Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors

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 4.0 https://creativecommons.org/licenses/by/4.0/ Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors © 2024 Olkinuora, Mäki-Nevala, Ukwattage, Ristimäki, Ahtiainen, Mecklin and Peltomäki. Published version Olkinuora, Alisa; Mäki-Nevala, Satu; Ukwattage, Sanjeevi; Ristimäki, Ari; Ahtiainen, Maarit; Mecklin, Jukka-Pekka; Peltomäki, Päivi Olkinuora, A., Mäki-Nevala, S., Ukwattage, S., Ristimäki, A., Ahtiainen, M., Mecklin, J.-P., & Peltomäki, P. (2024). Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors. Frontiers in Oncology, 14, Article 1378392. https://doi.org/10.3389/fonc.2024.1378392 2024 Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors Alisa Olkinuora 1 *, Satu Mäki-Nevala 1 , Sanjeevi Ukwattage 1 , Ari Ristimäki 2,3 , Maarit Ahtiainen 4 , Jukka-Pekka Mecklin 5,6 and Päivi Peltomäki 1,7 1 Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland, 2 Department of Pathology, HUSLAB, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, HUS, Helsinki, Finland, 3 Applied Tumor Genomics Research Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 4 Department of Pathology, Wellbeing Services County of Central Finland, Jyväskylä, Finland, 5 Department of Education and Science, Nova Hospital, Central Finland Health Care District, Jyväskylä, Finland, 6 Faculty of Sports and Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 7 HUSLAB Laboratory of Genetics, HUS Diagnostic Center, HUS, Helsinki University Hospital, Helsinki, Finland Background: Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Aim: Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. Methods: We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. Results: All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic Frontiers in Oncology frontiersin.org01 OPEN ACCESS EDITED BY Shulan Tian, Mayo Clinic, United States REVIEWED BY Florica Sandru, Carol Davila University of Medicine and Pharmacy, Romania Dongbo Yang, The University of Chicago, United States *CORRESPONDENCE Alisa Olkinuora alisa.olkinuora@helsinki.fi RECEIVED 29 January 2024 ACCEPTED 01 April 2024 PUBLISHED 25 April 2024 CITATION Olkinuora A, Mäki-Nevala S, Ukwattage S, Ristimäki A, Ahtiainen M, Mecklin J-P and Peltomäki P (2024) Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors. Front. Oncol. 14:1378392. doi: 10.3389/fonc.2024.1378392 COPYRIGHT © 2024 Olkinuora, Mäki-Nevala, Ukwattage, Ristimäki, Ahtiainen, Mecklin and Peltomäki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 25 April 2024 DOI 10.3389/fonc.2024.1378392 mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Conclusion: Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS. KEYWORDS Lynch syndrome, exome sequencing, panel sequencing, multiple adenomas, RNF43 1 Introduction LS is a relatively common disorder (affects one in 250-400 individuals) with a high lifetime risk of cancer caused by the deficiency of one of the main four DNA mismatch repair genes: MLH1,MSH2,MSH6,orPMS2, or by transcriptional silencing of MSH2 via 3’deletion of EPCAM. Typically, LS families display autosomal dominant inheritance of colorectal cancer (CRC) (and various extracolonic cancers such as endometrial cancer) with a relatively young age at onset (< 50 years) (1,2). The Amsterdam criteria, used as an aid in the clinical diagnosis of LS, draws heavily on the family history of CRC, but additionally states the need for the absence of polyps to distinguish LS from familial adenomatous polyposis (FAP) (3,4). Although the cumulative lifetime adenoma burden of LS patients generally stays below ten, recent data has shown that LS may manifest with an elevated polyp count, and that individuals with pathogenic germline variants in different genes may undergo gene-specific tumorigenesis (5–9), creating difficulties for diagnosis. LS patients with an elevated adenoma count are additionally significantly more likely to have an advanced colorectal neoplasia (8). The adenoma-carcinoma pathway of CRC is grouped into three distinct subtypes: the chromosomal instability (CIN) pathway, the MSI pathway, and the CIMP pathway. LS-associated tumorigenesis is generally thought to arise via MSI pathway, but recently the possibility of copy-neutral loss of heterozygosity for carriers of MLH1 germline variants as the cause of cancer has been discussed (10). Of all CRCs, about 10% follow an alternative pathway wherein the adenoma precursor is replaced with a serrated polyp. Although the MSI pathway can sometimes give rise to serrated-type tumors, the CIMP adenoma-carcinoma pathway produces adenomas that resemble MSI carcinomas due to hypermethylation of MLH1 but lack the hallmark genetic disruption in APC; instead, tumors form in association with BRAF and KRAS mutations driven by an exclusive WNT activating RNF43 mutation in sporadic CRC (11, 12). Once RNF43 is inactivated in the serrated lesions, they may advance into mucinous adenocarcinomas with a high likelihood of metastasis. Although frequently observed in sporadic CRC, the mechanisms of the accelerated tumor progression and metastatic spread are poorly understood. We undertook this investigation to explore the molecular background of colorectal tumor multiplicity in LS, the existing knowledge of which is scarce. We determined the constitutional and somatic molecular profiles of three LS cases with multiple adenomas and compared the findings to a cohort of LS patients with no such colorectal tumor multiplicity. We describe distinct tumor profiles that may be associated with increased colon tumor burden in LS. 2 Materials and methods 2.1 Patients and samples This investigation was carried out on a total of 26 index cases with molecularly confirmed LS (Figure 1)fromFinland.The patients were ascertained from the Finnish Lynch Syndrome Research Registry. DNA was extracted from blood or archival formalin-fixed paraffin-embedded (FFPE) samples following the protocol presented in Isola et al. (13). Case LS-298 Our index individual initially presented with a 25 mm mucinous cecal adenocarcinoma and a 40 mm moderately differentiated adenocarcinoma of the ascending colon at 52 years of age. The ascending colon housed a few adenomatous polyps, as well. After right sided hemicolectomy, he developed several tumors in the descending colon as well as renal carcinoma (Table 1). Abbreviations: CCP, Comprehensive Cancer Panel; CIMP, CpG Island Methylator Phenotype; CIN, Chromosomal Instability; CMMRD, Constitutional Mismatch Repair Deficiency; CpG, 5’- Cytosine –phosphate – Guanine-3’; CRC, colorectal cancer; ES, Exome sequencing; FAP, Familial adenomatous polyposis; FFPE, Formalin-Fixed Paraffin-Embedded; IBD, Inflammatory Bowel Disease; ID, Insertion-deletion; IGV, Integrative Genomics Viewer; IHC, Immunohistochemistry; LINE, Long Interspersed Elements; LS, Lynch syndrome; LOH, Loss of Heterozygosity; MSI, Microsatellite Instability; MLPA, Multiplex Ligation-dependent Probe Amplification; MMR, Mismatch Repair; MS-MLPA, Methylation-Specific Multiplex Ligation-dependent Probe Amplification; SBS, Single Base Substitution; TMB, Tumor MutationalBurden;VUS,Variantof Unknown Significance. Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org02 Analysis of the germline tissue revealed a pathogenic variant in MLH1, c.677G>T, p.(Gln197Argfs*8) (14). Case F1001 The index of F1001 had a personal history of several adenomas, leading to the initial suspicion of attenuated familial adenomatous polyposis. He underwent subtotal colectomy at 47 years of age due to the presence of multiple seemingly primary carcinomas with mucinous, and signet cell/mixed histology as well as several adenomatous polyps. Subsequent analysis of the case revealed a 27 bp deletion of MSH2 c.1140_1166del, p.(Leu381_Arg389del) in the MSH3/MSH6 interaction domain (6). MSI and IHC analyses complied with MSH2-associated Lynch syndrome. Case LS-202 The case LS-202 was initially diagnosed with a moderately differentiated rectum carcinoma and several sigmoid polyps at 57 years of age. He underwent an anterior resection During routine follow-up screens, adenomas were observed at a regular interval, and at the age of 64, he developed two cecal pT2N0 adenocarcinomas alongside few adenomas. Despite being subjected to right-sided hemicolectomy, several adenomas were observed at routine screens. Molecular analysis of germline tissue revealed a likely pathogenic germline alteration in MSH6, c.900dup p.(Lys301Glufs*11) (15). Control LS cohort Twenty-five LS-tumors (ten adenomas and 15 carcinomas) from 23 LS cases with a typical disease expression were subjected to panel sequencing using the Comprehensive Cancer Panel (CCP) as previously described (15). Somatic mutational data for this cohort is available online by Porkka et al. (15). All control LS patients were confirmed carriers of pathogenic or likely pathogenic germline variants in the MMR genes. Written informed consent preceded study participation and sample donation. This study was approved by the Institutional Review Boards of the Helsinki University Central Hospital (466/E6/ 01) and Central Finland Health Care District (10U/2011) approved this study. The National Supervisory Authority for Welfare and Health (Dnro 1272/04/044/07 and Dnro 10741/06.01.03.01/2015) approved the collection of archival specimens. 2.2 Exome sequencing and variant prioritization Exome sequencing (ES) was performed as previously described by Olkinuora et al., or by preparing the library and enriching components with the Twist Core Exome + RefSeq kit and run on Novaseq S1 system at the Institute for Molecular Medicine Finland, FIMM (Supplementary Table S1)(6). Sequences were aligned to the human reference genome GRCh37/hg19 using the Burrows- Wheeler Aligner version 0.6.2. Quality control was performed as described by Sulonen et al. (16). Germline and somatic variant data was annotated using ANNOVAR (17). Variants fulfilling the following selection criteria were selected for further analyses: gnomAD allele frequency < 0.001, nonsynonymous (frameshift, stop gained/lost, missense, disrupting donor/acceptor site variants) and predicted pathogenic with at least five of six programs assessing protein function in silico (for missense changes). DNA methyltransferases FIGURE 1 Outline of this investigation. Study cases and cohorts as well as methodological approaches and rationales behind them are shown. Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org03 TABLE 1 Molecular features of the case LS-298 in chronological context. Carcinomas Adenomas Sample ID carcinoma 1 carcinoma 2 carcinoma 3 carcinoma 4 carcinoma 5 adenoma 1 adenoma 2 adenoma 3 adenoma 4 adenoma 5 adenoma 6 adenoma 7 adenoma 8 adenoma 9 adenoma 10 same tumor same tumor same tumor Years since dx 0 0 0 0 21 12 15 15 15 18 18 21 21 21 21 Tumor information Tumor location ascendens ascendens caecum caecum transversum NA rectosigmoid rectum rectum rectum rectum transversum rectum rectum transversum Histology NA NA Mucinous Mucinous Mucinous, signet cell Villous Tubular Tubular Tubular Tubuvillous Tubuvillous Tubular Tubular Tubular Tubular Dysplasia moderate moderate high moderate moderate moderate high high high low MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI MSI 2nd hit or LOH No No No No Yes Yes Yes No Yes Yes No Yes No No No TMB (mut/Mb) 34.39 53.03 13.52 22.76 25.39 30.30 39.27 38.55 33.27 32.79 34.64 41.64 42.21 33.79 28.06 Oncogenic event APC –miss –miss –trunc ––––– – –trunc – CTNNB1 ––miss miss miss ––––––––miss – KRAS trunc – – – –– ––––––––– TP53 trunc ––trunc –– –––––trunc –– – RNF43 hotspot trunc trunc ––trunc –trunc trunc trunc trunc trunc trunc trunc –trunc MBD4 ––––––trunc trunc ––– – –trunc trunc SBS96 No Weak No No Weak Weak Yes Yes Yes Yes Yes Yes Yes Yes Yes CIMP (Weisenberger) Yes Yes No NA No Yes Yes No No No No Yes No Yes No LINE-1 1m 1.06 0.66 0.75 0.89 0.98 0.76 0.96 0.92 0.95 0.71 0.73 0.95 0.83 0.86 0.78 LINE-1 2m 1.13 0.63 0.73 0.78 0.74 0.82 0.90 0.81 0.89 0.58 0.66 0.94 0.80 0.79 0.79 LINE-1 3m 0.81 0.78 0.79 0.71 0.79 0.73 0.71 0.94 0.90 0.71 0.61 0.73 0.93 0.75 0.91 %C>T at CpG 36.3% 27.9% 14.2% 19.5% 42.2% 48.3% 56.6% 58.3% 56.8% 56.6% 56.8% 52.2% 50.5% 53.8% 54.6% CIMP was evaluated using the Weisenberger criteria. trunc, truncating; miss, missense; NA, Not Available. Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org04 and genes with the methyl-CpG binding domain according to the HGNC database (https://www.genenames.org/) were taken for closer inspection. The clinical enrichment analysis of MAFtools was used to compare germline variant distribution between groups (18). The input variants were restricted to the CCP panel, and all output genes were manually confirmed for sufficient coverage using Integrative Genomics Viewer (IGV). 2.3 Somatic variant profiling VarScan2 variant detection algorithm version 2.3.2 was applied to tumor-normal pairs to identify non-synonymous somatic variants from ES data. SnpEff version 4.0 with the Ensembl v68 annotation database (https://www.ensembl.org)wasusedto annotate the variant data. Due to the limited number of genes in the CCP used in the original analysis of the control LS dataset, the somatic exome data was filtered to match the sequenced regions of CCP for downstream analyses comparing two cohorts. Variants with a VarScan2-derived somatic p-value less than 0.01 were selected for somatic mutational signature analysis, which was carried out using the R package MutationalPatterns (19). The single-base substitution (SBS) somatic mutation matrix was additionally repaired for FFPE-derived artefacts using the FFPEsig python package (20). The signatures present in the tumors were evaluated by running NMF factorization rank estimation on the mutational matrices with the NMF R package to draw de novo signatures with 1000 iterations. The resulting de novo signatures were then mapped against the SBS signatures by Degasperi et al. and 18 insertion-and-deletion (ID) signatures recognized by the COSMICv3.1 database (cancer.sanger.ac.uk) (21). Tumor heterogeneity and driver mutation analyses were carried out using the R package MAFtools (18). 2.4 Loss of heterozygosity (LOH) Possible loss of heterozygosity was evaluated on germline mutation loci based on either the results ES or fragment analyses by comparing the allele or peak ratios of tumor samples to the corresponding ratios in the normal sample by applying the following formula: LOH (or allelic imbalance) ratio (R) = (A/B)T/ (A/B)N. LOH ratios 1.67 ≥R≤0.60 were considered indicative of strict LOH, and R= 0.6 –0.8 and R= 1.25 –1.67 putative LOH (22). 2.5 Microsatellite instability analysis Mononucleotide markers BAT25 and BAT26 were used to classify samples MSI or MSS. Samples were considered MSI when at least one marker showed instability. Recurrent frameshift mutations at coding microsatellites identified by the analysis of tumor tissue were confirmed in adjacent normal tissues with the following fluorescent markers: CASP5_fwd, 5’-AACTCT TTAAGCTGTGCCCA-3’;CASP5_rev,5’-TCTACCAAGATC AGGGCCTT-3’; LTN1_fwd, 5’-GAAGCTGATGTTGAGTCCGT- 3’;LTN1_rev,5’-GCTTTCAAGTATCTCATCAGCA-3’; MARCKS_fwd, 5’-CCGCCTCCTCGACTTCTT-3’, MARCKS_rev, 5’-CCGCTCAGCTTGAAAGACTT-3’;NCAM1_fwd,5’-TACTC AGCCTGGCAATTGTC-3’;NCAM1_rev,5’-ATTGTAATCT GCTGGCTGGG-3’;RNPC3_fwd,5’-GCAAAAGAGCAAGAT CGAGT-3’;RNPC3_rev,5’-ACTTGCTAGTCTGAAAACAA-3’; SLC22A9_fwd, 5’-TGCAGTCAACTCACTTCTCA-3’; SLC22A9_rev, 5’-CGTAAAGGACAGGAGGGAGA-3’; TAF1B_fwd, 5’-CTGCAGAGATATCAGGAAGTTACA-3’; TAF1B_rev, 5’-CATCATGAAGGTGAAAGATGTGA-3’; USP48_fwd, 5’-CTTTAGCAAAGCAAGAAAAGC-3’; USP48_rev, 5’-TGGAAACTCAGGAGCCTTTG-3’; RNF43_Arg117fs_fwd, 5’- TCTGGAGCCTGGATTCATCA-3’; RNF43_Arg117fs_rev, 5’- GCGAAGTGTGAGTCTACCTT-3’; RNF43_Gly659fs_fwd, 5’- CTCTCTGCCCGACACCCA-3’; RNF43_Gly659fs_rev, 5’- TTGCATCCTGGGGCCGAG-3’.Fragmentanalysiswas performed at the Institute for Molecular Medicine Finland FIMM Genomics unit supported by HiLIFE and Biocenter Finland. 2.6 Germline and somatic methylation analyses Possible presence of CIMP was evaluated by methylationspecific multiplex ligation-dependent probe amplification (MSMLPA) using SALSA MS-MLPA probemix ME042-C2 (MRC Holland, Amsterdam, the Netherlands) as previously described (23). CIMP+ status was given when three out of five genes exhibited dosage ratios above a threshold level for hypermethylation as defined by the Weisenberger panel (24). Thresholds for hypermethylation were calculated as described previously (23). The methylation status of LINE-1 was determined using custom MS-MLPA probes described by Pavicic et al. using commercially available reagents from MRC Holland (25). 2.7 Genomic rearrangements in MMR genes Blood or normal mucosa FFPE -derived DNA of the study cases were examined for large genomic rearrangements using multiplex ligation-dependent probe amplification (MLPA) according to manufacturer’s(MRC-Holland,Amsterdam,theNetherlands) instructions. SALSA MLPA P003-D1 and SALSA MLPA P072-D1 were used for MLH1/MSH2 and MSH6/MUTYH, respectively, whereas PMS2 was investigated by SALSA MLPA P008-C1. The results from fragment analysis were analyzed by Coffalyser™ (MRC-Holland, Amsterdam, the Netherlands). Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org05 2.8 Immunohistochemistry for MMR protein expression Archival FFPE tissue specimens from index cases were stained using anti-MLH1 (clone G168-15; Pharmingen), anti-MSH2 (clone FE-11; Calmiochem/Oncogene Research), and anti-MSH6 (clone 44; Transduction Laboratories) antibodies according to Thiel et al. (26). Dako Envision+ System, DAB Peroxidase was applied according to manufacturer’s instructions for visualization. 2.9 Statistical analysis Analysis of statistical significance between groups were carried out by Fisher’s Exact or two-tailed ANOVA for normally distributed data, and by Kruskal-Wallis analysis for nonparametric data. Tukey HSD or Dunn’s test was used for post hoc analyses for ANOVA and Kruskal-Wallis, respectively. All analyses were conducted on R v.4.2.3. 3 Results We sought to discriminate whether the underlying reason for the multiplicity of colorectal tumors in case LS-298 and two other similar cases (Figure 1)wasdueto1)metastaticdiseaseor nonradical removal of initial lesions; 2) mutagenic stressors, e.g. colibactin; 3) modifying germline variants resulting in ultrahypermutability; or 4) somatic mosaicism, e.g. potential constitutional mismatch repair deficiency syndrome (CMMRD) or involvement of APC that might have escaped detection. 3.1 Somatic mutation profiling of tumors All colorectal tumors from LS-298 were MSI although somatic mutation or loss of the wild type allele of MLH1 was detectable in only 7 tumors (58.3%). The tumors investigated from F1001, LS- 202, and the control LS cohort were likewise microsatellite unstable. To rule out metastatic disease, we compared somatic variants identified by our tumoral analyses (Supplementary Table S2) in all available samples. While the number of shared variants occurred at the same rate in the tumors of LS-298 as in the available controls, by ES analyses, we observed several frameshift-type variants shared across tumor samples of our index case. The rate of shared frameshift variants was not significantly different from the unrelated control LS tumors, however, which indicated that the frameshift variants likely represented mutational hotspots typical of LS. As the number of shared missense and synonymous somatic variants was low and occurred at a similar rate as the control group, the likelihood of metastatic disease or shared clonal origin was low. When considering driver genes for colorectal neoplasia recognized by the COSMIC database (cancer.sanger.ac.uk/ cosmic), most frameshift variants were shared at equal overall proportions in our sample cohorts. Although control LS adenomas acquired variants in APC at a higher rate than the tumors of LS-298 (48% (12/25) versus 26.67% (4/15), respectively), the difference was not statistically significant. Two of the three (66.7%) tumors from LS-202 also harbored somatic variants in APC (Supplementary Figure S1B). Interestingly, the adenomas and carcinomas of LS-298 frequently showed frameshift variants in RNF43 with high variant allele frequency (median VAF= 44.4; range = 15.69-62.3; Supplementary Table S2). As these variants mostly targeted the recognized mutational hotspots of the gene, G659 (G 7 repeat) and R117 (C 6 repeat), we screened the control LS datasets for the hotspot mutations as the CCP panel does not cover the RNF43 gene (27). Frameshift mutations in RNF43 hotspots occurred significantly more frequently in our study case and LS individuals with multiple tumors (p= 0.0001 by Fisher’s Exact Test; Supplementary Table S3); 73.3% (11/15) tumor samples of LS-298, and 60.0% (9/15) of F1001 tumors, versus 10% (2/15) of tumors from control LS with sufficient DNA for testing. The single tumor of LS-202 with enough DNA for testing did not carry any RNF43 hotspot mutations. Due to the high VAFs of several frameshift variants in coding microsatellite regions of genes in our initial analyses (Supplementary Table S2), we analyzed several normal mucosa samples for the possibility of MSI in non-malignant tissue. Although we failed to capture MSI using the typical BAT25/26 markers in most of the normal mucosa samples, we observed instability at selected markers (Supplementary Table S4), which could indicate “field defects”of hypermutated hotspot loci, previously reported to occur healthy mucosa of patients with sporadic CRC (28). No RNF43 hotspot variants were observed in normal tissue, suggesting later involvement in tumorigenesis despite the very high VAF in tumor tissue. Although our normal mucosa samples were evaluated by a pathologist, we cannot eliminate the possibility of tumor contamination or presence of individual cells with neoplastic potential within the sample. Typical of LS, majority of the tumors were hypermutated (with 10 somatic variants/Mb as a cut-off) and the rate of somatic variants at C>T context was elevated for adenomas and carcinomas for both LS-298 and control LS dataset (Supplementary Figure S1A; Supplementary Table S2 (15);). The adenomas of LS-298 displayed higher average tumor mutational burden (TMB) than control LS adenomas or carcinomas (p= 0.01748 and p= 0.00093, respectively, by Mann-Whitney U-test), while the carcinomas of LS- 298 did not significantly differ from control LS adenomas or carcinomas. However, when mapping the somatic C>T variants in context of CpG regions, we noticed an elevated rate of C>T variants at CpGs in the tumors of LS-298, being particularly pronounced in the adenomas (p= 0.0025 and p= 0.000031 vs control LS adenomas and carcinomas, respectively, by Wilcoxon pairwise test). When comparing TMB and the %C>T at CpG of samples with RNF43 hotspot mutations to those without, we noticed an increase in the mutational load and the percentage of variants occurring at CpG sites (Figures 2A,B). While the differences are notable, we were unable to attain statistical significance due to the small number of samples. Mutational Signature analyses refitted to the consensus signatures (COSMIC v3.1., cancer.sanger.ac.uk) showed typical Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org06 MSI-associated signatures in all groups including a very strong relative contribution of SBS1 (Supplementary Figure S2). 3.2 Analysis of sample methylation status Among the 58 tumor samples where testing was possible, CIMP was observed at a high rate: 50% (29/58) tumors were CIMP positive according to the Weisenberger criteria (24). CIMP was more frequent in carcinomas than in adenomas (p= 0.03), and particularly pronounced in the case F1001 (75%, 12/16 of samples were CIMP positive) and LS-202 (100%, 3/3 tumors were CIMP positive). Tumors of LS-298 did not differ significantly from the control LS cases (Table 1). However, the overall Dm ratios of the genes covered by the MS-MLPA kit used were higher in all tissue types of LS-298 compared to the matching tissue type of the control A B C FIGURE 2 Effect of RNF43 mutation status on molecular tumor features of LS-298 vs. control LS cohort. Tumor mutational burden (A), percentage of C to T transitions at CpG sites (B), and mutational signatures (C) are illustrated. RNF43 hotspot mutation status is indicated with a solid square. Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org07 LS cases. The Dm ratios of F1001 were comparable to those of LS- 298, whereas LS-202 did not differ significantly from tissuematching control LS group. (Figure 3). RNF43 hotspot-mutation positive samples had no significant correlation to CIMP status contrary to previous studies (29). Comparison of the genes included in the CCP panel did not reveal a difference between our study cases and controls in their somatic mutational load in genes with a primary function in epigenetic regulation (data not shown). However, prompted by the propensity of somatic variants to occur at the CpG regions, frequent CIMP positivity, and the predominance of SBS1 in our study case’s tumors when comparing the mutational data to COSMIC consensus signatures (Table 1;Supplementary Figure S2), we compared sample signatures to a specific subset of hypermutator signatures associated with MSI and spontaneous demethylation of C>T to see whether the tumor phenotypes were indicative of MBD4-associated tumorigenesis as described by Degasperi et al. (21). The subset signature analysis revealed that majority of tumors of LS-298 harbored the SBS96 signature (Figure 2C), whereas tumors from control LS cases unanimously harbored only SBS1 or SBS1 and SBS95. Interestingly, A B FIGURE 3 Relationship between CIMP status and tumor multiplicity. Colorectal tumors from LS patients with multiple tumors (LS-289, F1001, and ME16) are compared to those without (LS control group). (A) Box plots of distributions of DNA methylation (Dm) values at CIMP marker loci. (B) Pairwise Wilcoxon analysis of Dm differences for statistical significance between the sample sets. Statistical significance is indicated with bolded text. P-values were adjusted for multiple testing by Bonferroni correction. Olkinuora et al. 10.3389/fonc.2024.1378392 Frontiers in Oncology frontiersin.org08