Lynch syndrome genetics and clinical implications

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/ Lynch syndrome genetics and clinical implications © 2023 The Author(s). Published by Elsevier Inc. on behalf of the AGA Institute. Published version Peltomäki, Päivi; Nyström, Minna; Mecklin, Jukka-Pekka; Seppälä, Toni, T. Peltomäki, P., Nyström, M., Mecklin, J.-P., & Seppälä, T. (2023). Lynch syndrome genetics and clinical implications. Gastroenterology, 164(5), 783-799. https://doi.org/10.1053/j.gastro.2022.08.058 2023 Lynch Syndrome Genetics and Clinical Implications 1 Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland; 2 Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland; 3 Department of Education and Science, Nova Hospital, Central Finland Health Care District, Jyväskylä, Finland; 4 Faculty of Sports and Health Sciences, University of Jyväskylä, Jyväskylä, Finland; 5 Department of Surgery, Helsinki University Hospital, Helsinki, Finland; 6 Applied Tumor Genomics Research Programs Unit, University of Helsinki, Helsinki, Finland; and 7 Faculty of Medicine and Health Technology, Tampere University and Tays Cancer Center, Tampere University Hospital, Tampere, Finland Lynch syndrome (LS) is one of the most prevalent hereditary cancer syndromes in humans and accounts for some 3% of unselected patients with colorectal or endometrial cancer and 10%–15% of those with DNA mismatch repair–deficient tumors. Previous studies have established the genetic basis of LS predisposition, but there have been significant advances recently in the understanding of the molecular pathogenesis of LS tumors, which has important implications in clinical management. At the same time, immunotherapy has revolutionized the treatment of advanced cancers with DNA mismatch repair defects. We aim to review the recent progress in the LS field and discuss how the accumulating epidemiologic, clinical, and molecular information has contributed to a more accurate and complete picture of LS, resulting in genotypeand immunologic subtype–specific strategies for surveillance, cancer prevention, and treatment. Keywords: Lynch Syndrome; Colorectal Cancer; Endometrial Cancer; DNA Mismatch Repair; Genetic Testing; Cancer Prevention. Lynch syndrome (LS) represents an autosomal dominant predisposition to colorectal carcinoma (CRC), endometrial carcinoma (EC), and other cancers because of defective DNA mismatch repair (dMMR). The history of the syndrome dates back to 1895, when Dr Warthin started to collect information on the first LS family later, designated Family G. 1 The stringent Amsterdam criteria, 2,3 which require 3 or more family members diagnosed with an LS-associated cancer at an early age, were formulated to guide the selection of families for molecular studies. These and the less stringent Bethesda criteria 4 facilitate recognition of LS in the clinical setting. The discovery of the 4 LS-associated DNA mismatch repair (MMR) genes—MSH2,MLH1,MSH6, and PMS2—in 1993–1995 marks the beginning of the molecular era of LS. 5 Definitive diagnosis of LS requires the identification of a pathogenic or likely pathogenic constitutional variant affecting one of the MMR genes (or EPCAM), and the term LS is currently restricted to cases fulfilling this molecular definition. Prevalence dMMR often results in absent MMR protein(s) and microsatellite instability (MSI) in tumor tissue, providing valuable shortcuts to the identification of LS among consecutive patients with CRC or EC. This so-called universal tumor screening followed by constitutional testing has led to an estimate of some 3% of CRCs 6 and a roughly similar proportion of ECs 7 being attributable to LS. Recent studies applying hereditary cancer panel testing to patients with unselected CRC 8 or EC 9 without prior tumor-based screening arrived at estimates of 3% and 6% of LS among all CRCs and ECs, respectively. Using nuclear families of nearly 6000 incident CRC cases recruited irrespective of family history from population-based cancer registries, Win et al 10 estimated that 1 in 279 individuals (0.359%) could be carriers of pathogenic variants of any MMR gene in the US, Canadian, and Australian populations. In a gene-specific analysis, PMS2 and MSH6 were associated with the highest population prevalences, 1 in 714 (0.140%) and 1 in 758 (0.132%), respectively, compared with MLH1 (1 in 1946 [0.051%]) and MSH2 (1 in 2841 [0.035%]). 10 The finding reflects lower penetrance of PMS2 and MSH6 compared to MLH1 and MSH2. Accordingly, enrichment of founder Päivi Peltomäki 1 Minna Nyström 2 Jukka-Pekka Mecklin 3,4 Toni T. Seppälä 5,6,7 Abbreviations used in this paper: CMMRD, constitutional mismatch repair deficiency; CRC, colorectal carcinoma; dMMR, defective DNA mismatch repair; EC, endometrial cancer; FCCTX, familial colorectal cancer, type X; h, human; LLS, Lynch-like syndrome; LS, Lynch syndrome; MMR, DNA mismatch repair; MSI, microsatellite instability; MSI-H, microsatellite instability of high degree; MSS, microsatellite stable; PLSD, Prospective Lynch Syndrome Database; pMMR, mismatch repair proficiency/proficient. Most current article © 2023 The Author(s). Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 0016-5085 https://doi.org/10.1053/j.gastro.2022.08.058 Gastroenterology 2023;164:783–799 variants in PMS2 and MSH6 resulted in a high LS carrier frequency (1 in 226 [0.442%]) in the Icelandic population. 11 Population frequencies of even up to 1 in 100 have been suggested. 12 These figures make LS the most common form of hereditary CRC and probably the most prevalent singlegene cause of cancer predisposition overall. Clinical Phenotypes Phenotypes associated with constitutional variants of MMR genes depend on heterozygosity vs homozygosity for the predisposing defects (Table 1). Heterozygous variants of MLH1,MSH2,MSH6,andPMS2 underlie LS predisposition. 13,14 Rarely, genomic deletions of the 30end of EPCAM may cause LS predisposition through epigenetic inactivation of a structurally intact MSH2. 15 Muir–Torre syndrome may accompany LS. 16 LS also covers part of Turcot syndrome 17 asaphenotypicvariant(Table 1). In rare instances (w200 cases reported), pathogenic constitutional variants in any 1 of the 4 LS-associated MMR genes (or 30untranslated region deletions of EPCAM) may occur at a homozygous or compound heterozygous state. This results in a distinct syndrome called constitutional mismatch repair deficiency (CMMRD). 18–20 The predominant genes underlying CMMRD are PMS2 and MSH6, possibly reflecting higher population prevalence and lower penetrance (better tolerability) of their variants compared to MLH1 and MSH2. 18 Lynch Syndrome Genes and Functions of DNA Mismatch Repair Proteins DNA Mismatch Repair Mechanism The primary responsibility of the MMR system is to correct errors that arise during DNA replication and recombination—a function critical for cancer avoidance. 21 In humans, 5 MutS homologues (MSH2, MSH6, MSH3, MSH4, and MSH5) and 4 MutL homologues (MLH1, PMS2, PMS1, and MLH3) exist, 6 of which function in MMR (Figure 1). The main mismatch-binding factor in humans (h) is hMuSa, a heterodimer of MSH2 and MSH6. Another mismatch-recognition complex is hMutSbformed by MSH2 and MSH3. MSH6 is required for the correction of single base mispairs and 1–2 nucleotide insertion–deletion loops, and both MSH3 and MSH6 may participate in the correction of insertion–deletion loops larger than 2 nucleotides. The MSH proteins are in the resting state in the adenosine diphosphate–bound form. Adenosine triphosphate–bound hMutSaor hMutSbundergoes a conformational change into a clamp that moves along the DNA to signal to the additional components of the MMR machinery. 22 MLH1 and PMS2 (the latter is a homologue of yeast PMS1) form hMutLa, which coordinates the interplay between the mismatch-recognition complex and other proteins necessary for MMR (Figure 1). Although hMutLais the main hMutL heterodimer, MLH1 can also complex with MLH3 (hMutLg) and PMS1 (hMutLb). PMS2 is primarily required for the correction of single base mispairs, whereas MLH3 may contribute to the repair of insertion–deletion loops and, additionally, the correction of mismatches if PMS2 is absent. 23 The hMutLbcomplex does not seem to participate in MMR. Unlike Escherichia coli, whose MMR is methyl directed (a transient lack of methylation identifies the nascent strand), DNA replication-associated daughter strand nicks that direct asymmetric loading of proliferating cell nuclear antigen likely mediate strand discrimination in eukaryotes. 24 Upon encountering a strand discontinuity, the hMutS–hMutL complex recruits excision machinery, and Table 1.LS and Related Phenotypes Syndrome MIM number a Susceptibility genes Mode of inheritance Clinical features LS b 609310 MLH1 (41%) AD Colonic and extracolonic cancers of a defined spectrum 3 and occurring earlier than in the average population (at w40–60 years of age) 120435 MSH2 (36%) 614350 MSH6 (18%) 614337 PMS2 (5%) 185535 EPCAM (rare) Muir–Torre syndrome 158320 Mostly MSH2 and MLH1 AD Multiple sebaceous gland adenomas co-occurring with visceral malignancies, such as colorectal carcinoma Turcot syndrome —See LS and CMMRD AD or AR c Primary brain tumors co-occurring with multiple colorectal adenomas CMMRD 276300 Mostly PMS2 and MSH6 AR Childhood cancers, mainly hematologic malignancies and/or brain tumors, signs of neurofibromatosis type 1 (café-au-lait spots), combined with early-onset colorectal cancers and polyposis AD, autosomal dominant; AR, autosomal recessive; MIM, Mendelian Inheritance in Man. a Phenotype number in MIM. 14 b Shares of MLH1,MSH2,MSH6, and PMS2 variants are based on a total of 2105 variants of classes 3–5 reported in Thompson et al. 13 c Turcot syndrome may arise as a variant of LS (dominant) or CMMRD (recessive). It may also arise from constitutional defects of APC (MIM no. 175100), in which case the transmission pattern is dominant. 784 Peltomäki et al Gastroenterology Vol. 164, Iss. 5 degradation of the error-containing fragment and synthesis of a new strand follow. Microsatellite Instability as a Hallmark of Lynch Syndrome Defective MMR results in length variation of short tandem nucleotide repeats, microsatellites (MSI). MSI is a hallmark of LS and up to 30% of sporadic cancers of various organs. 25 Different substrate preferences of individual MMR proteins may explain different MSI phenotypes resulting from MMR gene defects. 26 MSH2,MLH1,orPMS2 inactivation is associated with high-degree MSI (MSI-H) with mononucleotide, dinucleotide, and other short tandem repeats affected. MSH6 inactivation mainly results in mononucleotide repeat instability. MSH3 (hMutSb) dysfunction may lead to a distinct form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats, also seen in tumors from carriers of biallelic MSH3 constitutional defects. 27 Whether MLH3 inactivation results in a specific type of MSI or not is unclear. Tumors from biallelic MLH3 variant carriers showed no instability at mono-, di-, tri-, or tetranucleotide repeats. 28 Other Functions of DNA Mismatch Repair Proteins If correction of replication errors is not possible, MMR proteins signal DNA damage to cell cycle arrest or apoptosis. 29 The MMR system also blocks recombination between related but nonidentical (homeologous) sequences, acting as a barrier to chromosomal rearrangements. 30 Under certain circumstances, MMR proteins can promote sequence alterations. Inferred from yeast studies, the MLH1–MLH3 complex (hMutLg) and the MSH4–MSH5 complex (hMutSg) facilitate meiotic crossover between homologous chromosomes. 31 PMS1 (yeast MLH2), too, functions in meiosis. The MLH1–PMS1 complex (hMutLb) limits the length of the gene conversion tract in meiotic recombination. 31 When the MMR system recognizes mismatches outside replication, strand discrimination between the old and new DNA is lost, and the MMR proteins can act Figure 1. Function of the human MMR system in the correction of single-base mismatches (G to T, left) and insertion–deletion loops (TG insertion, right) that have arisen as replication errors in the newly synthesized strand (red). See text for details. April 2023 Lynch Syndrome Genetics and Clinical Implications 785 mutagenically and contribute to trinucleotide repeat expansion. 32,33 Counterintuitively, trinucleotide repeat expansion requires MMR proficiency (pMMR). Constitutional Defects Underlying Lynch Syndrome Predisposition Genetic Alterations MLH1 and MSH2 are the most important predisposing genes for LS (Table 1), which is compatible with the fact that their products are obligatory components in all types of MMR protein heterodimers, whereas MSH6 is redundant with MSH3 and PMS2 is redundant with MLH3 (Figure 1). There is no convincing evidence that heterozygous variants of MSH3 or MLH3 would underlie LS predisposition. Interestingly, homozygous variants of these genes cause susceptibility to adenomatous polyposis with possible features of CMMRD, highlighting the dosage dependency of phenotypes associated with MMR gene defects. 27,28 There are no reports of LS-associated constitutional defects of PMS1, MSH4,orMSH5, which agrees with the primary role of these genes in meiotic recombination rather than MMR. A functional MMR system needs to produce MMR proteins, transport them to the nucleus, form appropriate protein complexes at the site of the DNA mismatch, and perform the actual MMR. LS-predisposing MMR gene alterations are pathogenic through the loss of any one of these functions (often several of them), which typically results from nonsense or frameshift changes. The share of missense alterations that lead to single amino acid substitutions is also significant (30%–60%) for all 4 LS-associated MMR genes. 34 Nonsense and frameshift alterations, canonical splice site changes, and deletions of a single exon or multiple exons generally disrupt gene function and are therefore pathogenic. Although multiple in silico tools exist to predict the pathogenicity of missense alterations, 35 verification by laboratory assays, 36,37 and including tests for aberrant splicing, 38 is necessary. Characteristics of a sequence variant combined with clinical and family features have led to a 5-tiered classification 13,39 to interpret sequence variants of disease-associated genes for clinical purposes. Variants belonging to class 1 (benign) or 2 (likely benign) are considered harmless and require no special attention. Class 3 is for variants of uncertain significance, and clinical management is case by case. Class 4 (likely pathogenic) or 5 (pathogenic) indicates that the variant is deleterious, warranting surveillance according to high-risk guidelines and enabling predictive testing of at-risk relatives. LS individuals typically inherit their predisposing variants from one of their parents, and de novo alterations are rare (2.3%). 40 Ancestral founding changes predominate in some populations and account for more than half of all LS families. 41 The extent of haplotype conservation in carriers of founding changes provides a tool to estimate the age of such alterations. Thus, a 3.5-kb genomic deletion of MLH1 exon 16 unique to Finnish LS families started to spread 400–1075 years ago. 42 A 20-kb deletion in MSH2 exons 1–6 characteristic of North American LS families may be 500 years old. 43 These 2 founder changes additionally illustrate richness of the MMR gene regions in Aluand other repeats. Recombination events mediated by such repeats explain why some 10%–20% of all LS-associated changes are large genomic rearrangements. 44 All EPCAM alterations responsible for LS predisposition consist of large genomic deletions of the 30end of the gene leading to the removal of the stop codon, and Alu-mediated recombination plays a major role in their origin. 45 Constitutional Epimutations Constitutional hypermethylation at the promoter of one allele of MLH1 or MSH2 can lead to silencing of expression from that allele in all main somatic tissues, causing susceptibility to colorectal and extracolonic cancers typical of LS. Epimutation can be primary (no apparent cause for hypermethylation identifiable) or secondary (induced by genetic alteration). Primary and secondary epimutations of MLH1 may account for 1%–10% of unexplained Lynchsuspected families with silenced MLH1 expression in tumors. 46–49 Constitutional MLH1 epimutations are rare among unselected CRC patients. 50 Secondary epimutation caused by deletions of the 30end of the upstream EPCAM gene is the only known type of constitutional epimutation for MSH2. After removal of the stop codon, transcription of EPCAM reads into the adjacent, structurally normal MSH2 gene, inducing its promoter methylation. 15 Secondary epimutations of MSH2 may be responsible for a variable percentage of unexplained Lynch-suspected families (0%–40% depending on possible founder effects). 15,47 There are no reports of LS-associated constitutional epimutations for MMR genes other than MLH1 and MSH2. 51 Epigenetic changes are subject to erasure when they pass through the germline. Therefore, primary constitutional epimutations segregate in a nonmendelian fashion and are seldom associated with any remarkable family history of cancer. Families of primary epimutation carriers may exhibit mosaic epigenetic inheritance, reversion of the methylated allele to the normal active state, or apparent heritability. 52–55 Variability in transmission patterns requires appropriate consideration in genetic counseling. In contrast, secondary epimutations of MLH1 or MSH2 give rise to classical LS families and cosegregate with their cis-acting genetic changes as dominant mendelian traits. 15,56 Nevertheless, the basic mechanism of transmission may differ from that of a genetic alteration. Hitchins et al 57 showed that MLH1 c.-27C>A–associated secondary epimutation underwent erasure in spermatozoa, followed by reestablishment in somatic cells of the next generation. No Constitutional Defect Found—Phenotypic Confounders Neither fulfilment of the clinical (Amsterdam) criteria nor dMMR in tumor tissue is specific for LS. Table 2 summarizes some key characteristics of LS and the various entities that mimic it. He MMR status of tumor tissues divides families meeting the Amsterdam criteria into 2 786 Peltomäki et al Gastroenterology Vol. 164, Iss. 5 subcategories: dMMR (LS) and pMMR (familial colorectal cancer type X [FCCTX]). 58 Recent genome-wide studies have identified several putative candidate genes for FCCTX, 59 but each accounts for only a small proportion of families, and the genetic basis of FCCTX remains largely unknown, as “type X”implies. Acquired MLH1 promoter methylation is by far the most frequent cause of dMMR in CRC (10%–20 %), whereas LS accounts for 1%–3% of all CRCs (Table 2). MMR-defective cancers that neither MLH1 methylation nor pathogenic constitutional variants of MMR genes explain represent Lynch-like syndrome (LLS). The share of all CRCs that LLS is responsible for is comparable to LS. 60,61 Double somatic events consisting of pathogenic sequence variants of MLH1,MSH2,MSH6,orPMS2 or loss of heterozygosity are detectable in more than half of LLS tumors. 61–63 It is reasonable to treat the double somatic subgroup as sporadic CRC for their heritability. However, a proportion of LLS cases displays a family history for LS-associated cancers, the basis of which is unknown. 60,64 Although certain clinicopathologic differences between LS and its phenotypic confounders are evident (Table 2), a reliable distinction between the individual conditions is achievable only by molecular methods. Tumorigenesis in Lynch Syndrome Two-Hit Paradigm and Haploinsufficiency The LS genotype is heritable in an autosomal dominant pattern. A carrier of a pathogenic constitutional variant is at increased risk of cancer, but the penetrance of the phenotype is not 100%. The loss of ability to repair DNA mismatches requires, in accordance with Knudson’s 2-hit hypothesis, 65 that a somatic second hit disable the functional allele of the MMR gene. Even in the event of the predisposing MMR gene retaining its wild-type allele in somatic cells, the total amount of gene product may not be sufficient for normal function (haploinsufficiency). Heterozygous MLH1 and MSH2 transgenic mice display decreased expression of MMR proteins 66 and increased levels of genomic frameshift deletions in the colon. 67 Reduced dose of effective MMR molecules may be tissue specific and explain some of the phenotypic variation (eg, age at disease onset) characteristic of LS. 66 Moreover, different functions may require different amounts of MMR protein. For example, DNA damage signaling requires a higher dosage of MLH1 than DNA MMR. 68 As another example, decreased messenger RNA expression of Mlh1 and other chromosomal segregation genes in normal colonic mucosa of Mlh1 þ/– and Mlh1 þ/þ mice is associated with predisposition to pMMR, chromosomally unstable CRC. 69 Developmental Pathways and Dependence on Somatic Alterations Normal bowel mucosa 70 and normal endometrium 71,72 of pathogenic MMR variant carriers contain dMMR niches. The role of these dMMR crypt foci has been studied more in the colorectum, but it remains unclear if they may give rise to neoplasia or not. Whole-genome sequencing analysis by Lee et al 73 showed that the vast majority of histologically normal epithelial crypts from LS individuals are genomically Table 2.Differential Diagnosis of LS Clinicopathologic characteristic LS FCCTX LLS MLH1 Methylated Share of all CRCs, %1–3w1? 2–310–20 Average age at cancer onset, y 45 50–60 60 75 Tumor spectrum Colonic and extracolonic cancers Mainly site-specific CRC CRC a CRC a Preferential location of CRC Proximal Distal Variable Proximal Transmission pattern Autosomal dominant Autosomal dominant None (some are familial) b None (sporadic) Predisposing genes MLH1,MSH2,MSH6, PMS2,EPCAM Mostly unknown, candidate genes exist c None? None Tumor characteristics MMR deficient and hypermutant MMR proficient and mostly nonhypermutant MMR deficient and hypermutant MMR deficient and hypermutant BRAF V600E negative BRAF V600E unknown BRAF V600E negative BRAF V600E positive (mostly) CIMP may be present Global hypomethylation present CIMP often present CIMP present CIMP, CpG island methylator phenotype. a LLS and MLH1-methylated subgroups also exist among ECs and several other cancers. b Family history of cancer may occur; the subgroup with double somatic MMR gene variants is sporadic. c GALNT12,RPS20,BRF1,FAN1,FAF1,SEMA4A, and other candidate genes have been proposed. 59 April 2023 Lynch Syndrome Genetics and Clinical Implications 787 stable; however, a dMMR crypt with an elevated mutation burden and dMMR-associated signature was identified that could represent a very early stage of LS colorectal tumorigenesis. Together with a well-established ineffectiveness of even the most meticulous colonoscopy surveillance to prevent colorectal cancers 74,75 rather than improve survival, 76 dMMR crypts have raised several hypotheses for unsuccessful cancer prevention by endoscopic removal of preceding adenomas. 77 The proposed mechanisms for the unsuccessful prevention despite successful colonoscopies include an evolutionary model depicted in Figure 2. Some of the cancers arising from dMMR crypts may develop as flat lesions not detectable or removable endoscopically early enough as opposed to the traditional adenoma–carcinoma sequence with a distinct temporal order of somatic hallmark variants. 78 Lesions escaping colonoscopy surveillance may more often be associated with MLH1 pathogenic variants, because adenomas are clearly more frequently detectable in MSH2 carriers despite the similarly high CRC incidence. 79 Somatic APC variants are more common in MSH2 than MLH1deficient cancers, whereas CTNNB1 variants predominate in MLH1-deficient tumors. 79 Moreover, developmental pathways may differ between screen-detected and non– screen-detected CRCs. This is plausible because incident cancers detected in regular colonoscopy surveillance rarely show KRAS codon 12 or 13 changes, and sequence alterations of APC have developed downstream of MMR deficiency based on mutational signatures. 80 An initial hypothesis postulated that CTNNB1 variants are involved in nonpolypous carcinogenesis. 81 A study comparing PMS2and MLH1-deficient CRCs detected no somatic CTNNB1 variants (0/20 [0%]) in PMS2-associated CRCs, whereas MLH1-associated CRCs carried a significant number of CTNNB1 variants (14/24 [58%]). Moreover, KRAS alterations appeared to precede PMS2 deficiency in adenomas and/or CRCs from PMS2 variant carriers. 82 This seems to fit well with the clinical and epidemiologic observation that no PMS2-associated early-onset CRCs are encountered during colonoscopy surveillance 83 and suggests that the dMMR crypt foci pathway does not play a role in PMS2-defective carcinogenesis. 84 Neoantigens and Immunogenicity LS-associated cancers are very immunogenic and labeled as “hot”tumors that the immunomodulative therapeutics cure more frequently than tumors with lower immune cell density. 85 Abundance of effector and memory T cells in the tumor microenvironment both in the tumor core and in the invasive front presumably results from the hypermutated genomic profile. MSI-H tumors usually present with sequence alteration burden above the indicative threshold of 10 changes per megabase in sequencing. There is more to it, however. Although it is not entirely clear if acetylic salicylate modulates the immune environment on normal bowel mucosa, preventively administered acetylic salicylate 600 mg per day for 2–4 years reduced the Figure 2. Alternative pathways to CRC in LS. 78,79 LS CRC may develop through pMMR adenoma with secondary MMR inactivation (pathway 1) or from dMMR crypts with or without adenoma formation (pathways 2 and 3, respectively). Pathway 2 involves formation of a polypoid lesion after APC inactivation and is associated with MSH2 pathogenic germline variants. CTNNB1 activation triggers an “immediate invasive”(pathway 3), which is a feature of CRCs from MLH1 variant carriers. 788 Peltomäki et al Gastroenterology Vol. 164, Iss. 5 incidence of CRC almost by half in more than 10-year follow-up 86 with a number needed to treat of 24. This double-blind randomized clinical trial showed a delayed onset of prevention, suggesting a slow onset with a lengthy legacy effect. Furthermore, either 220 mg or 440 mg of naproxen per day for 6 months induced significant molecular changes in patient-derived and mouse models of normal bowel mucosa of pathogenic MMR variant carriers. Naproxen activated different resident immune cells, reduced prostaglandin E2 levels, and promoted downregulation of stem cell markers and up-regulation of epithelial differentiation markers. 87 Finally, LS individuals with and without cancer have different immune profiles in their normal colorectal mucosa. Pathogenic MMR variant carriers without CRC showed elevated CD3-, FOXP3-, and CD8-positive T-cell densities compared with non-LS control individuals and LS patients with CRC. Moreover, the relative immune cell density on normal bowel seems to reduce when coming closer to the diagnosis of cancer. 88 The successes of nonsteroidal anti-inflammatory drug–based chemoprevention and immune therapy by checkpoint inhibitors indicate that the epithelial immune microenvironment is a modifiable risk factor. It may even serve as a possible risk stratification biomarker for intensified surveillance and risk reduction. Because the microsatellites often develop on the same genomic positions in microsatellite unstable cancers regardless of which tissue type the tumor arises from, the subsequent frameshifts also take place similarly, causing truncating proteins as neopeptides to form across different tumor types. This results in immunoediting by counterselection of cell clones with the most immunogenic frameshift peptides and depends on the HLA haplotype. This dependence is lost in tumors that have acquired a somatic b 2 microglobulin alteration that reduces counterselection. 89 The shared frameshift alterations in MSI tumors open an avenue for successful immunotherapy and immune prevention by using these peptides as neoantigens for adaptive immunity (see Seth et al 90 and Cerretelli et al 91 for overviews and the “Vaccine-Based Immunotherapy and Immunoprevention”section). Phenotypic Correlations of Molecular Alterations Cancer Risks Associated With Constitutional Defects of Individual DNA Mismatch Repair Genes Large observational data recorded in the Prospective Lynch Syndrome Database (PLSD) have consistently shown that the cancer risks associated with each MMR gene differ from one another. 83,92,93 Pathogenic variants in MLH1 and MSH2 produce a lifetime risk of any cancer of approximately 80% despite surveillance efforts, with the median age of onset being 48–54 years with little difference between sexes. However, lifetime cancer risk in MSH6 pathogenic variant carriers is 29% in males and 55% in females at a median age of onset of 56–57 years, with a substantial sex-limited trait because of the high risk of EC. Early-onset cancers do not generally take place in PMS2 pathogenic variant carriers, but there is prospective observational evidence of CRC and EC at a median age of 66–70 and 61 years, respectively. PMS2 findings from the PLSD comply with observations by Senter et al 94 of unremarkable family histories of carriers of heterozygous variants ascertained through isolated loss of PMS2 in tumor tissue. It is unknown whether and to what extent features of the MMR mechanism, type and location of the predisposing variant, or somatic alterations directed by the constitutional defects may explain the associated phenotypic differences between the 4 main LS predisposition genes. Tissue-specific reduction in the dose of MMR protein may explain some of the variation between the functional protein deficiencies by gene (see the “Tumorigenesis in Lynch Syndrome”section). Observational data comparing truncating pathogenic MLH1 and MSH2 variant carriers to nontruncating variants has not demonstrated differences in lifetime penetrance of cancer. 95 Prognostic Correlations The major LS-associated cancer types are associated with excellent survival: 10-year crude survival was 88% for cancer of the colon, 70% for rectum, 89% for endometrium, and 84% for ovary according to the PLSD data. 83 Early detection due to increased awareness and regular surveillance, combined with inherent biological properties of LS tumors, likely contribute to the good prognosis. In CRC, MSI is a generally favorable prognostic sign. 96 LS-associated CRCs are immunologically active because of their inherent “mutator phenotype.” 97 Metastatic MMR-deficient cancers from LS and other patients overexpress immune checkpoint ligands, which makes them responsive to immune checkpoint inhibitors. 98 The favorable prognosis of LS-associated ovarian carcinomas compared to those from the general population is especially striking. Underrepresentation of serous cancers in LS does not alone provide a satisfactory explanation, 99 and unique molecular 100 and immunologic 101 properties of LS ovarian tumors may serve as additional prognostic contributors. Lynch Syndrome Tumor Spectrum The Amsterdam II criteria 3 acknowledge cancers of the colon and rectum, endometrium, small bowel, ureter, and renal pelvis as LS-associated cancers because these are significantly more frequent in LS compared to the average population. Later studies have consistently reported significantly increased risks for additional cancers, including cancers of the stomach, ovaries, pancreas, and several other organs, in LS carriers vs the general population or noncarriers. 93,102–104 Urothelial and prostate cancers are especially associated with the MSH2 genotype. 83 MSI and/or extinct MMR protein expression in tumor tissue provides an additional tool to evaluate if the predisposing defect contributes to tumor development. Among cancers from pathogenic MMR gene (mainly MLH1) variant April 2023 Lynch Syndrome Genetics and Clinical Implications 789 carriers from a nationwide registry, 34 immunohistochemical analysis regularly showed absent MMR protein(s), whereas MSI-H in the same tumor types varied from 80%–100% (stomach, ovary, colon, and ureter) to approximately 50% (bladder, endometrium, and kidney) and even less (35% for breast and 0% for brain tumors). Different growth patterns (clonal heterogeneity) may offer one possible explanation for the varying frequencies of MSI among different tumor types from LS individuals. Latham et al 105 investigated more than 15,000 tumors (over 50 cancer types) for MSI and analyzed matched constitutional DNA for alterations of MMR genes and EPCAM regardless of MSI status. Pathogenic constitutional variants (ie, LS) were identified in 16.3%, 1.9%, and 0.3% of patients with MSI-H, MSI-indeterminate, and microsatellite-stable (MSS) tumors, respectively. Two observations relevant to the LS tumor spectrum stand out. First, among LS patients with MSI-H (plus indeterminate) tumors, half had tumors other than CRC or EC, including tumor types not previously or regularly connected to LS, such as mesothelioma, melanoma, soft tissue sarcoma, and prostate cancer. Second, 36% of LS patients had MSS tumors, and these were predominantly non-CRC/ECs. Reduced penetrance of MMR gene variants (MSH6) or low tumor purity might explain MSS. 105 MSS tumors could also arise through non-MMR functions of MMR proteins (see the “Lynch Syndrome Genes and Functions of DNA Mismatch Repair Proteins”section) or be truly sporadic. Breast cancer illustrates a cancer type whose relationship to LS is controversial, but at least a subset seems etiologically linked to the predisposing MMR defects based on dMMR tumor profiles 106–108 and may respond to anti-PD1/PD-L1 immunotherapy. 108 Detection of Lynch Syndrome Predisposition Population Screening Multiple guidelines recommend universal tumor screening, that is, testing any new CRC 109,110 or EC 110,111 for deficient MMR protein expression and/or MSI, to select potential cases of LS for constitutional testing. Universal screening is cost-efficient for LS identification alone, 112,113 with further major implications for overall cancer management after the checkpoint inhibition therapy became available. Although immunohistochemical analysis for MMR protein expression and MSI testing have similar sensitivities and specificities and a generally good concordance, 6,112,114,115 neither method is 100% accurate. Some LS-predisposing missense (eg, MLH1 P28L 116 and MSH2 T33P 117 ) and even truncating (MSH6 V131fs*2 114 ) variants may give rise to stable but nonfunctional protein. Conversely, immunohistochemical analysis may show abnormal protein expression in some LS tumors that remain MSS because of, for example clonal heterogeneity or low tumor purity. Compared to MSI testing, immunohistochemical analysis has an additional advantage of identifying the specific MMR protein with aberrant expression, thus pinpointing the gene likely altered constitutionally. Dependence of the secondary protein partner (MSH6 or PMS2) on the primary protein partner (MSH2 or MLH1) for stability results in characteristic immunohistochemical patterns, where negative nuclear staining results for both MSH2 and MSH6 suggest an MSH2 alteration, whereas the lack MLH1 and PMS2 proteins indicates an MLH1 change. The absence of MSH6 alone points to defective MSH6 and isolated loss of PMS2 to an altered PMS2 gene. However, exceptions to these basic rules exist (for example, patients with isolated loss of MSH6 may show a predisposing variant in MSH2 62,118 ), suggesting that constitutional testing should not be restricted to only the gene predicted from the immunohistochemical pattern but should cover other relevant alternatives as well. In patients with CRC and absent MLH1 and PMS2, tumor testing for MLH1 promoter methylation and/or BRAF V600E is useful to rule out likely sporadic cases before proceeding to constitutional testing. Detection of a somatic BRAF V600E change is closely associated with MLH1 hypermethylation, which accounts for approximately 70% of consecutive MMR-deficient CRCs. 60,61 The presence of BRAF V600E and methylation of MLH1 promoter (affecting region C specifically 119 ) in colon cancer strongly argue against LS. 91,120 This prediction is not without exceptions. In a literature review by Parsons et al, 120 4 of 550 CRCs (1.4%) from known MMR gene variant carriers and in another large cohort, 121 15 of 969 (1.6%) of LS CRCs showed BRAF V600E. Moreover, somatic MLH1 promoter methylation may, although rarely, accompany a pathogenic MLH1 constitutional variant as a “second hit.” 122 In EC, oncogenic BRAF variants are rare, 123 and it is not possible to use BRAF V600E as a proxy for somatic MLH1 hypermethylation in that (or other noncolorectal) context. Next-generation tumor sequencing may provide an alternative approach to the universal screening method. 124 Tumor sequencing alone had better sensitivity than MMR protein expression analysis plus BRAF and MSI plus BRAF and equal specificity to MMR protein expression analysis plus BRAF and MSI plus BRAF. Constitutional sequencing of the respective MMR gene(s) needs to confirm the results from next-generation tumor sequencing. In addition to heritable changes of MMR genes, tumor sequencing followed by constitutional analysis could detect double somatic MMR gene alterations responsible for LLS (Table 2). Finally, tumor sequencing could reveal actionable therapeutic targets, such as KRAS,NRAS,orBRAF mutations, that could inform chemotherapy choice. 124 In patients with suspected LS but no tumor sample available for analysis, clinical prediction models such as PREMM (PREdiction Model for gene Mutations) 125 may be useful when assessing the need for constitutional testing. 109,110 Recently, it has become possible to test MMR in nonneoplastic tissue and, thus, diagnose LS regardless of clinical affection status or family history. Early small-pool polymerase chain reaction analyses were able to detect MSI in peripheral blood leukocytes from LS patients. However, the labor intensiveness of the method and ambiguity of the results at low and high extremes (false negatives in LS patients and false positives in older healthy individuals) prevent applying the method to clinical diagnostics. 126 790 Peltomäki et al Gastroenterology Vol. 164, Iss. 5 105. Latham A, Srinivasan P, Kemel Y, et al. Microsatellite instability is associated with the presence of Lynch syndrome pan-cancer. J Clin Oncol 2018;37:286–295. 106. Lotsari JE, Gylling A, Abdel-Rahman WM, et al. Breast carcinoma and Lynch syndrome—molecular analysis of tumors arising in mutation carriers, non-carriers, and sporadic cases. Breast Cancer Res 2012;14(3):R90. 107. Porkka N, Olkinuora A, Kuopio T, et al. Does breast carcinoma belong to the Lynch syndrome tumor spectrum?—Somatic mutational profiles vs. ovarian and colorectal carcinomas. Oncotarget 2020;11:1244–1256. 108. Schwartz CJ, da Silva EM, Marra A, et al. Morphologic and genomic characteristics of breast cancers occurring in individuals with Lynch syndrome. Clin Cancer Res 2022;28:404–413. 109. Rubenstein JH, Enns R, Heidelbaugh J, et al. American Gastroenterological Association institute guideline on the diagnosis and management of Lynch syndrome. Gastroenterology 2015;149:777–782. 110. Stjepanovic N, Moreira L, Carneiro F, et al. Hereditary gastrointestinal cancers: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2019;30:1558–1571. 111. Crosbie EJ, Ryan NAJ, Arends MJ, et al. The Manchester International Consensus Group recommendations for the management of gynecological cancers in Lynch syndrome. Genet Med 2019;21:2390–2400. 112. Snowsill T, Coelho H, Huxley N, et al. Molecular testing for Lynch syndrome in people with colorectal cancer: systematic reviews and economic evaluation. Health Technol Assess 2017;21:1–238. 113. Snowsill TM, Ryan NAJ, Crosbie E. Cost-effectiveness of the Manchester approach to identifying Lynch syndrome in women with endometrial cancer. J Clin Med 2020;9(6):1664. 114. Goodfellow PJ, Billingsley CC, Lankes HA, et al. Combined microsatellite instability, MLH1 methylation analysis, and immunohistochemistry for Lynch syndrome screening in endometrial cancers from GOG210: an NRG Oncology and Gynecologic Oncology Group study. J Clin Oncol 2015;33:4301–4308. 115. Coelho H, Jones-Hughes T, Snowsill T, et al. A systematic review of test accuracy studies evaluating molecular micro-satellite instability testing for the detection of individuals with Lynch syndrome. BMC Cancer 2017;17:836. 116. Raevaara TE, Korhonen MK, Lohi H, et al. Functional significance and clinical phenotype of nontruncating mismatch repair variants of MLH1. Gastroenterology 2005;129:537–549. 117. Ollila S, Sarantaus L, Kariola R, et al. Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein. Gastroenterology 2006;131:1408–1417. 118. Olkinuora A, Gylling A, Almusa H, et al. Molecular basis of mismatch repair protein deficiency in tumors from Lynch suspected cases with negative germline test results. Cancers 2020;12(7):1853. 119. Deng G, Chen A, Hong J, et al. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res 1999; 59:2029–2033. 120. Parsons MT, Buchanan DD, Thompson B, et al. Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification. J Med Genet 2012;49:151–157. 121. Bläker H, Haupt S, Morak M, et al. Age-dependent performance of BRAF mutation testing in Lynch syndrome diagnostics. Int J Cancer 2020;147:2801–2810. 122. Rahner N, Friedrichs N, Steinke V, et al. Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome. J Pathol 2008; 214:10–16. 123. Metcalf AM, Spurdle AB. Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review. Fam Cancer 2014;13:1–12. 124. Hampel H, Pearlman R, Beightol M, et al. Assessment of tumor sequencing as a replacement for Lynch syndrome screening and current molecular tests for patients with colorectal cancer. JAMA Oncol 2018;4:806–813. 125. Kastrinos F, Uno H, Ukaegbu C, et al. Development and validation of the PREMM5 model for comprehensive risk assessment of Lynch syndrome. J Clin Oncol 2017; 35:2165–2172. 126. Coolbaugh-Murphy MI, Xu J-P, Ramagli LS, et al. Microsatellite instability in the peripheral blood leukocytes of HNPCC patients. Hum Mutat 2010;31:317–324. 127. Kansikas M, Kasela M, Kantelinen J, Nyström M. Assessing how reduced expression levels of the mismatch repair genes MLH1, MSH2, and MSH6 affect repair efficiency. Hum Mutat 2014;35:1123–1127. 128. Kasela M, Nyström M, Kansikas M. PMS2 expression decrease causes severe problems in mismatch repair. Hum Mutat 2019;40:904–907. 129. Kansikas M, Vähätalo L, Kantelinen J, et al. Tumor-independent detection of inherited mismatch repair deficiency for the diagnosis of Lynch syndrome with high specificity and sensitivity. Cancer Res Commun 2023; 3:361–370. 130. Karam R, Conner B, LaDuca H, et al. Assessment of diagnostic outcomes of RNA genetic testing for hereditary cancer. JAMA Network Open 2019;2(10): e1913900. 131. Landrith T, Li B, Cass AA, et al. Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes. NPJ Precis Oncol 2020;4:4. 132. Conner BR, Hernandez F, Souders B, et al. RNA analysis identifies pathogenic duplications in MSH2 in patients with Lynch syndrome. Gastroenterology 2019; 156:1924–1925. 133. Yurgelun MB, Allen B, Kaldate RR, et al. Identification of a variety of mutations in cancer predisposition genes in patients with suspected Lynch syndrome. Gastroenterology 2015;149:604–613. 134. Levine MD, Pearlman R, Hampel H, et al. Up-front multigene panel testing for cancer susceptibility in patients with newly diagnosed endometrial cancer: a multicenter April 2023 Lynch Syndrome Genetics and Clinical Implications 797 prospective study. JCO Precis Oncol 2021; 5:1588–1602. 135. Espenschied CR, LaDuca H, Li S, et al. Multigene panel testing provides a new perspective on Lynch syndrome. J Clin Oncol 2017;35:2568–2575. 136. Uson PLS Jr, Riegert-Johnson D, Boardman L, et al. Germline cancer susceptibility gene testing in unselected patients with colorectal adenocarcinoma: a multicenter prospective study. Clin Gastroenterol Hepatol 2022;20(3):e508–e528. 137. Gupta S, Weiss JM, Axell L, et al. NCCN Guidelines, Genetic/Familial High-Risk Assessment: Colorectal, Version 2. 2022. Available at: https://www.nccn.org/professionals/ physician_gls/pdf/genetics_colon.pdf. Accessed February 8, 2023. 138. Seppälä TT, Latchford A, Negoi I, et al. European guidelines from the EHTG and ESCP for Lynch syndrome: an updated third edition of the Mallorca guidelines based on gene and gender. Br J Surg 2021; 108:484–498. 139. Monahan KJ, Bradshaw N, Dolwani S, et al. Guidelines for the management of hereditary colorectal cancer from the British Society of Gastroenterology (BSG)/Association of Coloproctology of Great Britain and Ireland (ACPGBI)/United Kingdom Cancer Genetics Group (UKCGG). Gut 2020;69:411–444. 140. Herzig DO, Buie WD, Weiser MR, et al. Clinical practice guidelines for the surgical treatment of patients with Lynch syndrome. Dis Colon Rectum 2017;60:137–143. 141. Lonati C, Necchi A, Rivas JG, et al. Upper tract urothelial carcinoma in the Lynch syndrome tumour spectrum: a comprehensive overview from the European Association of Urology–Young Academic Urologists and the Global Society of Rare Genitourinary Tumors. Eur Urol Oncol 2022;5:30–41. 142. Soer EC, Leicher LW, Langers AMJ, et al. Equivalent Helicobacter pylori infection rates in Lynch syndrome mutation carriers with and without a first-degree relative with gastric cancer. Int J Colorectal Dis 2016; 31:693–697. 143. Boland CR, Yurgelun MB, Mraz KA, Boland PM. Managing gastric cancer risk in Lynch syndrome: controversies and recommendations. Fam Cancer 2022; 21:75–78. 144. Kamiza AB, Hsieh LL, Tang R, et al. Risk factors associated with colorectal cancer in a subset of patients with mutations in MLH1 and MSH2 in Taiwan fulfilling the Amsterdam II criteria for Lynch syndrome. PLoS One 2015;10(6):e0130018. 145. Dashti SG, Hardikar SS, Glombicki SE, et al. Physical activity and the risk of colorectal cancer in Lynch syndrome. Int J Cancer 2018;143:2250–2260. 146. Sievänen T, Törmäkangas T, Laakkonen E, et al. Body weight, physical activity and risk of cancer in Lynch syndrome. Cancers (Basel) 2021;13(8):1849. 147. Coletta AM, Peterson SK, Gatus LA, et al. Energy balance related lifestyle factors and risk of endometrial and colorectal cancer among individuals with lynch syndrome: a systematic review. Fam Cancer 2019; 18:399–420. 148. Movahedi M, Bishop DT, Macrae F, et al. Obesity, aspirin, and risk of colorectal cancer in carriers of hereditary colorectal cancer: a prospective investigation in the CAPP2 study. J Clin Oncol 2015;33:3591–3597. 149. Renkonen-Sinisalo L, Seppälä TT, Järvinen HJ, Mecklin J-P. Subtotal colectomy for colon cancer reduces the need for subsequent surgery in Lynch syndrome. Dis Colon Rectum 2017;60:792–799. 150. Sargent DJ, Marsoni S, Monges G, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 2010;28:3219–3226. 151. André T, de Gramont A, Vernerey D, et al. Adjuvant fluorouracil, leucovorin, and oxaliplatin in stage II to III colon cancer: updated 10-year survival and outcomes according to BRAF mutation and mismatch repair status of the MOSAIC study. J Clin Oncol 2015; 33:4176–4187. 152. Zhang X, Yang Z, An Y, et al. Clinical benefits of PD-1/ PD-L1 inhibitors in patients with metastatic colorectal cancer: a systematic review and meta-analysis. World J Surg Oncol 2022;20:93. 153. Diaz A Jr, Shiu K-K, Kim T-W, et al. Pembrolizumab versus chemotherapy for microsatellite instability-high or mismatch repair-deficient metastatic colorectal cancer (KEYNOTE-177): final analysis of a randomised, openlabel, phase 3 study. Lancet Oncol 2022;23:659–670. 154. Benson AB, Venook AP, Al-Hawary MM, et al. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines ® ), Colon Cancer, Version 3.2023. Available at: https://www.nccn.org/professionals/physician_gls/ pdf/colon.pdf. Accessed February 8, 2023. 155. Edwards P, Monahan KJ. Diagnosis and management of Lynch syndrome. Frontline Gastroenterol 2022; 13(e1):e80–e87. 156. TherkildsenC,JensenLH,RasmussenM,BernsteinI.An update on immune checkpoint therapy for the treatment of Lynch syndrome. Clin Exp Gastroenterol 2021;14:181–197. 157. Overman M, Fakih M, Le D, et al. Phase I interim study results of NOUS-209, an off-the-shelf immunotherapy, with pembrolizumab, for the treatment of tumors with a deficiency in mismatch repair/microsatellite instability (dMMR/ MSI). J Immunother Cancer 2021;9(Suppl 2):A1–A105. 158. Kloor M, Reuschenbach M, Paulig C, et al. A frameshift peptide neoantigen-based vaccine for mismatch repairdeficient cancers: a phase I/IIa clinical trial. Clin Cancer Res 2020;26:4503–4510. 159. Gebert J, Gelincik O, Oezcan-Wahlbrink M, et al. Recurrent frameshift neoantigen vaccine elicits protective immunity with reduced tumor burden and improved overall survival in a Lynch syndrome mouse model. Gastroenterology 2021;161:1288–1302. 160. Brand RE, Dudley B, Karloski E, et al. Detection of DNA mismatch repair deficient crypts in random colonoscopic biopsies identifies Lynch syndrome patients. Fam Cancer 2020;19:169–175. 161. Buglyó G, Styk J, Pös O, et al. Liquid biopsy as a source of nucleic acid biomarkers in the diagnosis and management of Lynch syndrome. Int J Mol Sci 2022; 23(8):4284. 798 Peltomäki et al Gastroenterology Vol. 164, Iss. 5 162. Mathers JC, Elliott F, Macrae F, et al. Cancer prevention with resistant starch in Lynch syndrome patients in the CAPP2-randomized placebo controlled trial: planned 10year follow-up. Cancer Prev Res (Phila.) 2022; 15:623–634. 163. Pylvänäinen K, Lehtinen T, Kellokumpu I, et al. Causes of death of mutation carriers in Finnish Lynch syndrome families. Fam Cancer 2012;3:467–471. Author names in bold designate shared co-first authorship. Received August 4, 2022. Accepted August 30, 2022. Correspondence Address correspondence to: Päivi Peltomäki, MD, PhD, Department of Medical and Clinical Genetics, Medicum, PO Box 63 (Haartmaninkatu 8), FI-00014 University of Helsinki, Finland. e-mail: paivi.peltomaki@helsinki.fi. CRediT Authorship Contributions Päivi Peltomäki, MD, PhD (Conceptualization: Lead; Funding acquisition: Lead; Supervision: Lead; Writing –original draft: Lead; Writing –review & editing: Lead). Minna Nyström, PhD (Conceptualization: Equal; Funding acquisition: Equal; Writing –original draft: Equal; Writing –review & editing: Equal). Jukka-Pekka Mecklin, MD, PhD (Conceptualization: Equal; Funding acquisition: Equal; Writing –original draft: Equal; Writing –review & editing: Equal). Toni T. Seppälä, MD, PhD (Conceptualization: Lead; Funding acquisition: Equal; Writing –original draft: Equal; Writing –review & editing: Equal). Conflicts of interest These authors disclose the following: Minna Nyström and Päivi Peltomäki are inventors of the patent PCT/EP2012/062708. Minna Nyström is a shareholder and board member of LS CancerDiag Ltd. The remaining authors disclose no conflicts. Funding This work was supported by grants from the Jane and Aatos Erkko Foundation (Päivi Peltomäki, Minna Nyström, Jukka-Pekka Mecklin, Toni T. Seppälä), the Academy of Finland (grant no. 330606 to Päivi Peltomäki), Cancer Foundation Finland sr (Päivi Peltomäki, Jukka-Pekka Mecklin, Toni T. Seppälä), and the Sigrid Juselius Foundation (Päivi Peltomäki, Toni T. Seppälä). April 2023 Lynch Syndrome Genetics and Clinical Implications 799