR-P NS NS 0·009 NS NS NS NS NS NS Normal – MMR-D NS <0·0001 0·009 NS NS NS NS 0·045 NS MMR-P – MMR-D NS NS NS NS NS NS NS NS NS 3 14 333 10 3300 42 50 0 8 42 25 33 8 10 79 36 14 7 36 14 31 8 0 20 40 60 80 100 CACNA1G IGF2 NEUROG1 RUNX3 SOCS1 CDKN2A MLH1 CRABP1 CIMP(+) % Normal (n = 29) MMR-P (n = 12) MMR-D (n = 42) Normal – MMR-P NS 0·049 NS NS NS NS <0·0001 Normal – MMR-D NS <0·0001 NS NS NS NS <0·0001 MMR-P – MMR-D NS NS NS NS NS NS NS 17 17 21 24 0 14 17 33 60 27 33 7 40 87 30 70 19 33 2 35 72 0 20 40 60 80 100 DKK1 SFRP2 CDH1 HOXD1 SFRP5 SLC5A8 SFRP1 % Normal (n = 29) MMR-P (n = 15) MMR-D (n = 43) a b Fig. 1. Frequencies of methylated genes in normal colon, and MMR-P and MMR-D adenoma specimens. a. Hypermethylation frequencies of CIMP marker genes and frequencies of CIMP positive phenotype. b. Hypermethylation frequencies of candidate tumor suppressor genes. Bonferroni corrected two-sided pvalues are presented for pairwise comparisons. Abbreviations: MMR-D, MMR deficient; MMR-P, MMR proficient; NS, non-significant. 283S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 MSI status was determined by the mononucleotide markers BAT25 and BAT26. We found a clear association between IHC and MSI status (pb0·0001). All tested carcinomas and all adenomas with absent MMR protein showed MSI, with one exception of adenoma with low dysplasia having stable microsatellites. All adenomas with intact MMR protein expression for whichenough DNA was available for MSI analysis (n= 11) were MSS. Throughout this paper, adenomas with absent MMR protein or presence of MSI or both are considered MMRdeficient (MMR-D, n= 49) and those with neither abnormality MMRproficient (MMR-P, n=18). The existence of significant MMR-proficient subsets of LS-adenomas indicates the necessity of other somatic events to initiate malignant Normal n = 29 MMR-P n = 15 MMR-D n = 43 SFRP1 (Dm) SFRP2 (Dm) Normal n = 29 MMR-P n = 15 MMR-D n = 43 bc p<0·0001 p< 0·0001 p= 0·011 p< 0·0001 1·0 0·8 0·6 0·4 0·2 0·0 1·0 0·8 0·6 0·4 0·2 0·0 Probe Normal – MMR-P Normal – MMR-D MMR-P – MMR-D Kruskal-Wallis pvalue MLH1 III NS 0·060 NS 0·046 IGF2 I 0·005 <0·0001 NS <0·0001 IGF2 II NS 0·023 NS 0·028 IGF2 III 0·005 <0·0001 NS <0·0001 NEUROG1 I NS 0·021 NS 0·017 NEUROG1 II NS 0·024 NS 0·016 NEUROG1 III NS 0·021 NS 0·026 NEUROG1 IV NS 0·008 NS 0·009 NEUROG1 VI 0·047 NS NS 0·045 CDKN2A IV 0·028 0·001 NS 0·001 CRABP1 III 0·040 NS NS 0·034 CACNA1G III NS 0·034 NS 0·034 MMR-D (n = 42) MMR-P (n = 12) Normal (n = 29) CACNA1G III CRABP1 III CDKN2A IV NEUROG1 VI NEUROG1 IV NEUROG1 III NEUROG1 II NEUROG1 I IGF2 III IGF2 II IGF2 I MLH1 III Methylation (Dm) 1·0 0·8 0·6 0·4 0·2 0·0 a Fig. 2. Comparison of the average degrees of methylation in normal colon, and MMR-P and MMR-D adenoma specimens. a. The CIMP probes showing statistical significance (pb0·05) with Kruskal-Wallis test. b. Average degree of methylation of SFRP1. c. Average degree of methylation of SFRP2. Bonferroni corrected two-sided pvalues b0·10 are presented for the pairwise comparisons. The upper and lower edges of the boxes are the 75th and 25th percentiles, the horizontal line inside the box denotes the median, the whiskers indicate the highest and lowest values, and the asterisks and open circles stand for outliers. Abbreviations: D m , methylation dosage; MMR-D, MMR deficient; MMR-P, MMR proficient. 284 S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 transformation, leading us to investigate DNA methylation changes and somatic mutations in LS-associated adenomas (see below). 3.2. DNA methylation changes 3.2.1. CIMP markers Methylation analysis of eight genes (CACNA1G,IGF2,NEUROG1, RUNX3,SOCS1,CDKN2A,MLH1 and CRABP1) linked to CIMP was used to assess the frequency of CIMP, a high-degree coordinated methylation of CpG islands that are normally unmethylated. CIMP(+) was defined according to the Weisenberger panel [30]. The frequency of CIMP(+) increased along with the dysplasia grade, occurring in 0% (0/29), 11% (4/37), 17% (3/18), and 45% (10/22) of normal mucosae, adenomas with low-grade dysplasia, adenomas with high-grade dysplasia, and carcinomas, respectively. Altered methylation was evaluated in two ways, by determining the frequencies of hypermethylated samples based on defined cut-off methylation dosage (D m ) values for hypermethylation for each marker gene with normal mucosa as a reference (see Materials and Methods) and alternatively, by treating the degree of methylation (by D m values) as a continuous variable in the samples. Fig. 1ashowsthefrequenciesof hypermethylation for eight CIMP markers in adenomas stratified by MMR status, with normal mucosae as a reference. Hypermethylation frequency of NEUROG1 was increased already in MMR-P adenomas compared to normal colon (p= 0·009), and methylation frequency was higher than in MMR-D adenomas, although statistical significance was not obtained between MMR-P and MMR-D cases. MMR-D adenomas showed significantly elevated frequencies of hypermethylation relative to normal mucosa at IGF2 (pb0·0001), NEUROG1 (p=0·009)and CRABP1 (p= 0·045) loci. Among adenomas with MLH1 methylation depicted in Figs. 1aand2a, only one, a MMR-D adenoma with highgrade dysplasia, revealed hypermethylation with the probe targeting the promoter region “C”of MLH1 (MLH1-I probe); hypermethylation of this region is associated with loss of MLH1 protein [33]. In the remaining cases, other regions of the MLH1 promoter (MLH1-II, III, and IV probes), not associated with protein expression, were involved. When examining the average D m values for the individual probes contributing to each CIMP marker gene (Fig. 2a), at least one probe of the following marker genes revealed a significant increase already in MMR-P adenomas vs. normal colon: IGF2,NEUROG1,CDKN2A,and CRABP1. Of those, almost all probes showed significantly increased methylation also in MMR-D adenomas vs. normal colon. There were two probes, one for CRABP1 and one for NEUROG1, which showed significantly elevated methylation solely in MMR-P adenomas (vs. normal colon). 3.2.2. Candidate tumor suppressor genes We studied seven candidate TSGs (DKK1,SFRP2,CDH1, HOXD1, SFRP5,SLC5A8, and SFRP1) associated with early colon oncogenesis and being downregulated by promoter methylation in an experimental mouse model for LS [23]. Similarly to CIMP marker analysis, we first compared the hypermethylation frequencies of each gene in normal colon, MMR-P and MMR-D adenomas. Here, both SFRP1 and SFRP2 showed significantly increased frequency of hypermethylation in both MMR-P and MMR-D tumors compared to normal colon (Fig. 1b). Likewise, in the analysis of average D m values, SFRP1 and SFRP2 showed increased methylation levels in both MMR-P and MMR-D adenomas compared to normal colon (Fig. 2b, c). 3.2.3. LINE-1 The average degree of methylation of LINE-1, a marker for global hypomethylation, decreased along with dysplasia (p= 0·005) (Fig. 3a). Statistically significant difference relative to normal colon was observed for carcinoma, but not for adenomas with highor low-grade dysplasia, suggesting that LINE-1 methylation is a rather late event in LSassociated tumorigenesis. When comparing adenomas stratified by MMR status with normal colon, significantly decreased methylation level was observed for MMR-D adenomas (Fig. 3b), indicating that LINE-1 hypomethylation is likely to occur after the loss of MMR protein. 4. Effect of dysplasia grade on methylation results To produce the results given in Figs. 1–3, adenomas with highand low-grade dysplasia were combined. The same analyses were performed on adenomas with low-grade dysplasia only and are available in Supplementary Materials (Supplementary Fig. S1–S3). Adenomas with high-grade dysplasia included too few MMR-P cases (Table 2) and could not be studied as an independent group. As evident from Supplementary Figs. S1–S3, the results remained essentially similar to those derived from adenomas not stratified by dysplasia grade. Moreover, to exclude the effect of dysplasia grade on the results, we compared adenomas with high-grade dysplasia to those with low-grade dysplasia LINE-1 (Dm) Normal n = 16 AdLGD n = 45 AdHGD n = 21 Carcinoma n = 24 p= 0·002 p= 0·079 ba LINE-1 (Dm) Normal n = 16 p= 0·010 MMR-P n = 17 MMR-D n = 48 1·1 1·0 0·8 0·7 0·6 0·5 0·9 1·1 1·0 0·8 0·7 0·6 0·5 0·9 Fig. 3. Average degree of LINE-1 methylation. a. LINE-1 methylation levels in all the specimens sorted by histology. b. LINE-1 methylation in normal colon, and MMR-P and MMR-D adenoma specimens. Bonferroni corrected two-sided pvalues b0·10 are presented in the figure. The upper and lower edges of the boxes are the 75th and 25th percentiles, the horizontal line inside the box denotes the median, the whiskers indicate the highest and lowest values, and the asterisks and open circles stand for outliers. Abbreviations: AdHGD, adenoma with high-grade dysplasia; AdLGD, adenoma with low-grade dysplasia; D m , methylation dosage; MMR-D, MMR deficient; MMR-P, MMR proficient. 285S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 regardless of MMR status, and no significant differences were observed (data not shown). 4.1. Somatic mutations Adenomas were studied for somatic mutations targeting the mutational hotspots in 22 cancer-associated genes (see Materials and Methods). As the amount of sample material of adenoma specimens was limited, this panel served as an excellent option to study somatic mutations even in those samples with a very low DNA content. In total, 59 out of initial 63 samples that were eligible for the sequencing study produced good quality sequencing library and sequencing results, including 16 MMR-P adenomas, out of which a majority (n=14)were adenomas with low-grade dysplasia. For the obtained sequencing data, mean values for the read depth, the mapping uniformity between the amplicons, and the percentage of reads mapped on targets were 1379 (± 176 CI 95%), 94% (± 2 CI 95%), and 92% (± 1 CI 95%) respectively. Here, we present non-synonymous mutations that have previously been reported in the COSMIC database [32], emphasizing their cancerassociated nature. The most frequently mutated genes were TP53, followed by KRAS,SMAD4,andCTNNB1, found in 24% (n = 14), 22% (n = 13), 19% (n= 11), and 15% (n= 9) of the adenomas, respectively. Overall, 54% (32/59) of adenomas harbored at least one mutation. One MMR-P sample (249Ad) included clearly more mutations (n=7) than other MMR-P adenomas (Fig. 4). This particular case was the only CIMP(+) MMR-P adenoma. In total, there were five CIMP(+) SampleID MMR status CIMP status MSI status Dysplasia grade Gene mutated No. of mutations AKT1 ALK BRAF CTNNB1 DDR2 EGFR ERBB2 ERBB4 FBXW7 FGFR1 FGFR2 FGFR3 KRAS MAP2K1 MET NOTCH1 NRAS PIK3CA PTEN SMAD4 STK11 TP53 249Ad P + MSS? LOW MLH1 7000 0 010000001000010103 missense mutation 686Ad P - MSS HIGH MSH6 2000 0 000000001000000001 nonsense mutation 264Ad P - MSS? LOW MLH1 2000 0 000000001000000100 in-frame deletion 670Ad P ND ND LOW MSH2 2000 0 000010001000000000 frameshift deletion 187Ad P - MSS LOW MSH6 1000 1000000000000000000 248Ad P - MSS? LOW MSH2 1000 0 000000001000000000 268Ad P - MSS LOW MLH1 1000 1000000000000000000 677Ad P - ND HIGH MLH1 0000 0 000000000000000000 148Ad P - MSS LOW MLH1 0 164Ad P ND MSS? LOW MLH1 0 197Ad P ND ND LOW MLH1 0 259Ad P - MSS LOW MLH1 0 271Ad P - MSS LOW MLH1 0 672Ad P - ND LOW MSH6 0 690Ad P - ND LOW MLH1 0 693Ad P - MSS LOW MLH1 0 689Ad D - ND LOW MSH6 821311111102 101Ad D + MSI LOW MLH1 6 112211151 260Ad D ND ND LOW MLH1 2132312151 151Ad D ND ND LOW MLH1 311121101 261Ad D ND ND HIGH MLH1 12121119 100Ad D + MSI HIGH MLH1 21137 169Ad D - MSI HIGH MLH1 4 1111 170Ad D - MSI HIGH MLH1 2 11 270Ad D - MSI HIGH MLH1 112 674Ad D - MSI HIGH MLH1 112 680Ad D - MSI LOW MLH1 211 685Ad D - MSI LOW MLH1 211 695Ad D - MSI LOW MSH2 112 247Ad D - MSI HIGH MSH2 1 1 265Ad D - MSI HIGH MLH1 11 272Ad D - MSI HIGH MLH1 11 671Ad D ND MSI HIGH MSH2 1 1 682Ad D - ND HIGH MLH1 11 106Ad D - MSI LOW MLH1 1 1 168Ad D - MSI LOW MLH1 1 1 171Ad D - MSI LOW MLH1 1 1 256Ad D + MSI LOW MLH1 11 258Ad D - MSI LOW MSH2 1 1 266Ad D + MSI LOW MLH1 11 684Ad D - ND LOW MLH1 1 1 165Ad D - MSI HIGH MLH1 0 253Ad D - MSI HIGH MLH1 0 263Ad D - MSI HIGH MLH1 0 679Ad D ND ND HIGH MLH1 0 687Ad D - MSI HIGH MSH6 0 160Ad D - MSS LOW MSH2 0 188Ad D - MSI LOW MSH6 0 195Ad D - MSI LOW MLH1 0 196Ad D - MSI? LOW MLH1 0 250Ad D - MSI LOW MLH1 0 251Ad D - MSI LOW MSH2 0 252Ad D - MSI LOW MLH1 0 254Ad D - MSI LOW MLH1 0 255Ad D - MSI LOW MLH1 0 257Ad D - MSI? LOW MLH1 0 675Ad D - MSI LOW MLH1 0 691Ad D - MSI LOW MLH1 0 692A d D - MSI LO W MLH1 0 Total (n = 59) 1.7 8.5 15.3 1.7 10.2 3.4 5.1 8.5 1.7 3.4 1.7 22.0 1.7 1.7 5.1 10.2 18.6 3.4 23.7 5.215.213.63.133.63.65.21)61=n(P-RMM MMR-D (n = 43) 2.3 11.6 16.3 2.3 11.6 4.7 7.0 9.3 2.3 4.7 18.6 2.3 2.3 4.7 14.0 20.9 4.7 28.0 AdLGD (n = 41) 2.4 9.8 9.8 2.4 12.2 2.4 4.9 9.8 2.4 2.4 19.5 2.4 2.4 4.9 9.8 19.5 4.9 19.5 3.337.611.116.58.726.56.56.56.56.58.726.5)81=n(DGHdA Fig. 4. Mutation status of adenomas stratified by their MMR status and number of mutations. Mutations presented in this table are reported in the COSMIC database (v71). Numbers in the colored cells indicate the number of mutations affecting a given gene in the sample in question, and color distinguishes the type of mutation. Table below shows mutation frequencies in adenomas stratified by MMR status or dysplasia grade. Note: question mark after MSI or MSS status indicates that only one probe produced an interpretable result (for MSS?) or due to poor quality, the result was weakly interpretable (MSI?). Abbreviations: AdHGD, adenoma with high-grade dysplasia; AdLGD, adenoma with low-grade dysplasia; CIMP, CpG island methylator phenotype; D, deficient; MMR, DNA mismatch repair; MMR-D, MMR deficient; MMR-P, MMR proficient; ND, not done; P, proficient. 286 S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291 adenomas and three of those harbored several mutations in multiple genes (Fig. 4). A vast majority of the mutations were missense mutations (Fig. 4). The full list of our COSMIC mutations is available in Supplementary Table S3. We observed a somewhat higher average number of mutations in MMR-D adenomas compared to MMR-P adenomas (2·4 vs. 1·0), although no statistical difference was found (p= 0·323). As the most intriguing finding, KRAS mutations were clearly more prevalent in MMR-P adenomas (Fig. 4) (5/16, 31% vs. 8/43, 19%), although the difference is statistically non-significant. Considering only KRAS mutations in the codons 12, 13, 61, or 146 regarded pathogenic [34–36], these were clearly more prevalent in MMR-P tumors (5/16, 31% vs. 7/43, 16%). The observation remained the same when only adenomas with low-grade dysplasia were included: the frequency of pathogenic KRAS mutations was 29% (4/14) in MMR-P cases vs. 15% (4/27) in MMR-D cases. Detailed molecular characteristics of MMR-P adenomas are presented case by case in Table 3. Mutational frequencies between adenomas with low-grade and high-grade dysplasia were mostly similar, with no statistically significant differences observed (Fig. 4). However, multiple genes, such as BRAF (4/41, 10% vs. 1/18, 6%) and SMAD4 (8/41,20% vs. 3/18, 17%) were slightly more frequently mutated in adenomas with low-grade dysplasia than in tumors with high-grade dysplasia. Instead, mutations in CTNNB1 (5/18, 28% vs. 4/41, 10%), KRAS (5/18, 28% vs. 8/41, 20%), and TP53 (6/18, 33% vs. 8/41, 20%) occurred more often in advanced stage adenomas. One adenoma with low-grade dysplasia (256Ad) carried the BRAF V600E mutation (VAF 5%). The BRAF mutation was the only mutation detected in this specimen. Other BRAF missense mutations that were found in four cases occurred in the codons 455, 467, and 469. The BRAF V600E mutant tumor was characterized by MMR-D, CIMP(+), MSI, and germline point mutation in MLH1 (c.543C NG). Moreover, this tumor harbored hypermethylation in multiple promoters, including all three probes of IGF2, two probes of NEUROG1,oneCRABP1,allthree probes of RUNX3,SFRP1,HOXD1,andSFRP2. We also addressed the mutation signatures by Alexandrov et al. [37], taking the MMR deficiency related signature 6 as a reference. The adjacent bases for single base pair substitutions were determined and mutation patterns constructed for adenomas based on the relative frequencies of mutation types among all mutations found (Fig. 5a, b). In this analysis, in addition to the COSMIC mutations, we also included other variants fulfilling the filtering criteria (see Materials and Table 3 Molecular characteristics of Lynch syndrome adenoma specimens with retained MMR protein expression, undergone somatic mutation analysis. Patient ID Sample ID Dysplasia grade Germline mutation Hypermethylated CIMP markers Hypermethylated candidate TSGs Somatic mutations (VAF %) MSI status CIMP status 73 148Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) IGF2 None None MSS (−) 3 164Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) ND ND None MSS a ND 16 187Ad LOW MSH6 c.900dup NEUROG1 SFRP1 CTNBB1 p.Ser45Phe (37) MSS (−) 95 197Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) ND SFRP1 None ND ND 13 248Ad LOW MSH2 c.1667_1671del CDKN2A SFRP2,SLC5A8,SFRP1 KRAS p.Gly12Asp (7) MSS a (−) 17 249Ad LOW MLH1 c.546-2A NGMLH1, IGF2, NEUROG1, CDKN2A, CRABP1 SFRP2,HOXD1,SFRP1 EGFR p.Ala767Val (5), KRAS p.Gly12Val (8), PIK3CA p.Arg693His (5), SMAD4 p.Pro514Ser (5), TP53 p.Arg282Trp (6); p.Pro250Leu (5); p.Gly244Ser (5) MSS a (+) 45 259Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) NEUROG1 SFRP2,SFRP1 None MSS (−) 76 264Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) NEUROG1, CDKN2A, CRABP1 SFRP1 KRAS p.Gly12Val (29), SMAD4 p.Pro102Leu (18) MSS a (−) 95 268Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) MLH1, IGF2, NEUROG1, CRABP1 none CTNNB1 p.Thr41Ala (5) MSS (−) 101 271Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) none SFRP1 None MSS (−) 12 670Ad LOW MSH2 c.1552_1553_del ND SFRP2,CDH1,HOXD1, SLC5A8,SFRP1 FBXW7 p.Arg465His (7), KRAS p.Ala146Thr (10) ND ND 16 672Ad LOW MSH6 c.900dup ND SFRP2,CDH1,HOXD1, SLC5A8, SFRP1 None ND ND 73 677Ad HIGH MLH1 c.1731 + 2247_1897-402del (Mutation 1) None SFRP2,SFRP1 None ND (−) 112 686Ad HIGH MSH6 c.3195_3199del IGF2, NEUROG1, CDKN2A, CRABP1 SFRP2,SLC5A8, SFRP1 KRAS p.Ala146Thr (45), TP53 p.Glu180Lys (5) MSS (−) 120 690Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) None SFRP2,CDH1,HOXD1, SOCS1,SLC5A8,SFRP1 None ND (−) 123 693Ad LOW MLH1 c.1731 + 2247_1897-402del (Mutation 1) IGF2, NEUROG1, CDKN2A SFRP2,CDH1,HOXD1, SFRP5,SOCS1, SLC5A8,SFRP1 None MSS (−) Abbreviations: CIMP, CpG island methylator phenotype; MSS, microsatellite stable; MSI, microsatellite instable; ND, not done; TSGs, tumor suppressor genes; VAF, variant allele frequency. a Only one of the two microsatellite markers produced an interpretable result. 287S. Mäki-Nevala et al. / EBioMedicine 39 (2019) 280–291