Perspectives

PERSPECTIVES

The origins of LS TIMELINE The history of LS begins in 1895 with Warthin, a world-renowned pathologist at Milestones of Lynch syndrome: the University of Michigan, USA. Warthin became greatly moved by the story of his 1895–2015 seamstress, who attributed her depression to the many deaths throughout her family due to cancer, particularly of the colorec- Henry T. Lynch, Carrie L. Snyder, Trudy G. Shaw, Christopher D. Heinen and tum, stomach and uterus. Warthin began Megan P. Hitchins documenting her medical and cancer family history, as well as the pathology findings Abstract | Lynch syndrome, which is now recognized as the most common of cancer in the family. He assembled her hereditary colorectal cancer condition, is characterized by the predisposition to a pedigree, which he called Family G, along spectrum of cancers, primarily colorectal cancer and endometrial cancer. with two other cancer-prone pedigrees from We chronicle over a century of discoveries that revolutionized the diagnosis and records at the University of Michigan School clinical management of Lynch syndrome, beginning in 1895 with Warthin’s of Medicine and, in 1913, published his find- ings1. He also noted that transmission of observations of familial cancer clusters, through the clinical era led by Lynch and the cancer phenotype within these families the genetic era heralded by the discovery of causative mutations in mismatch was consistent with Mendel’s proposal of repair (MMR) genes, to ongoing challenges. autosomal dominant inheritance2. He continued to follow Family G, and an update More than 100 hereditary cancer-prone syn- homologue 1 (MLH1), mutS homologue 2 was published in the mid‑1930s by Warthin’s dromes have been discovered, and many of (MSH2), MSH6 or postmeiotic segrega- colleagues Hauser and Weller3. The reports these harbour well-defined cancer-causing tion increased 2 (PMS2) — which result in on Family G were one of the first compre- germline mutations. Observational stud- loss of function of the encoded protein. In hensive recorded observations of the familial ies in cancer-prone families have enabled LS‑associated cancers, which arise following clustering of cancer, which laid the founda- clinicians, molecular geneticists and genetic the somatic loss of function of the remaining tion for the discovery of an inherited genetic counsellors to identify individuals who are wild-type allele of the affected MMR gene, basis to cancer and the disease now referred at enormous lifetime risk of developing can- downstream genetic mutations accumulate. to as LS. cer and to offer cancer prevention surveil- These cancers typically manifest micro In 1962, during his internal medicine res- lance, whereas family members without the satellite instability (MSI) — alterations in the idency, Henry Lynch encountered a patient causative mutation have general population length of tandem repeats within microsatel- from Nebraska with a family history similar risk for the syndrome-associated cancers. lite repeat regions — a molecular phenotype to that of Warthin’s seamstress. The proband, The knowledge accrued from studying that is a direct consequence of impaired while recovering from delirium tremens, told affected families has laid the foundation for MMR activity. Our current understanding of Lynch that he drank because he was con- population-based screening to identify those this cancer-prone condition has culminated vinced that he would die of colorectal cancer at the highest risk of cancer. Genetic coun- from more than a century of multidiscipli- (CRC), as “everybody” in the family died selling will be key to the clinical translation nary discoveries from observational studies of this disease. Lynch completed a detailed of the research. in affected families; pathology and molecu- family history, which showed excessive One of the first of these hereditary lar studies of their cancers; basic biology cases of CRC transmitted through multiple cancer-prone syndromes to be recognized, studies of the MMR system; and molecular generations. Lynch’s immediate thoughts Lynch syndrome (LS), is also one of the most genetic and epigenetic studies. Our objective focused on familial adenomatous polypo- prevalent. LS is now firmly established as an in this Timeline article is to describe the sis (FAP), as heretofore this was the most inherited condition of defective DNA mis- history of LS and chart the major contribu- favoured diagnosis of CRC-prone families. match repair (MMR) — the post-replicative tions that have collectively transformed the However, an intensive review of medical and proofreading and editing system that ensures clinical management of affected families, pathology records of family members failed genome integrity. LS is defined as the predis- from Aldred Warthin’s original discoveries to show evidence of multiple colonic adeno- position to a spectrum of cancers, primarily that particular cancers cluster in the family mas, a hallmark of FAP. Therefore, Lynch of the colorectum and endometrium, which of his seamstress in 1895 to the present day questioned whether this could be an unde- exhibit impaired MMR activity. This can- (FIG. 1). Looking ahead, we present some scribed syndrome with a segregating pattern cer predisposition is caused by autosomal of the challenges faced by the community of of CRC predisposition that is consistent with dominant heterozygous germline mutations LS patients, clinical management teams and an autosomal dominant mode of genetic in one of the four key MMR genes — mutL researchers, which warrant further research. transmission but without the presence of the

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• MSI described in LS tumours34 and • Accelerated carcinogenesis • Advantage of subtotal colectomy shown to be due to MMR deficiency37 described38 Survival advantage of • Evidence for over segmental resection shown in • MSH2 identified as first LS locus45–48 • AFAP described185 LS-associated CRC compared prophylactic • Call for universal LS: reduction of synchronous and 179 • Human MSH2–MSH6 with sporadic CRC41 • Listing of LS gynaecological testing of CRC metachronous CRC heterodimer shown to bind to mutations surgery174 for LS; 1 in 35 of • MSH2 and MLH1 shown to have Prevalence of Muir–Torre Increased instability at DNA mismatches195,196 published198 • Heritability of CRCs are LS133 synthetically lethal interactions with Warthin Lynch publishes proximal colonic syndrome microsatellite repeats • Increased instability observed in Amsterdam II First case of MLH1 • Bethesda MSH2 • Recommendation BER DNA polymerases167 begins study pedigrees of tumours in LS identified as a observed in MMR-mutant simple repeat sequences within Criteria epimutation Guidelines epimutation for urological of Family G Families N and M4 described13 variant of CFS18 bacterial strains31 TGFBR2 in MSI+ tumours103 published24 identified74 revised27 shown80 screening in LS17 Excess polyps shown in LS10

1895 1913 1966 1971 1977 1978 1981 1984 1987 1991 1993 1994 1995 1997 1998 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010 2013 2014

Warthin CSF terminology Recommendation HNPCC9 and • Early description of extracolonic • MSH6 identified as • Conversion of diploidy Status risk • FCC-X described186,188 • Heritability of MLH1 EPCAM deletion • Standardized 5-tiered publishes study coined7 of prophylactic Lynch tumour spectrum in LS194 LS locus53 to haploidy identifies change due • Reconstitution of epimutation shown199 identified as an system for classification on Family G1 gynaecological syndrome8 • Amsterdam I Criteria published23 • Bethesda Guidelines the causative to genetic human MMR • X-ray crystal structure underlying cause of pathogenicity of surgery173 terminologies published26 mutation in Family G63 testing197 reaction82,83 of MSH2–MSH6 of MSH2 MMR variants145 coined • MLH1 and PMS2 identified as LS loci49–51 • Silencing of MLH1 • First extended study • MMR-deficient human complex bound to epimutation81 • Technology use in • Pathology of LS-associated CRC by promoter shows efficacy of cells shown to have DNA mismatch genetic counselling described40 hypermethylation colonoscopy in LS172 increased resistance solved146 summarized193 • Full colonoscopy to cecum reported in sporadic to 5-FU159 recommended14 MSI+ cancers137

Figure 1 | Historical timeline of LS. 5‑FU, 5‑fluorouracil; AFAP, attenuated familial adenomatous polyposis; BER, base excision repair; CFS, cancer family syndrome; CRC, colorectal cancer; EPCAM, epithelial cell adhesion molecule; FCC‑X, familial colorectal cancer type X; HNPCC, hereditary non-polyposis colorectal cancer; LS, Lynch syndrome; MLH1, mutL homologue 1; MMR, mismatch repair; MSH, mutS homologue; MSI, microsatellite instability; PMS2, postmeiotic segregation increased 2; TGFBR2, transforming growth factor‑β type II receptor.

Nature Reviews | Cancer multiple colonic adenomas found in FAP. The term CFS was coined in 1971 to incidence of adenomas in patients with LS. Soon after, other cancers, particularly of the describe this familial clustering of can- By consensus, this cancer predisposition endometrium, were recognized as syndro- cers7. However, despite these and numer- syndrome is now referred to as LS11,12. mal throughout this family, which Lynch ous subsequent reports of comparable labelled Family N (for Nebraska). cancer-prone families, which consistently Clinicopathological features of LS Marjorie Shaw of the University of showed an autosomal dominant pattern Phenotypic spectrum in LS. One of the first Michigan told Lynch that she had a family of inheritance of the cancer phenotype, recognized clinical features of LS (then with striking clinical and pathology findings its existence as a hereditary cancer syn- termed CFS), which was reported in 1977, that were comparable to those of Family N; drome remained largely unaccepted by was the high percentage of colonic tumours this family was labelled Family M (for the medical community until the ‘genet- that were located in the proximal colon13, Michigan). The pedigrees from both families ics era’ (1993 to present), whereupon its mandating full colonoscopy to the cecum, were published in 1966 (REF. 4). Although genetic aetiology was firmly established. as urged by Lanspa et al.14. In fact, the interest in this phenomenon rapidly Nevertheless, these early reports launched cecum was found to harbour about one- emerged, funding agencies were reluctant to the ‘clinical era’ in the history of LS, which third of syndromic CRCs. Rondagh et al.15 accept the likelihood of this ‘cancer family provided a more comprehensive picture showed that colorectal neoplasms in LS are syndrome’ (CFS) as being genetic in aetiol- of the LS phenotype and led to the estab- more likely to be non-polypoid and there- ogy, given the presiding dogma of that era lishment of clinical diagnostic criteria fore pose a challenge to the endoscopist. A that environmental factors, which were often and management guidelines for affected high rate of synchronous and metachronous shared within families, were solely responsi- families (see below). This strong clinical tumours is seen. Along with excess cancers ble for cancer causation. The fact that both foundation enabled subsequent molecular of the colorectum and endometrium, which families were part of Midwestern farming studies of the genetic aetiology and patho- had been recognized since the syndrome communities that experienced exposure to genesis of LS‑associated cancers in clini- was first identified, a study in 1994 by pesticides and other potential carcinogens in cally well-defined familial cases. In 1984, Watson and Lynch16 identified significantly the agricultural industry seemed consistent CFS was renamed LS by Boland8. The increased frequencies of cancers of the with this concept. term hereditary non-polyposis colorectal stomach, small bowel, hepatobiliary system, The then-chairperson of pathology cancer (HNPCC) was also used from 1984 upper urologic tract and ovary. A follow‑up at the University of Michigan School of (REF. 9) onwards to differentiate the pheno- study in 2008 (REF. 17) added glioblastomas Medicine invited Lynch to study Warthin’s type from FAP (which is associated with to the list and found that urologic tract and Family G. This led to an extensive review multiple colonic adenomatous polyps). ovarian cancers occurred frequently enough of family records. Lynch and Anne Krush, However, as cancers at multiple extraco- in some LS subgroups to justify trials of a medical social worker, then visited lonic sites had been recognized from the preventive measures. Muir–Torre syndrome, Germany where most of Family G origi- initial descriptions as integral to this syn- which is characterized by sebaceous and nated and accumulated further evidence drome, HNPCC was considered a misno- other skin tumours, was identified as a vari- of cancer predisposition, which they pub- mer. Furthermore, Kalady et al.10 recently ant of LS in 1981 (REF. 18). More recently, lished in 1971 (REF. 5); a subsequent update demonstrated that the term HNPCC is cancers of the pancreas19, breast20 and pros- was published in 2005 (REF. 6). inaccurate when they identified increased tate21, as well as the rare adrenocortical22

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1895 1913 1966 1971 1977 1978 1981 1984 1987 1991 1993 1994 1995 1997 1998 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010 2013 2014

tumours, have been considered to be over- US National Cancer Institute hosted a meet- mechanism of pathogenesis of LS-associated represented in patients with LS. Conversely, ing to develop criteria for selecting CRCs cancers and their genetic aetiology. The a given patient with a germline hetero that warranted MSI testing, which led to the key discoveries of a mutator phenotype that zygous MMR gene mutation may develop development of the Bethesda Guidelines25,26 manifested MSI as a consequence of a faulty only a single cancer late in life or die with- (BOX 1). In 2004, the Revised Bethesda MMR system were initiallyNature made Reviews in lower | Cancer out ever having had a cancer diagnosis, Guidelines provided recommendations for organisms. The recognition that tandem which affects the penetrance estimate for a the clinical selection of cases and for the pro- repeat sequences were prone to increased given type of tumour. Although the typical cess of molecular evaluation of tumours and numbers of frameshift mutations was case of LS involves early onset of CRC or germline DNA for the identification of LS, noted in the 1970s in studies of bacterio- other LS‑spectrum tumours, more pheno- including a consensus MSI testing panel27. phage28,29. Streisinger and Owen30 proposed typic variation, including later age of cancer that, upon denaturation, tandem sequence onset, has become evident since the imple- MSI as a marker of defective MMR in repeats were prone to reanneal ‘out of reg- mentation of ‘reflex testing’ for LS-associated LS-associated cancers. In parallel with the ister’ (that is, become misaligned), leading cancers among population-based cancers. clinical era of LS, a series of basic science to bases that bulged out from the duplex discoveries were made, which subsequently DNA. Frameshift mutations in microsatel- Standardized clinical guidelines to aid proved to be crucial to unravelling the lite repeats occurred even more frequently the diagnosis of LS. In 1991, the then International Collaborative Group on HNPCC devised standardized clinical cri- Glossary teria to aid the diagnosis of LS23 (BOX 1). The Consanguineous Mutator phenotype initial impetus for these guidelines was to Related by blood, such as in a marriage between A cancer with a high burden of somatic mutations across standardize the inclusion criteria for patients cousins. the genome (>12 mutations per 106 bases). in multicentre collaborative research studies that were aimed at unravelling the aetiology Constitutional epimutation Neurofibromatosis An epigenetic aberration within normal somatic An autosomal dominant inherited syndrome with and pathogenesis of LS. These criteria, which cells that predisposes to disease but neither neurofibromin 1 (NF1) mutation and the presence of are now referred to as the Amsterdam I precludes nor dictates that its origin is in the neuropathological findings of gliomas, neuroblastomas, Criteria, focused on a strong family history germ line or that it is distributed evenly throughout ‘café au lait’ spots and multiple neurofibromas. of CRC at a young age of onset (with FAP somatic tissues. excluded). In 1999, the Amsterdam I Criteria Proband Delirium tremens The key family member who is cooperative with respect to were amended to take into account the A condition in which an individual with chronic alcoholic his or her diagnosis and who is a signature member of the 24 extracolonic manifestations of LS . These history exhibits neurodegenerative features, which cancer-prone family. guidelines further evolved as the rationale include occasional hallucination, fever and other behind them became more focused on the neuroderangement. Signet cell features Features of signet ring cells, which have intracytoplasmic identification of suspected LS cases on a Founder mutations mucin-filled vacuoles that cause lateral displacement of population basis and as new knowledge Mutations that are common to multiple families within a the nuclei. on the clinicopathological and molecular given population, with one or more ancestors carrying the characteristics of LS emerged. The discovery mutation. Reflex testing of MSI in LS tumours in 1993 (see below) As performed in the context of colorectal cancer (CRC), the Muir–Torre syndrome automatic testing of all incidence of CRC for microsatellite revolutionized the diagnosis of LS patients. A variant of Lynch syndrome characterized by cutaneous instability and/or immunohistochemical loss of mismatch In recognition of the importance of MSI as signs (sebaceous adenomas and carcinomas, as well as repair activity in order to identify cases that warrant a characteristic of LS tumours, in 1996 the multiple keratoacanthomas). mutation testing for Lynch syndrome.

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Box 1 | Amsterdam I and Amsterdam II Criteria, and Bethesda Guidelines whereas sporadic CRC takes 6–10 years to evolve. Further investigations of the pathol- Amsterdam I Criteria ogy of LS‑associated CRC found near-diploid 23 For a diagnosis of Lynch syndrome (LS), the Amsterdam I Criteria require at least three relatives DNA content39, an excess of mucinous and with histologically verified colorectal cancer (CRC): poorly differentiated (that is, medullary) cell 1. One is a first-degree relative of the other two; types, villous components and lymphocytic 2. At least two successive generations are affected; infiltration40. The presence of tumour- 3. At least one of the relatives with CRC is diagnosed at <50 years of age; 4. Familial adenomatous polyposis (FAP) has been excluded. infiltrating lymphocytes may help to account for the known survival advantage Amsterdam II Criteria of patients with LS‑associated CRC com- For a diagnosis of LS the Amsterdam II Criteria24 require at least three relatives with an LS-associated cancer (that is, CRC and cancers of the endometrium, stomach, ovary, ureter or renal pared with stage-adjusted sporadic CRC 41,42 pelvis, brain, small bowel, hepatobiliary tract and skin (sebaceous tumours)): patients . Signet cell features are also found 1. One is a first-degree relative of the other two; in LS‑associated CRCs, which are unusual in 2. At least two successive generations are affected; FAP-associated and sporadic CRCs. Recent 3. At least one of the LS‑associated cancers should be diagnosed at <50 years of age; reports by Hamilton43 and Bartley et al.44 4. FAP should be excluded in any CRC cases; further emphasize the pathological features 5. Tumours should be verified by pathology whenever possible. that may be more distinctive to LS-associated Bethesda Guidelines for testing of colorectal tumours for microsatellite instability (MSI) CRC than its sporadic CRC counterpart. To justify MSI testing, the Bethesda Guidelines27,171 require: 1. CRC diagnosed in a patient who is <50 years of age; Molecular genetics 2. Presence of synchronous or metachronous colorectal or other LS‑associated tumours*, 1993 marked the end of more than a cen- regardless of age; tury of mystery as to the cause of LS and ‡ § || 3. CRC with MSI-high (MSI‑H) histology diagnosed in a patient who is <60 years of age ; the beginning of the genetic era that revolu- ¶ 4. CRC or LS‑associated tumour* diagnosed <50 years of age in at least one first-degree relative ; tionized its diagnosis (BOX 1). When the first 5. CRC or LS‑associated tumour* diagnosed at any age in two first- or second-degree relatives¶. genetic locus for LS was mapped by link- *LS‑associated tumours include colorectal, endometrial, stomach, ovarian, pancreas, ureter or renal pelvis, age analysis and MSI was simultaneously biliary tract and brain (usually glioblastoma) tumours, sebaceous gland adenomas and keratoacanthomas in identified in LS‑associated tumours, the Muir–Torre syndrome, and carcinoma of the small bowel. ‡MSI‑H in tumours refers to changes in two or more of the five US National Cancer Institute-recommended panels of microsatellite markers. §MSI-H histology genetic cause for LS was soon identified as refers to the presence of tumour-infiltrating lymphocytes, Crohn disease-like lymphocytic reaction, inactivating mutations within MMR genes. mucinous or signet-ring differentiation, or medullary growth pattern. ||There was no consensus among the Workshop participants on whether to include the age criteria in guideline 3; participants voted to keep Identification of the MMR genes with an <60 years of age in the guidelines. ¶Criteria 4 and 5 have been reworded to clarify the Revised Bethesda Guidelines27. aetiological role in LS. While the recognition was dawning that LS‑associated tumours shared the MSI phenotype with lower organisms that were defective in MMR, other when these sequences were introduced into were due to expansions or contractions groups were concurrently searching for the the mutS and mutL Escherichia coli strains31. in the number of tandem repeats within LS locus through traditional linkage analy- These strains were deficient in MMR, a path- these microsatellite loci. The discovery sis in families meeting the Amsterdam I way known to repair base-pairing errors in in LS tumours of a molecular phenotype Criteria (BOX 1). In 1993, Peltomäki et al.45 bacteria and fungi32. In 1993, Strand et al.33 reminiscent of that previously found in lower mapped the first genetic locus responsible demonstrated that mutations in the organisms led the basic scientists who were for LS to chromosome 2p21 using polymor- Saccharomyces cerevisiae genes MSH2 (a studying MMR to immediately focus on phic microsatellite repeat markers in two MutS homologue), PMS1 or MLH1 (a MutL identifying a homologous MMR pathway in large pedigrees from different continents. homologue) led to 100–700‑fold increases human cells. Parsons et al.37 demonstrated This provided the first definitive evidence in repeat tract instability. Upon sequencing that LS cancer cells lacked the ability to repair for a genetic basis to cancer predisposi- of the altered tracts, they determined that small insertions or deletions in tandem repeat tion in LS. Lindblom et al.46 subsequently the deletions or insertions differed by one sequences, as well as single-base mispairs, mapped a second LS locus to chromosome or two repeat units in length in the mutant which is consistent with defective MMR. 3p21–23 in family members that exhibited strains. This observation was consistent with These findings provided the first evidence MSI+ tumours. However, not all LS families the strand misalignment model put forward that LS was a disease of defective MMR. showed linkage to these loci, indicating previously by Streisinger and Owen30. Following the recognition that genome- genetic heterogeneity in the aetiology of LS34. MSI was simultaneously identified wide MSI was a key characteristic of LS Armed with the knowledge that a human in human cancers by Aaltonen et al.34, tumours, MSI testing was subsequently homologue of a MMR gene was a likely Ionov et al.35 and Thibodeau et al.36 in 1993, adopted to identify cancers with an increased candidate, Fishel et al.47 identified MSH2, including LS‑associated cancers from cases likelihood of being associated with LS. the human homologue of the bacterial mutS meeting the Amsterdam I Criteria34. While and S. cerevisiae MSH genes, and concluded exploiting polymorphic microsatellite mark- Pathological features of LS-associated cancers. that mutations in this gene were likely to ers to test for genetic loss of heterozygosity A key feature of LS‑associated CRC is acceler- be causative of LS. Using positional cloning in cancer, they observed additional fragment ated carcinogenesis, which was first described techniques, Leach et al.48 mapped MSH2 to lengths within a proportion of tumours by Jass in 1994 (REF. 38); a tiny colonic ade- the first LS‑linked 2p21 region and identi- (which were absent in germline DNA) that noma can form a CRC in as little as 2–3 years, fied deleterious mutations within MSH2 that

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segregated with cancer in LS families. In 1994, epidemiology studies. However, for mis- mutations involves sequence analysis of all Papadopoulos et al.49 and Bronner et al.50 sense variants, which are collectively termed coding exons and exon–intron boundaries cloned and mapped the human homologue variants of uncertain significance (VUS), the and MLPA, given the broad distribution of of the yeast MUTL MMR gene, MLH1, to the pathogenic importance still remains in ques- mutations throughout these genes. In the second linked 3p21–23 region, and identified tion. Occasional sequence analysis of cDNAs case of PMS2, highly conserved pseudogenes deleterious mutations in several LS families. led to the detection of large exonic deletions complicate mutation detection, but the use In 1994, Nicolaides et al.51 identified PMS1 within MLH1 and MSH2 (REFS 60,61). In 1998, of long-range PCR, cDNA sequencing and and PMS2 on 2q31–33 and 7p22, respectively, Wijnen et al.62 showed, by Southern blotting, novel MLPA strategies circumvents this on the basis of their sequence conservation that genomic rearrangements of MSH2 in issue69–72. Many laboratories are adopting with the yeast homologues and described two particular — including interstitial deletions diagnostic testing strategies based on LS patients with a germline mutation in either and duplications that result in loss of an intact targeted next-generation sequencing73. gene. However, subsequent studies argued protein — were a frequent cause of LS. In In 2004, a database of all known against a role for PMS1 in LS51,52. In 1997, 2000, Yan et al.63 described the technique of cancer-causing mutations in LS was assem- Miyaki et al.53 reported a deleterious muta- conversion of diploidy to haploidy, in which a bled, which is curated and continuously tion of MSH6 in an LS family with multiple patient’s chromosome homologues could be updated by the International Society for affected members, although this family did separated from one another. This technique Gastrointestinal and Hereditary Tumours not meet the Amsterdam I Criteria owing to enabled genetic analyses to be performed on (InSiGHT)66. In 2012, the database showed the predominance of extracolonic cancers and the two genetic alleles individually and led LS‑associated mutation contributions of 42% an age of first cancer onset above 50 years. to the discovery of previously unidentified for MLH1, 33% for MSH2, 18% for MSH6 Miyaki et al.53 at that time proposed pheno- cryptic point mutations and large genomic and 7.5% for PMS2 (REF. 66). typic heterogeneity in LS according to which rearrangements, including the identification of the MMR genes contained the causative of the MSH2 c.646‑3T→G mutation within Alternative causes of LS. Despite the mutation, a concept that was subsequently the splice acceptor site of exon 4 (REF. 63), improvements in genetic screening tech- substantiated through collective epidemiolog- which is responsible for LS in Family G6. nologies, germline mutations of the MMR ical studies (TABLE 1). In 2000, a further MMR With the advent of PCR-based dosage genes remained undetected in up to 30% of gene, MLH3 on 14q24, was identified through analysis that evolved into multiplex ligation- families with a clinical suspicion of LS. In biochemical analyses of the MMR system54. dependent probe amplification (MLPA) 2002, Gazzoli et al.74 identified a case with However, few carriers of MLH3 mutations that is currently in use, large copy number the epigenetic defect now referred to as a have been identified, and either these have variants, primarily within MSH2 and MLH1, constitutional epimutation of MLH1, which is had no family history or their cancers do not were found to be common in LS64,65. Most characterized by monoallelic methylation and exhibit MSI, so there currently is no defini- of these variants were attributable to mal- transcriptional loss of expression throughout tive evidence for the role of MLH3 mutations recombination events between Alu repeats, normal somatic tissues, thereby serving as an in LS55–58. which are present at especially high density alternative cause for LS in MMR gene muta- across MSH2. A number of founder mutations tion-negative cases75. Hitchins et al.76 showed MMR mutations. The rate and nature of have also been identified among LS families that MLH1 epimutations tended to arise mutations detected in LS have reflected tech- within particular populations66–68. Current de novo, which provided an explanation for nological advances over the years. Mutation genetic screening for germline MMR the lack of a family history in most carriers. detection initially involved sequence analyses of exons in genomic DNA, and intron–exon boundaries were subsequently incorporated. Table 1 | Phenotypic heterogeneity by germline MMR gene defect Numerous point mutations have been iden- Causative mechanism Associated phenotypic heterogeneity tified, including frameshift, nonsense and Heterozygous MLH1 LS: CRC predominance; extracolonic cancers less frequent than with splicing mutations that result in nonsense- mutation MSH2 mutations mediated mRNA decay of the transcripts and/or truncated or altered protein structure. Heterozygous MSH2 LS: greater frequency of extracolonic cancers mutation The loss of MMR protein expression in LS‑associated tumours has led to the rou- Heterozygous MSH6 LS: predominance of endometrial cancer; tumours sometimes exhibit mutation low-level MSI tine use of immunohistochemistry (IHC) to determine the status of MMR proteins Heterozygous PMS2 LS: may contain excess colonic polyps; lower frequency of cancer both as a biomarker to identify potential mutation LS‑associated cancers and as a guide to Heterozygous EPCAM LS: silences MSH2 expression; often lower risk of extracolonic uncover the gene that is most likely to con- deletion cancers, although if the deletion is close to the MSH2 gene, risk for endometrial cancer increases tain the germline mutation (see below). Numerous missense variants that result in Monoallelic MLH1 LS: phenotypic expression seems to be similar to that of MLH1 epimutation mutation carriers; a proportion of cases with MLH1 epimutation are amino acid substitutions have also been inherited but, more commonly, this epigenetic defect arises de novo identified. A proportion have been shown to be pathogenic through a combination of Biallelic mutation in any CMMR‑D syndrome: very early-onset (paediatric) haematological, of the four MMR genes colorectal, urinary tract and brain (glioblastoma) cancers, and functional studies demonstrating that they neurofibromatosis alter the MMR capability of the encoded 59 CMMR‑D, constitutional mismatch repair deficiency; CRC, colorectal cancer, EPCAM, epithelial cell variant protein and that they segregate adhesion molecule; LS, Lynch syndrome; MLH1, mutL homologue 1; MMR, mismatch repair; MSH, mutS with the cancer phenotype in families and homologue; MSI, microsatellite instability; PMS2, postmeiotic segregation increased 2.

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The likelihood of vertical transmission of non-Mendelian pattern. In 2011, Hitchins providing lesion recognition specificity, the epimutations to the next generation was et al.77 and Morak et al.78 reported families formation of the two MSH heterodimers pro- hotly debated for the next few years. In with autosomal dominant inheritance of vides some functional redundancy87,88. This 2007, Hitchins et al.199 reported the verti- MLH1 epimutation due to the presence redundancy may underlie the distribution cal transmission of an MLH1 epimutation of an underlying genetic alteration in the of MMR gene mutations identified in LS, from mother to son that occurred in a vicinity of the MLH1 gene, including a in which MSH2 mutations are much more private promoter sequence variant and a prevalent than MSH6 mutations66. Upon large duplication encompassing MLH1 and recognition of a single-base-pair mismatch GGGG G neighbouring genes. An ancestral haplo- in the presence of ATP, the MSH2–MSH6 C 10 CCCC CCCCC type underlying a dominant form of MLH1 heterodimer undergoes a conformational Strand epimutation has now been described in a change to form a sliding clamp that can dif- denaturation collection of European families79. Thus, epi- fuse along DNA89,90 and recruits a second GGGG mutations of MLH1 have been shown to be heterodimer comprised of MLH1 and PMS2 GGG heritable but with distinct Mendelian and (REFS 28,91,92). MLH1 also pairs with MLH3, C10 CCCCCCCCCC Strand non-Mendelian patterns depending on the which again may provide some functional misalignment underlying mechanism. MLH1 epimutation redundancy that could provide an explana- G has been estimated to account for up to 10% tion for the reduced frequency of mutations GGGG G of MMR gene mutation-negative cases of LS within PMS2 in LS compared with MLH1 C 10 CCCCCCCCCC with MLH1‑deficient tumours and for a simi- (REF. 93). Together, the MSH2–MSH6 and Replication lar proportion of incidental CRC cases with MLH1–PMS2 complexes regulate the down- proceeds MLH1‑hypermethylated tumours in patients stream steps of repair. The DNA processiv- G below 60 years of age75. ity factor proliferating cell nuclear antigen G 11 GGGGGGGGGG In 2006, Chan et al.80 reported an auto- (PCNA) is also loaded near the site of the C10 CCCCCCCCCC somal dominant LS family with a heritable mismatch and can stimulate a latent endo- MMR- MMR- proﬁcient deﬁcient MSH2 epimutation characterized by methyl- nuclease activity of PMS2, which introduces ation of the MSH2 promoter within normal single-strand nicks into the daughter DNA G G tissues. In 2009, Ligtenberg et al.81 demon- strand94,95. These nicks facilitate the removal G G 11 GGGGGGGGGG GGGGGGGGGGG 11 strated that MSH2 epimutations in some LS of a segment of the errant daughter strand C10 CCCCCCCCCC CCCCCCCCCCC C11 families resulted from germline deletions that encompasses the sequence error, either Misaligned Next of the terminal end of the adjacent gene, by exonuclease 1 (EXO1)-dependent exci- strand round of 96,97,82 excised replication epithelial cell adhesion molecule (EPCAM; sion or by other mechanisms that may also known as TACSTD1). EPCAM deletions include polymerase-induced strand G GG GGGGGGGGGGG 11 encompass the transcription termination displacement synthesis98. C CCCCCCCCCC CCC 10 signal, resulting in abnormal transcriptional elongation from EPCAM into MSH2. As The mutator phenotype and cancer develop G G 99 10 GGGGGGGGGG GGGGGGGGGGG 11 EPCAM is expressed exclusively in epithelial ment. Hemminki et al. showed that loss C C 10 CCCCCCCCCC CCCCCCCCCCC 11 tissues, the downstream methylation and of the remaining wild-type allele of the MSS MSI silencing of MSH2 manifests only within mutated MMR gene was a feature of LS PCNA EXO1 PMS2 epithelial tissues. This has clinical ramifica- tumours, which is consistent with Knudson’s 100 Polδ MLH1 MSH2 MSH6 tions in terms of the relative risk of cancer two-hit model of carcinogenesis . The development. Screening for EPCAM dele- loss of function of both alleles in somatic Figure 2 | Molecular mechanism of MSI. During tions that are associated with MSH2 epimu- cells results in loss of MMR function and replication of tandem repeat sequences (C10), Nature Reviews | Cancer tation by MLPA has now been incorporated the establishment of a mutator pheno- DNA strand denaturation may occur, resulting in into routine molecular genetic testing for LS. type101. The idea of a mutator phenotype strands reannealing ‘out of register’ (that is, was first proposed in 1974 by Loeb et al.102, becoming misaligned). This may lead to the addi‑ tion (or subtraction) of one or more nucleotides MMR function in normal cells. While who argued that defects in DNA replica- during replication (G11). The extra nucleotide knowledge of the MMR pathway in lower tion or repair would enhance the mutation bulge is recognized by the mismatch repair organisms aided the discovery of the human frequency in cancer cells and increase the (MMR) heterodimer mutS homologue 2 (MSH2)– MMR genes, several biochemical experi- chances of a mutation in an important MSH6 which, together with MLH1–postmeiotic ments carried out in the decades since — oncogene or tumour suppressor gene. Loss segregation increased 2 (PMS2), promotes exci‑ most notably, the complete reconstitution of MMR would result in DNA polymerase sion of this portion of the errant daughter strand. of the MMR reaction in vitro82,83 — have errors remaining intact. Upon the next However, in the absence of MMR activity, the further expanded our understanding of this round of DNA replication, an incorrectly extra nucleotide remains. During the next round pathway. The MMR pathway repairs single- inserted base or an extra repeat sequence of DNA replication, the G11 strand becomes the base-pair mismatches and small insertion bulge would be replicated and permanently template strand. Successful replication of this (FIG. 2) errant strand results in permanent fixation of the or deletion loops (IDLs) that form when the fixed in the genome . This phenom- additional nucleotide and the generation of a polymerase attempts to replicate small repeat enon underlies the presence of MSI and the 84,85 new allele (C11). EXO1, exonuclease 1; MSI, sequences . MSH2 and MSH6 form hetero several hundred-fold increase in mutation microsatellite instability; MSS, microsatellite sta‑ dimers that recognize both of these types of frequency observed in MMR-deficient bility; PCNA, proliferating cell nuclear antigen; errors, whereas an MSH2–MSH3 hetero cells85. The recognition that microsatellite Polδ, DNA polymerase-δ. dimer recognizes larger IDLs86. In addition to sequences were particularly susceptible

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a Cellular phenotype Carcinoma with mutator phenotype

Normal colonic Polyp <8 mm Polyp ≥8 mm epithelium

• Loss of MMR by both IHC and MSI • Frameshift peptides probably attract tumour- • MMR function intact • MMR usually IHC loss of MMR inﬁltrating lymphocytes (normal by IHC) normal by IHC and MSI • MSS • MSS Accelerated polyp-to-carinoma sequence b Parallel intracellular molecular events

• MMR haploinsuﬃciency? • Somatic driver mutation?

Somatic loss of wild-type Accumulation of somatic allele with consequent MMR mutations, including frameshift • Heterozygous germline loss results in replication mutations within TGFBR2, BAX, mutation encodes defective errors being unrepaired APC and other genes protein and predisposes to cancer • Wild-type allele encodes normal protein Chromosome Other Intact DNA MMR Deleterious • MMR protein function containing MMR gene chromosome protein heterodimer heterozygous intact germline mutation Loss of Somatic Normal transcription heterozygosity mutations and translation

Figure 3 | CRC development in individuals with LS. A schematic represen‑ transformation. However, given that polyps are moreNature prevalent Reviews in individuals | Cancer tation of the mechanism of development of colorectal cancer (CRC) via pre- with LS than in the general population, they are also presumably predisposed malignant colorectal polyps in individuals with Lynch syndrome (LS) is shown, to the development of polyps (and thereby CRC). This implicates haplo with the cellular phenotype (part a) above the molecular sequence of events insufficiency (that is, reduced levels or availability) of MMR protein (or pro‑ that occur in parallel (part b). The normal colonic epithelium in carriers of a teins) in initiating pre-neoplastic lesions. This insufficiency could result in the heterozygous germline mismatch repair (MMR) gene mutation is MMR- occurrence of one or more de novo somatic driver mutations, which initiate proficient and thus microsatellite stable (MSS)200. Individuals with LS develop the neoplastic process, before the apparent loss of MMR activity as detected pre-malignant colorectal polyps of mixed morphology, including adenoma‑ by IHC. Full-blown MSI is likely to represent a later manifestation of MMR tous, sessile serrated and hyperplastic polyps15,201. However, the early events deficiency that is only observed following multiple cell replications after the in polyp formation remain unknown. Only large polyps (≥8 mm in diameter) sequence errors are first introduced. The accelerated carcinogenesis from were consistently found to be MMR-deficient, by means of immunohisto‑ polyps found in LS‑associated tumour formation38 is likely to result from the chemistry (IHC)-detected reduction or loss of expression of the respective rapid accumulation of additional somatic mutations following the loss of MMR protein (or proteins), and large polyps also exhibit microsatellite insta‑ MMR activity. This gives rise to carcinoma exhibiting the mutator phenotype bility (MSI). Only a fraction of smaller polyps (<8 mm in diameter) were found and MSI. The prevalence of frameshift mutations, some of which are to have IHC-detected loss of MMR protein expression201. This suggests that expressed on the cell surface, is likely to give rise to tumour-specific antigens, somatic loss of function of the wild-type allele, which is evident in most which elicit immune reactivity and thereby attract high numbers of tumour- LS‑associated CRC99, may not always precede polyp formation but may infiltrating lymphocytes202. This provides one possible explanation for the occur as a late event as the polyp enlarges. It is possible that a somatic ‘driver’ improved prognosis of LS‑associated CRC relative to other incidental CRC42. mutation arises by chance independently of MMR status, which initiates APC, adenomatous polyposis coli; BAX, BCL2‑associated X protein; TGFBR2, polyp formation, and that subsequent MMR loss promulgates malignant transforming growth factor-β type II receptor. to a mutator phenotype led researchers to growth inhibition104. Frameshift mutations Genome Atlas (TCGA) colon cancer study, search for mutations within tandem repeat within coding mononucleotide repeats were which showed that MMR-deficient tumours sequences in the coding region of cancer subsequently identified in other tumour contain hundreds to thousands of somatic genes. In 1995, Markowitz et al.103 identi- suppressor genes in LS‑associated tumours, point mutations, including both frameshift fied an increased frequency of frameshift including adenomatous polyposis coli and nucleotide substitutions, well in excess mutations in a polyadenine tract in the (APC)105, BCL2‑associated X protein (BAX, of those observed in MMR-proficient transforming growth factor-β type II recep- which encodes a pro-apoptotic protein)106 tumours110. This accelerated accumulation tor (TGFBR2) gene. These mutations were and many others107–109. Further evidence of mutations in MMR-deficient cells may associated with loss of the receptor at the for a mutator phenotype in MMR-deficient underlie the rapid progression observed in cell surface and loss of TGFβ‑mediated cell cancer cells came recently from The Cancer LS tumours (FIG. 3).

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MMR genotype–phenotype heterogeneity. The complete inability to repair sequence carriers were found to be at the highest risk The widespread implementation of MSI and errors introduced during DNA replication of endometrial cancer, with a mean age of genetic testing has resulted in a better under- in individuals with CMMR‑D syndrome onset above 50 years118,119. Furthermore, the standing of the disease phenotype among explains the high penetrance and early onset tumours did not consistently display MSI120,121, MMR mutation carriers. While heterozygous of cancers. By contrast, patients with LS, who although instability specifically at mono MMR gene mutations, by definition, cause are carriers of heterozygous MMR gene muta- nucleotide repeat sequences was consistently LS, a more severe cancer phenotype known tions, are afforded some protection by the observed122. PMS2 mutation carriers typically as constitutional mismatch repair deficiency presence of one wild-type allele. The parents develop CRC with MSI but sometimes in the (CMMR‑D) syndrome is caused by biallelic of patients with CMMR‑D syndrome are absence of a family history or at a later age of mutations within the MMR genes. In 1999, also at risk of developing LS‑associated can- onset123–126. The higher penetrance and earlier Ricciardone et al.111 and Wang et al.112 concur- cers, given that they are obligate carriers of a mean age of cancer onset associated with rently reported the paediatric onset of can- heterozygous MMR mutation. MLH1 and MSH2 mutations than with MSH6 cers, including haematological malignancy Disease expression in LS is variable, and PMS2 mutations may be partly explained and neurofibromatosis in two sets of siblings. although particular phenotypic traits have by the functional redundancy with MSH3 and Both were from consanguineous families and been correlated with the MMR gene affected MLH3 noted above, whereas loss of MLH1 or found to be homozygous for an MLH1 muta- by a germline mutation (TABLE 1). Cases with MSH2 expression results in the destabilization tion. Subsequent case reports of individuals a germline mutation within MLH1 or MSH2 of their respective interaction partners. LS with biallelic germline MMR gene mutations typically develop a ‘classic LS’ phenotype that patients with EPCAM deletions were found have shown manifestations of CRC, haema- fulfils the Amsterdam I Criteria with a mean by Kempers et al.127 to be at a similar risk of tological malignancies, features of neuro age of CRC onset of 43–46 years and with developing CRC as their counterparts with fibromatosis, glioblastoma and urinary tract tumours exhibiting MSI, although cases with mutations within MSH2; however, they had a tumours, with age of onset as early as 6 years. MSH2 mutations tend to develop more extra- significantly reduced risk of endometrial can- Non-consanguineous cases of CMMR‑D colonic tumours114–116. MSH2 mutations pre- cer. This phenomenon was subsequently con- syndrome have also been described with dominate in the Muir–Torre variant of LS117. firmed by Lynch et al.128. Only patients with compound heterozygous MMR gene muta- By contrast, LS cases with germline muta- large EPCAM deletions that extended as far as tions within MSH6 and PMS2, including tions within MSH6 and PMS2 tend to develop the MSH2 promoter had an increased risk of point mutations and complex deletions113. an atypical LS phenotype. MSH6 mutation endometrial cancer. Variation in the LS phenotype has been observed even among cases with the same Box 2 | Screening and management germline mutation, which suggests that this is • Prediction of cancer risk is partially defined by clinical pathology findings in an extended influenced by additional factors. For example, pedigree, coupled with the presence or absence of a mismatch repair (MMR) gene mutation. Peltomäki et al.116 reported a variable age of • Colorectal cancer (CRC) prevention requires colonoscopy, which is initiated at age 18 and cancer onset among Finnish families carry- repeated biennially through age 40 and then annually thereafter. Järvinen et al.172 evaluated the ing the same founder mutations of MLH1. efficacy of colonoscopic surveillance in a 15‑year controlled study, which investigated 133 at‑risk Halvarsson et al.129 studied the pathological family members who underwent colonoscopy at 3‑year intervals and compared them with 119 features of CRCs and adenomas from 12 at‑risk family members who declined surveillance. Those who underwent colonoscopy had a 62% members of 2 LS families, and found that the reduced risk of CRC, and all CRCs in the surveillance cohort were local and caused no deaths, compared with 9 deaths caused by CRC in the control group. tumour morphology and IHC expression of β‑catenin (the nuclear localization of which • Screening for gynaecological cancer (endometrial and ovarian cancer) is limited, and there is almost no successful screening strategy for ovarian cancer. Option for prophylactic hysterectomy is a hallmark of cancers driven by chronic and bilateral salpingo-oophorectomy is performed at approximately age 35 (or when pregnancy activation of the WNT signalling pathway, is no longer an option) in germline MMR gene mutation carriers. This was first recommended by for example, owing to APC mutation) varied Lynch as early as 1978 (REF. 173). In 2006, Schmeler et al.174 reported strong evidence supporting extensively within families and even between the role of prophylactic hysterectomy and bilateral salpingo-oophorectomy in women with synchronous or metachronous CRCs from Lynch syndrome (LS)-associated germline mutations. Ketabi et al.175 indicated that based on the the same individual. Proximal CRCs more study of 19,334 women years of endometrial cancer surveillance in LS patients, such surveillance frequently showed poor tumour differentia- should be targeted at MMR gene mutation carriers. tion, expanding growth pattern and tumour- • Upper urological cancer involving urine cytology (which requires a highly experienced infiltrating lymphocytes, whereas distal CRCs pathologist) is coupled with ultrasound scans annually starting at age 30. often lacked distinct LS‑associated morpho- • Owing to evidence of increased synchronous and metachronous CRCs in LS — in which the risk is logical features. Interestingly, glioblastoma approximately 20–30% for metachronous cancer 10 years after initial CRC if less than a subtotal and kidney cancers in patients with either 176 177 colectomy is performed — in 1996 Church and Lynch suggested a role for prophylactic LS or CMMR‑D syndrome are more likely colectomy in LS patients. This must include genetic counselling and referral to one or more to be microsatellite stable, despite loss of the colorectal surgeons for their opinions; most importantly, this must involve LS‑confirmed patients relevant MMR protein as determined by IHC who have declined further colonoscopy. Scaife and Rodriguez-Bigas178 gave further support for both gynaecological and colorectal prophylactic surgeries in 2003. In 2010, Natarajan et al.179 and the finding of MSI in the CRC and other provided data that showed the importance of extended colectomy versus limited resection in LS tumours from the same individuals, suggest- patients with CRC. ing these LS tumours develop through dis- 130–132 • It is clear that in order for a clinical and prophylactic surgical management programme to be tinct pathways . The nature of the genes successful, there must be full patient compliance. in which somatic mutations accumulate as • Identification of a deleterious germline MMR gene mutation provides a definitive diagnosis. a consequence of the mutator phenotype is likely to contribute to some of the variation in

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tumour pathology. However, the reasons that Box 3 | Other syndromes involved in differential diagnosis certain tissues show a propensity for cancer in the context of both LS and CMMR‑D MYH-associated polyposis (MAP) 180 syndrome remain poorly understood. In 2002, Al‑Tassan et al. reported biallelic germline mutations of the mutY homologue (MYH) base excision repair gene in a family with multiple colorectal adenomas and colorectal cancer (CRC) Diagnosis and management that had increased somatic transversion mutations of the adenomatous polyposis coli (APC) gene within the tumours, thereby defining a new recessive CRC predisposition syndrome. In a 2010 Differential diagnosis. Diagnosis of LS is study, Morak et al.181 found a great deal of variability in the clinical phenotype of monoallelic and now based on a combination of clinical phe- biallelic MUTYH mutation carriers. It is estimated that monoallelic MUTYH mutation carriers have notype, routine tumour pathology and/or an approximately 2.5‑fold increased risk of CRC compared with the general population182,183. genetic screening practices. The Revised Morak et al.184 noted that the highly variable phenotype of MAP may overlap with the Lynch Bethesda Guidelines (BOX 1) remain the cur- syndrome (LS) phenotype. As an illustration of this, they described one patient with biallelic MUTYH rent clinical criteria for the identification of mutations who manifested CRC, urothelial carcinoma and sebaceous gland carcinoma. LS had been patients with an increased likelihood of hav- suspected because of a positive family history of CRC, as well as high levels of microsatellite ing LS27. However, there is now much evi- instability (MSI) and immunohistochemistry (IHC) results that showed mutS homologue 2 (MSH2) dence for the efficacy and cost effectiveness and MSH6 deficiency in the sebaceous gland carcinoma. It was found that two somatic, as opposed to germline, MSH2 mutations provided an explanation for the MSI and IHC results. of universal reflex testing for MSI and/or IHC for loss of MMR protein expression Familial adenomatous polyposis (FAP) of all CRCs133–135. The US Multi-Society Variants include: Task Force on Colorectal Cancer has issued • Gardner’s syndrome that contains extracolonic phenotypic aspects; guidelines that call for the use of reflex • Turcot’s syndrome that is associated with medulloblastoma (as opposed to the LS variant of testing135 while noting the difficulty of Turcot’s syndrome, which is associated with glioblastoma); implementation in the clinical setting. • Attenuated FAP (AFAP), which can pose considerable diagnostic problems because its phenotype The identification of MSI in LS‑associated overlaps with that of LS, as colonic adenomas are fewer, more frequently flat and more likely to tumours and the use of IHC to detect the be proximal than in classical FAP (which is associated with mainly distal adenomas). Diagnosis is heavily based on the presence of an APC germline mutation185. expression of MMR proteins have revolutionized the diagnosis of LS. Combinatorial IHC Familial colorectal cancer type X (FCC‑X) testing of all four MMR proteins can provide FCC‑X comprises families that meet the Amsterdam Criteria for diagnosis of LS but that lack an indication of the specific MMR gene that evidence of mismatch repair (MMR) defects. Clinical differences between LS and FCC‑X are as follows. is most likely to contain a pathogenic germ • Lindor et al.186 found only a twofold increased risk for CRC with an older average age of CRC line mutation135. For instance, specific loss onset (61 years) in FCC‑X compared with LS (49 years). of PMS2 or MSH6 protein expression impli- • In a German study187, two-thirds of the tumours in FCC‑X were left-sided, which is the reverse of cates a germline mutation within the PMS2 CRC in LS. There were fewer synchronous and metachronous CRCs than in LS, and there was a or MSH6 genes, respectively. However, dual greater adenoma:carcinoma ratio and a tendency towards more adenomas, which suggests loss of MLH1 and PMS2 protein expression slower adenoma–carcinoma progression. would suggest a germline mutation within • In a 2005 study of 100 individuals from 25 families fulfilling the Amsterdam Criteria188, 40% MLH1, as the PMS2 protein is destabilized showed normal DNA MMR and, in these individuals, 89% of the tumours were left-sided. None in the absence of MLH1. Similarly, IHC loss showed tumour-infiltrating lymphocytes, whereas half of the LS-associated CRCs showed this of both MSH2 and MSH6 staining implies a pathology phenomenon. germline mutation within MSH2, as MSH6 • In a 2007 study189, FCC‑X cases were found to be less likely to have mucinous tumours. is unstable in the absence of MSH2. A move The recent discovery by Nieminen et al. of the role of germline mutations within the bone towards more population-based testing morphogenetic protein receptor type 1A (BMPR1A) gene190 and the ribosomal protein S20 (RPS20) of incident cancers came with the revision of gene191 in FCC‑X families has provided new evidence of the genetic heterogeneity among FCC‑X the Bethesda Guidelines in 2004. In 2008, families and the distinction of FCC-X from LS. Hampel et al.133 studied 500 consecutive A summary of studies on FCC‑X was published by Lindor192 in 2009. patients with CRC and showed that 18 (3.5%) harboured MMR gene mutations, which accounted for 1 in 35 patients with CRC. The mucinous histology136. MSI+ sporadic CRCs MLH1 epimutation or methylation as a sec- demonstration that LS was more prevalent were found to be caused primarily by somati- ond hit during tumorigenesis from sporadic than previously assumed provided a strong cally acquired hypermethylation of both MSI+ cases. rationale for the implementation of more alleles of the MLH1 promoter, with resultant A definitive diagnosis of LS is provided widespread reflex testing of every incidental loss of MLH1 protein expression137–139, which by the identification of a deleterious germ case of CRC for the possibility of LS. was closely associated with the presence of the line mutation or epimutation affecting one of Although MSI is a characteristic feature of oncogenic BRAF‑V600E mutation140. Given the associated MMR genes. LS screening and LS-associated cancers, approximately 12% the comparative rarity of these two molecular management recommendations are discussed of sporadic CRCs also exhibit MSI, which events in LS tumours, BRAF‑V600E mutation in BOX 2. Molecular genetics-based diagnosis poses an additional challenge to identifying or MLH1 hypermethylation testing has been has also become crucial in providing a differ- LS‑associated cancers34–36. These sporadic used in reflex testing of CRC to differentiate ential diagnosis of LS, as there are additional tumours share similar clinicohistopatho- LS‑associated CRC from the more common cancer syndromes with clinically overlap- logical features with cases of LS‑associated sporadic MSI+ counterparts141,142. However, ping features, including FAP, mutY homo- CRC, including a tendency to develop in the MLH1 methylation testing in tumours will logue (MYH)-associated polyposis (MAP) proximal colon, and poorly differentiated and not distinguish LS cases with a constitutional and familial CRC type X (BOX 3). LS, FAP

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and MAP together account for as much as In 2014, InSiGHT reported the annotation important clues to their effect on function. 5% of the total CRC burden worldwide, with of their database of MMR genetic variants Furthermore, it is likely that a number of LS being the most frequent143. It is clear that according to their likely pathogenicity on variants identified by private genetic testing molecular genetics will have an increasing the basis of a five‑tiered scale145. This will companies are not listed on this database; role in the differential diagnosis of cancer assist in the diagnosis of patients; how- therefore, the full extent of genetic variation predisposition syndromes in the future. ever, much further work is still required to in the MMR genes remains unrealized. One of the major challenges that remain definitively determine the pathogenicity of for the molecular diagnosis of LS is deter- a considerable fraction of variants that still The impact of mutation identification on mining the importance of a large number remain VUS. For VUS mutations of MSH2 patient management. The identification of of identified VUS mutations144. Appropriate and MSH6, for example, mapping their loca- the causative mutation within a family is genetic counselling of patients must be tion on the crystal structure of the MSH2– instrumental in defining each blood rela- based on reliable assessment of the patho- MSH6 complex, which was resolved by tive’s risk for developing LS‑associated can- genicity of the MMR variant they carry. Warren et al. in 2007 (REF. 146), will provide cers. For example, in FIG. 4, individual V-2’s

1 2 I Eso62 d.41 d.66

AFF→OGC AFF→OGC AFF→OGC 1 2 3 II Ut37 Abd46 Des70 d.43 d.46 Bl79 Pro79 d.80 – + + – + – – + + AFF→OGC 50→0 AFF→100 AFF→100 50→0 AFF→100 50→0AFF 50→0 OGC→100 AFF→100 1 2 3 4 5 6 7 8 9 10 11 III En55 d.84 En61 Cx37 Pro62 Hepf54 84 Ov37 82 80 Asc50 d.58 100 Co55 Sk85 Pro65 Bl52 Ki65 Rec72 98 d.72 d.61 d.65 d.82 + + + + – + – + + + + – – – AFF→ AFF→ AFF→ 50→ AFF→ 50→ AFF→ AFF→ 25→ 25→ 50→ 50→ 50→ AFF→ AFF→ AFF→AFF→ 50→50→ 50→ OGC 100 100 25→0 25→0 50→0 100 100 25→0 0 OGC 100 0 0 50 50 0 100 100 100 100 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 IV 3 3 3 2 Ce25 Hepf53 Sig46 61 Mmel59 Li38 67 74 Asc40 65 En69 70 Pan23 Ki50 59 60 57 61 50 60 Ov40 En41 En38 Ov33 43 30 30 Co27 Ur56 d.47 79 d.39 Ki44 70 73 d.23 Ur50 59 58 58 54 48 44 35 Co37 Ki57 Ur44 68 61 62 51 Pro49 80 Bl44 Rec50 d.52 d.52 – + + + – – – + + – 50→ 50→ 50→ 50→ 50→ <25 <25 25 25 25→ <25 <25 25→ 50→ <25 <25 <25 25→ 25→ 25→ 50→ 50→ 0 100 AFF 100 100 0 →0 →0 →0 →0 100 →0 →0 0 100 →0 →0 →0 25 25 0 0 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 V 2 4 2 2 5 2 2 2 3 53 52 Rec19Tes31 48 50 33 58 53 41 33 43 44 35 39 51 46 50 36 32 47 26 28 35 31 35 32 27 26 32 31 26 34 30 d.20 51 49 28 60 48 44 37 39 28 33 33 54 40 28 59 43 42 + 25→ 25→ 25→ <25 25→ 0 50 50 →0 100 1 2 3 4 5 6 7 VI 3 2 2 Red High risk En Endometrium 32 24 ~22 25 33 40 Grey General population risk ~20 20 36 Eso Oesophagus ~20 Blue Undefined risk; not tested Hepf Hepatic flexure + Positive results for mutation Ki Kidney – Male Female Multiple primary cancers by medical Negative results for mutation Li Liver records or death certificates AFF Affected Mmel Malignant melanoma 1 2 Individual number OGC Obligate gene carrier Ov Ovary Unaffected Multiple primary cancers by pathology Pan Pancreas 33 29 Current age Cancer by death certificate or Abd Abdomen Pro Prostate Cancer by pathology medical records Asc Ascending colon Rec Rectal colon Li53 Bl45 Age at diagnosis Bl Bladder Sig Sigmoid colon 55 47 Current age 4 2 Number of unaffected children Ce Cecum colon Sk Skin Proband Co Colon Tes Testicular Cancer by family history Cx Cervix Ur Ureter d.54 d.86 Age at death No children Des Descending colon Ut Uterus

Figure 4 | Pedigree of an LS family. This is a pedigree of an extended member197. For example, <25 → 0 shows that the status of the family Nature Reviews | Cancer Lynch syndrome (LS) family, which depicts the importance of key indica‑ member changed from a <25% risk for carrying the mutation to a 0% risk tors by molecular genetic evidence of cancer-causing mutations or the because of the testing of another family member; AFF → OGC indicates lack thereof, determining high-risk versus low-risk individuals. Pairs of that a cancer-affected family member (AFF) was determined to be an numbers with arrows indicate the change in risk status that came not obligate gene carrier (OGC). Bold numbers denote family members men‑ from a test of that individual but from test results of another family tioned in the text.

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knowledge that she was positive for the Box 4 | Genetic counselling and testing family MLH1 mutation (exon 2; 1‑bp insertion mutation at codon 64 that results in • Detailed cancer family history with pathology documentation is required whenever possible. frameshift and stop codon at position 91) • Involvement of a knowledgeable physician, trained genetic counsellor or centre of medical enabled her to work closely with her physi- genetics expertise is needed. cians to develop a cancer screening and • Family history should be extended to four generations whenever possible and should be inclusive prevention plan. Conversely, individual III‑5 of both parents, progeny, maternal and paternal aunts and uncles, grandparents and their was found to be negative for the MLH1 siblings and descendants and, whenever possible, great-grandparents. mutation, meaning that all of his descend- • Trust, compassion and confidentiality are prerequisites to success. ants are automatically negative for this • Recognition of a hereditary cancer syndrome in a family poses a challenge in reaching and mutation; consequently, they are at general communicating with relatives. In most genetic counselling settings, information is limited and population risk for LS‑associated cancers forthcoming only for the proband’s nuclear family. More-distant relatives are rarely evaluated, and do not require intensive screening. which is unfortunate because DNA testing and surveillance screening could be lifesaving in patients with Lynch syndrome (LS) and other hereditary cancer syndromes. Lynch et al.193 Some individuals choose not to be tested summarized the type of information technology that can be effectively used for the for the family mutation and, therefore, their dissemination of susceptibility information and cancer risk knowledge to members of extended cancer risk remains defined by the family families. This involves an effective use of telephone and video counselling, which has been found history. For example, individual IV‑18 is at to be useful in contacting and counselling distant relatives. (FIG. 4) a 50% risk of inheriting the mutation • A detailed educational programme for the entire family membership is crucial. but has chosen not to be tested. Studies have shown that individuals who choose not to know their genetic result have a higher rate double-strand breaks, is generated, which on MMR deficiency in LS‑associated of anxiety than those who tested positive147. results in checkpoint activation and, tumours by treating these cancers with Genetic counselling implications for LS ultimately, cell death151,152,155. inhibitors of oxidative DNA damage are discussed by Eliezer et al.148 and Aktan- The relevance of this DNA damage repair166,167. Martin et al.166,167 showed that Collan et al.149; considerations regarding response function in tumorigenesis is MSH2‑deficient cancer cells were differen- genetic counselling and testing are presented unclear; however, it is likely to be an impor- tially susceptible to RNA interference-based in BOX 4. tant consideration for how MMR-deficient knockdown of DNA polymerase‑β, which tumours respond to chemotherapy. For is involved in the repair of 8‑oxoguanine. Pharmacogenetics and chemoprevention example, 5‑fluorouracil treatment may be Similarly, they showed that MLH1‑deficient Genetic factors have recently been shown to less effective in MMR-deficient CRC156,157, cells were sensitive to loss of DNA affect cancer therapeutics150. The relatively although how this relates specifically to polymerase‑γ, an enzyme involved in the new field of hereditary cancer therapeu- LS‑associated CRC is not yet known158. repair of these same lesions in mitochon- tics has potential to influence cancer care Preclinical studies have shown that treat- dria. These studies suggest that MMR has a through the elucidation of relationships ment of cells with 5‑fluorouracil leads to the back‑up role in the repair of oxidative dam- between a mutation and response to specific incorporation of 5‑fluoro‑2ʹ‑deoxyuridine age and that loss of both repair pathways chemotherapy and/or chemoprevention. into DNA, which can mispair with gua- leads to an overload of unrepaired lesions Continued pharmacogenetics research nine and be recognized by MSH2–MSH6 that affects cell viability. might yield a more tailored treatment for (REF. 159). In addition, 5‑fluorouracil also An initial study by Burn et al.168 found patients with LS based on a better under- decreases thymidine nucleotide pools, aspirin to be ineffective in preventing standing of the impact of a patient’s germline leading to increased incorporation of colonic adenomas and CRC in LS. However, mutation on therapeutic outcome. Basic deoxyuridine into DNA. This event leads when follow‑up time was increased, the science research has shown that the MMR to the production of U∙G mispairs, which chemopreventive effect on polyps and pathway is also involved in a global response also stimulate an MMR-dependent DNA CRC began to emerge169. These findings to certain forms of DNA damage that ulti- damage response. However, prolonged are consistent with observations in mice, mately result in the activation of cell cycle exposure to 5‑fluorouracil may also lead to which showed that aspirin and nitric oxide- checkpoints and cell death151,152. Karran and lesions that are repaired by the base excision donating aspirin (an aspirin derivative) Marinus153 first showed that, in addition repair (BER) pathway which, particularly in increase lifespan and delay tumour onset in to hypermutability, MMR-defective E. coli combination with agents that block down- an intestinal tumour model with MSI170. also display an increased resistance to the stream steps of BER, can result in MMR- cytotoxic effects of the DNA alkylating agent independent cytotoxicity160. Although MSI Conclusion N‑methyl-Nʹ‑nitro‑N‑nitrosoguanidine cancers may be less susceptible to 5‑fluoro We have catalogued the salient clinical, (MNNG). Kat et al.154 later determined uracil, some studies have indicated an pathological and molecular aspects of LS. that an isolated lymphoblastoid cell line improved response in tumours with MSI to The story of LS is exemplary in illustrating that survived the effects of MNNG lacked another key CRC chemotherapeutic agent, how the combination of clinical observa- MMR activity, whereas the MNNG-sensitive irinotecan161–163. An improved understand- tions and basic scientific discoveries was parental cell line maintained MMR func- ing of the role of the MMR pathway in integral to its evolution. The predisposition. Numerous studies have shown that the response to topoisomerase stabilizers such tion of patients with LS to develop multiple MMR proteins recognize lesions created by as irinotecan will be beneficial, as the selec- primary cancers in a single family and the MNNG and other DNA damaging agents; tive benefit of this treatment is not entirely genotypic heterogeneity of LS have, and however, in the attempt to repair these clear164,165. More recent studies suggest syn- will continue to, advance the elucidation of lesions, secondary DNA damage, including thetically lethal approaches that capitalize carcinogenic theory.

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This is an early report of effects of MYH variants on Department of Health and Human Services. Its contents are 160. Liu, A., Yoshioka, K., Salerno, V. & Hsieh, P. The CRC. solely the responsibility of the authors and do not necessarily mismatch repair-mediated cell cycle checkpoint 181. Morak, M., Laner, A.,, Bacher, U.,, Keiling, C. & represent the official views of the State of Nebraska or the response to fluorodeoxyuridine. Cell Biochem. 105, Holinski-Feder, E. MUTYH-associated polyposis — Nebraska Department of Health and Human Services. H.T.L.’s 245–254 (2008). variability of the clinical phenotype in patients with work is partially funded through the Charles F. and Mary C. 161. Kim, J. H. & Kang, G. H. Molecular and prognostic biallelic and monoallelic MUTYH mutations and report Heider Chair in Cancer Research, which he holds at Creighton heterogeneity of microsatellite-unstable colorectal on novel mutations. Clin. Genet. 78, 353–363 (2010). University. The Creighton work also receives funding from cancer. World J. Gastroenterol. 20, 4230–4241 (2014). 182. Jones, N. et al. Increased colorectal cancer incidence in “Kicks for a Cure”. The authors appreciate the intense and 162. Bertagnolli, M. M. et al. Microsatellite instability obligate carriers of heterozygous mutations in MUTYH. constant help provided by LS patients and their families. This predicts improved response to adjuvant therapy with Gastroenterology 137, 489–494 (2009). clearly has made their research possible. irinotecan, fluorouracil, and leucovorin in stage III colon 183. Win, A. K. et al. Risk of colorectal cancer for carriers of cancer: Cancer and Leukemia Group Protocol 89803. mutations in MUTYH, with and without a family history Competing interests statement J. Clin. Oncol. 27, 1814–1821 (2009). of cancer. Gastroenterology 146, 1208–1211 (2014). The authors declare no competing interests.

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