Milestones of Lynch Syndrome

More than 100 hereditary cancer-prone syn-dromes have been discovered, and many of these harbour well-defined cancer-causing germline mutations. Observational stud-ies in cancer-prone families have enabled clinicians, molecular geneticists and genetic counsellors to identify individuals who are at enormous lifetime risk of developing can-cer and to offer cancer prevention surveil-lance, whereas family members without the causative mutation have general population risk for the syndrome-associated cancers. The knowledge accrued from studying affected families has laid the foundation for population-based screening to identify those at the highest risk of cancer. Genetic coun-selling will be key to the clinical translation of the research.

One of the first of these hereditary cancer-prone syndromes to be recognized, Lynch syndrome (LS), is also one of the most prevalent. LS is now firmly established as an inherited condition of defective DNA mis-match repair (MMR) the post-replicative proofreading and editing system that ensures genome integrity. LS is defined as the predis-position to a spectrum of cancers, primarily of the colorectum and endometrium, which exhibit impaired MMR activity. This can-cer predisposition is caused by autosomal dominant heterozygous germline mutations in one of the four key MMR genes mutL

homologue1 (MLH1), mutS homologue2 (MSH2), MSH6 or postmeiotic segrega-tion increased2 (PMS2) which result in loss of function of the encoded protein. In LS-associated cancers, which arise following the somatic loss of function of the remaining wild-type allele of the affected MMR gene, downstream genetic mutations accumulate. These cancers typically manifest micro-satellite instability (MSI) alterations in the length of tandem repeats within microsatel-lite repeat regions a molecular phenotype that is a direct consequence of impaired MMR activity. Our current understanding of this cancer-prone condition has culminated from more than a century of multidiscipli-nary discoveries from observational studies in affected families; pathology and molecu-lar studies of their cancers; basic biology studies of the MMR system; and molecular genetic and epigenetic studies. Our objective in this Timeline article is to describe the history of LS and chart the major contribu-tions that have collectively transformed the clinical management of affected families, from Aldred Warthins original discoveries that particular cancers cluster in the family of his seamstress in 1895 to the present day (FIG.1). Looking ahead, we present some of the challenges faced by the community of LS patients, clinical management teams and researchers, which warrant further research.

The origins of LSThe history of LS begins in 1895 with Warthin, a world-renowned pathologist at the University of Michigan, USA. Warthin became greatly moved by the story of his seamstress, who attributed her depression to the many deaths throughout her family due to cancer, particularly of the colorec-tum, stomach and uterus. Warthin began documenting her medical and cancer family history, as well as the pathology findings of cancer in the family. He assembled her pedigree, which he called Family G, along with two other cancer-prone pedigrees from records at the University of Michigan School of Medicine and, in 1913, published his find-ings1. He also noted that transmission of the cancer phenotype within these families was consistent with Mendels proposal of autosomal dominant inheritance2. He con-tinued to follow Family G, and an update was published in the mid-1930s by Warthins colleagues Hauser and Weller3. The reports on Family G were one of the first compre-hensive recorded observations of the familial clustering of cancer, which laid the founda-tion for the discovery of an inherited genetic basis to cancer and the disease now referred to asLS.

In 1962, during his internal medicine res-idency, Henry Lynch encountered a patient from Nebraska with a family history similar to that of Warthins seamstress. The proband, while recovering from delirium tremens, told Lynch that he drank because he was con-vinced that he would die of colorectal cancer (CRC), as everybody in the family died of this disease. Lynch completed a detailed family history, which showed excessive cases of CRC transmitted through multiple generations. Lynchs immediate thoughts focused on familial adenomatous polypo-sis (FAP), as heretofore this was the most favoured diagnosis of CRC-prone families. However, an intensive review of medical and pathology records of family members failed to show evidence of multiple colonic adeno-mas, a hallmark of FAP. Therefore, Lynch questioned whether this could be an unde-scribed syndrome with a segregating pattern of CRC predisposition that is consistent with an autosomal dominant mode of genetic transmission but without the presence of the

T I M E L I N E

Milestones of Lynch syndrome: 18952015Henry T.Lynch, Carrie L.Snyder, Trudy G.Shaw, Christopher D.Heinen and Megan P.Hitchins

Abstract | Lynch syndrome, which is now recognized as the most common hereditary colorectal cancer condition, is characterized by the predisposition to a spectrum of cancers, primarily colorectal cancer and endometrial cancer. We chronicle over a century of discoveries that revolutionized the diagnosis and clinical management of Lynch syndrome, beginning in 1895 with Warthins observations of familial cancer clusters, through the clinical era led by Lynch and the genetic era heralded by the discovery of causative mutations in mismatch repair (MMR) genes, to ongoing challenges.

PERSPECTIVES

NATURE REVIEWS | CANCER VOLUME 15 | MARCH 2015 | 181

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HNPCC9 and Lynch syndrome8 terminologies coined

Early description of extracolonic tumour spectrum in LS194

Amsterdam I Criteria published23

MLH1 and PMS2 identified as LS loci4951 Pathology of LS-associated CRC

described40 Full colonoscopy to cecum

recommended14

MSH6 identified as LS locus53

Bethesda Guidelines published26

Silencing of MLH1 by promoter hypermethylation reported in sporadic MSI+ cancers137

Amsterdam II Criteria published24

First case of MLH1 epimutation identified74

Listing of LS mutations published198

Bethesda Guidelines revised27

Evidence for prophylactic gynaecological surgery174

Heritability of MSH2 epimutation shown80

Call for universal testing of CRC for LS; 1 in 35 of CRCs are LS133

Recommendation for urological screening in LS17

Advantage of subtotal colectomy over segmental resection shown in LS: reduction of synchronous and metachronous CRC179

MSH2 and MLH1 shown to have synthetically lethal interactions with BER DNA polymerases167

Excess polyps shown in LS10

Increased instability at microsatellite repeats observed in MMR-mutant bacterial strains31

MSI described in LS tumours34 and shown to be due to MMR deficiency37

MSH2 identified as first LS locus4548

Accelerated carcinogenesis described38

AFAP described185 Human MSH2MSH6

heterodimer shown to bind to DNA mismatches195,196

Increased instability observed in simple repeat sequences within TGFBR2 in MSI+ tumours103

Survival advantage of LS-associated CRC compared with sporadic CRC41

Conversion of diploidy to haploidy identifies the causative mutation in Family G63

First extended study shows efficacy of colonoscopy in LS172

Status risk change due to genetic testing197

FCC-X described186,188 Reconstitution of

human MMR reaction82,83

MMR-deficient human cells shown to have increased resistance to 5-FU159

Heritability of MLH1 epimutation shown199

X-ray crystal structure of MSH2MSH6 complex bound to DNA mismatch solved146

EPCAM deletion identified as an underlying cause of MSH2 epimutation81

Standardized 5-tiered system for classification of pathogenicity of MMR variants145

Technology use in genetic counselling summarized193

Nature Reviews | Cancer

Recommendation of prophylactic gynaecological surgery173

Warthin begins study of Family G

Lynch publishes pedigrees of Families N and M4

Prevalence of proximal colonic tumours in LS described13

MuirTorre syndrome identified as a variant of CFS18

1895 1913 1966 1971 1977 1978 1981 1984 1987 1991 19951993 1994 1997 1998 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010 2013 2014

CSF terminology coined7

Warthin publishes study on Family G1

multiple colonic adenomas found in FAP. Soon after, other cancers, particularly of the endometrium, were recognized as syndro-mal throughout this family, which Lynch labelled FamilyN (for Nebraska).

Marjorie Shaw of the University of Michigan told Lynch that she had a family with striking clinical and pathology findings that were comparable to those of FamilyN; this family was labelled FamilyM (for Michigan). The pedigrees from both families were published in 1966 (REF.4). Although interest in this phenomenon rapidly emerged, funding agencies were reluctant to accept the likelihood of this cancer family syndrome (CFS) as being genetic in aetiol-ogy, given the presiding dogma of that era that environmental factors, which were often shared within families, were solely responsi-ble for cancer causation. The fact that both families were part of Midwestern farming communities that experienced exposure to pesticides and other potential carcinogens in the agricultural industry seemed consistent with this concept.

The then-chairperson of pathology at the University of Michigan School of Medicine invited Lynch to study Warthins FamilyG. This led to an extensive review of family records. Lynch and Anne Krush, a medical social worker, then visited Germany where most of Family G origi-nated and accumulated further evidence of cancer predisposition, which they pub-lished in 1971 (REF.5); a subsequent update was published in 2005 (REF.6).

The term CFS was coined in 1971 to describe this familial clustering of can-cers7. However, despite these and numer-ous subsequent reports of comparable cancer-prone families, which consistently showed an autosomal dominant pattern of inheritance of the cancer phenotype, its existence as a hereditary cancer syn-drome remained largely unaccepted by the medical community until the genet-ics era (1993 to present), whereupon its genetic aetiology was firmly established. Nevertheless, these early reports launched the clinical era in the history of LS, which provided a more comprehensive picture of the LS phenotype and led to the estab-lishment of clinical diagnostic criteria and management guidelines for affected families (see below). This strong clinical foundation enabled subsequent molecular studies of the genetic aetiology and patho-genesis of LS-associated cancers in clini-cally well-defined familial cases. In 1984, CFS was renamed LS by Boland8. The term hereditary non-polyposis colorectal cancer (HNPCC) was also used from 1984 (REF.9) onwards to differentiate the pheno-type from FAP (which is associated with multiple colonic adenomatous polyps). However, as cancers at multiple extraco-lonic sites had been recognized from the initial descriptions as integral to this syn-drome, HNPCC was considered a misno-mer. Furthermore, Kalady etal.10 recently demonstrated that the term HNPCC is inaccurate when they identified increased

incidence of adenomas in patients with LS. By consensus, this cancer predisposition syndrome is now referred to as LS11,12.

Clinicopathological features of LSPhenotypic spectrum in LS. One of the first recognized clinical features of LS (then termed CFS), which was reported in 1977, was the high percentage of colonic tumours that were located in the proximal colon13, mandating full colonoscopy to the cecum, as urged by Lanspa etal.14. In fact, the cecum was found to harbour about one-third of syndromic CRCs. Rondagh etal.15 showed that colorectal neoplasms in LS are more likely to be non-polypoid and there-fore pose a challenge to the endoscopist. A high rate of synchronous and metachronous tumours is seen. Along with excess cancers of the colorectum and endometrium, which had been recognized since the syndrome was first identified, a study in 1994 by Watson and Lynch16 identified significantly increased frequencies of cancers of the stomach, small bowel, hepatobiliary system, upper urologic tract and ovary. A follow-up study in 2008 (REF.17) added glioblastomas to the list and found that urologic tract and ovarian cancers occurred frequently enough in some LS subgroups to justify trials of preventive measures. MuirTorre syndrome, which is characterized by sebaceous and other skin tumours, was identified as a vari-ant of LS in 1981 (REF.18). More recently, cancers of the pancreas19, breast20 and pros-tate21, as well as the rare adrenocortical22

Figure 1 | Historical timeline of LS. 5FU, 5fluorouracil; AFAP, attenuated familial adenomatous polyposis; BER, base excision repair; CFS, cancer family syndrome; CRC, colorectal cancer; EPCAM, epithelial cell adhesion molecule; FCCX, familial colorectal cancer type X; HNPCC, hereditary nonpolyposis 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 typeII receptor.

P E R S P E C T I V E S

182 | MARCH 2015 | VOLUME 15 www.nature.com/reviews/cancer


HNPCC9 and Lynch syndrome8 terminologies coined

Early description of extracolonic tumour spectrum in LS194

Amsterdam I Criteria published23

MLH1 and PMS2 identified as LS loci4951 Pathology of LS-associated CRC

described40 Full colonoscopy to cecum

recommended14

MSH6 identified as LS locus53

Bethesda Guidelines published26

Silencing of MLH1 by promoter hypermethylation reported in sporadic MSI+ cancers137

Amsterdam II Criteria published24

First case of MLH1 epimutation identified74

Listing of LS mutations published198

Bethesda Guidelines revised27

Evidence for prophylactic gynaecological surgery174

Heritability of MSH2 epimutation shown80

Call for universal testing of CRC for LS; 1 in 35 of CRCs are LS133

Recommendation for urological screening in LS17

Advantage of subtotal colectomy over segmental resection shown in LS: reduction of synchronous and metachronous CRC179

MSH2 and MLH1 shown to have synthetically lethal interactions with BER DNA polymerases167

Excess polyps shown in LS10

Increased instability at microsatellite repeats observed in MMR-mutant bacterial strains31

MSI described in LS tumours34 and shown to be due to MMR deficiency37

MSH2 identified as first LS locus4548

Accelerated carcinogenesis described38

AFAP described185 Human MSH2MSH6

heterodimer shown to bind to DNA mismatches195,196

Increased instability observed in simple repeat sequences within TGFBR2 in MSI+ tumours103

Survival advantage of LS-associated CRC compared with sporadic CRC41

Conversion of diploidy to haploidy identifies the causative mutation in Family G63

First extended study shows efficacy of colonoscopy in LS172

Status risk change due to genetic testing197

FCC-X described186,188 Reconstitution of

human MMR reaction82,83

MMR-deficient human cells shown to have increased resistance to 5-FU159

Heritability of MLH1 epimutation shown199

X-ray crystal structure of MSH2MSH6 complex bound to DNA mismatch solved146

EPCAM deletion identified as an underlying cause of MSH2 epimutation81

Standardized 5-tiered system for classification of pathogenicity of MMR variants145

Technology use in genetic counselling summarized193


Recommendation of prophylactic gynaecological surgery173

Warthin begins study of Family G

Lynch publishes pedigrees of Families N and M4

Prevalence of proximal colonic tumours in LS described13

MuirTorre syndrome identified as a variant of CFS18

1895 1913 1966 1971 1977 1978 1981 1984 1987 1991 19951993 1994 1997 1998 1999 2000 2002 2003 2004 2005 2006 2007 2008 2009 2010 2013 2014

CSF terminology coined7

Warthin publishes study on Family G1

tumours, have been considered to be over-represented in patients with LS. Conversely, a given patient with a germline hetero-zygous MMR gene mutation may develop only a single cancer late in life or die with-out ever having had a cancer diagnosis, which affects the penetrance estimate for a given type of tumour. Although the typical case of LS involves early onset of CRC or other LS-spectrum tumours, more pheno-typic variation, including later age of cancer onset, has become evident since the imple-mentation of reflex testing for LS-associated cancers among population-based cancers.

Standardized clinical guidelines to aid the diagnosis of LS. In 1991, the then International Collaborative Group on HNPCC devised standardized clinical cri-teria to aid the diagnosis of LS23 (BOX1). The initial impetus for these guidelines was to standardize the inclusion criteria for patients in multicentre collaborative research studies that were aimed at unravelling the aetiology and pathogenesis of LS. These criteria, which are now referred to as the AmsterdamI Criteria, focused on a strong family history of CRC at a young age of onset (with FAP excluded). In 1999, the Amsterdam I Criteria were amended to take into account the extracolonic manifestations of LS24. These guidelines further evolved as the rationale behind them became more focused on the identification of suspected LS cases on a population basis and as new knowledge on the clinicopathological and molecular characteristics of LS emerged. The discovery of MSI in LS tumours in 1993 (see below) revolutionized the diagnosis of LS patients. In recognition of the importance of MSI as a characteristic of LS tumours, in 1996 the

US National Cancer Institute hosted a meet-ing to develop criteria for selecting CRCs that warranted MSI testing, which led to the development of the Bethesda Guidelines25,26 (BOX1). In 2004, the Revised Bethesda Guidelines provided recommendations for the clinical selection of cases and for the pro-cess of molecular evaluation of tumours and germline DNA for the identification of LS, including a consensus MSI testing panel27.

MSI as a marker of defective MMR in LS-associated cancers. In parallel with the clinical era of LS, a series of basic science discoveries were made, which subsequently proved to be crucial to unravelling the

mechanism of pathogenesis of LS-associated cancers and their genetic aetiology. The key discoveries of a mutator phenotype that manifested MSI as a consequence of a faulty MMR system were initially made in lower organisms. The recognition that tandem repeat sequences were prone to increased numbers of frameshift mutations was noted in the 1970s in studies of bacterio-phage28,29. Streisinger and Owen30 proposed that, upon denaturation, tandem sequence repeats were prone to reanneal out of reg-ister (that is, become misaligned), leading to bases that bulged out from the duplex DNA. Frameshift mutations in microsatel-lite repeats occurred even more frequently

Glossary

ConsanguineousRelated by blood, such as in a marriage between cousins.

Constitutional epimutationAn epigenetic aberration within normal somatic cells that predisposes to disease but neither precludes nor dictates that its origin is in the germ line or that it is distributed evenly throughout somatic tissues.

Delirium tremensA condition in which an individual with chronic alcoholic history exhibits neurodegenerative features, which include occasional hallucination, fever and other neuroderangement.

Founder mutationsMutations that are common to multiple families within a given population, with one or more ancestors carrying the mutation.

MuirTorre syndromeA variant of Lynch syndrome characterized by cutaneous signs (sebaceous adenomas and carcinomas, as well as multiple keratoacanthomas).

Mutator phenotypeA cancer with a high burden of somatic mutations across the genome (>12 mutations per 106 bases).

NeurofibromatosisAn autosomal dominant inherited syndrome with neurofibromin 1 (NF1) mutation and the presence of neuropathological findings of gliomas, neuroblastomas, caf au lait spots and multiple neurofibromas.

ProbandThe key family member who is cooperative with respect to his or her diagnosis and who is a signature member of the cancer-prone family.

Signet cell featuresFeatures of signet ring cells, which have intracytoplasmic mucin-filled vacuoles that cause lateral displacement of the nuclei.

Reflex testingAs performed in the context of colorectal cancer (CRC), the automatic testing of all incidence of CRC for microsatellite instability and/or immunohistochemical loss of mismatch repair activity in order to identify cases that warrant mutation testing for Lynch syndrome.




when these sequences were introduced into the mutS and mutL Escherichia coli strains31. These strains were deficient in MMR, a path-way known to repair base-pairing errors in bacteria and fungi32. In 1993, Strandetal.33 demonstrated that mutations in the Saccharomyces cerevisiae genes MSH2 (a MutS homologue), PMS1 or MLH1 (a MutL homologue) led to 100700-fold increases in repeat tract instability. Upon sequencing of the altered tracts, they determined that the deletions or insertions differed by one or two repeat units in length in the mutant strains. This observation was consistent with the strand misalignment model put forward previously by Streisinger andOwen30.

MSI was simultaneously identified in human cancers by Aaltonen etal.34, Ionovetal.35 and Thibodeau etal.36 in 1993, including LS-associated cancers from cases meeting the Amsterdam I Criteria34. While exploiting polymorphic microsatellite mark-ers to test for genetic loss of heterozygosity in cancer, they observed additional fragment lengths within a proportion of tumours (which were absent in germline DNA) that

were due to expansions or contractions in the number of tandem repeats within these microsatellite loci. The discovery in LS tumours of a molecular phenotype reminiscent of that previously found in lower organisms led the basic scientists who were studying MMR to immediately focus on identifying a homologous MMR pathway in human cells. Parsons etal.37 demonstrated that LS cancer cells lacked the ability to repair small insertions or deletions in tandem repeat sequences, as well as single-base mispairs, which is consistent with defective MMR. These findings provided the first evidence that LS was a disease of defective MMR. Following the recognition that genome- wide MSI was a key characteristic of LS tumours, MSI testing was subsequently adopted to identify cancers with an increased likelihood of being associated withLS.

Pathological features of LS-associated cancers. A key feature of LS-associated CRC is acceler-ated carcinogenesis, which was first described by Jass in 1994 (REF.38); a tiny colonic ade-noma can form a CRC in as little as 23years,

whereas sporadic CRC takes 610years to evolve. Further investigations of the pathol-ogy of LS-associated CRC found near-diploid DNA content39, an excess of mucinous and poorly differentiated (that is, medullary) cell types, villous components and lymphocytic infiltration40. The presence of tumour- infiltrating lymphocytes may help to account for the known survival advantage of patients with LS-associated CRC com-pared with stage-adjusted sporadic CRC patients41,42. Signet cell features are also found in LS-associated CRCs, which are unusual in FAP-associated and sporadic CRCs. Recent reports by Hamilton43 and Bartley etal.44 further emphasize the pathological features that may be more distinctive to LS-associated CRC than its sporadic CRC counterpart.

Molecular genetics1993 marked the end of more than a cen-tury of mystery as to the cause of LS and the beginning of the genetic era that revolu-tionized its diagnosis (BOX1). When the first genetic locus for LS was mapped by link-age analysis and MSI was simultaneously identified in LS-associated tumours, the genetic cause for LS was soon identified as inactivating mutations within MMRgenes.

Identification of the MMR genes with an aetiological role in LS. While the recognition was dawning that LS-associated tumours shared the MSI phenotype with lower organ-isms that were defective in MMR, other groups were concurrently searching for the LS locus through traditional linkage analy-sis in families meeting the AmsterdamI Criteria (BOX1). In 1993, Peltomki etal.45 mapped the first genetic locus responsible for LS to chromosome 2p21 using polymor-phic microsatellite repeat markers in two large pedigrees from different continents. This provided the first definitive evidence for a genetic basis to cancer predisposi-tion in LS. Lindblom etal.46 subsequently mapped a second LS locus to chromosome 3p2123 in family members that exhibited MSI+ tumours. However, not all LS families showed linkage to these loci, indicating genetic heterogeneity in the aetiology ofLS34.

Armed with the knowledge that a human homologue of a MMR gene was a likely candidate, Fishel etal.47 identified MSH2, the human homologue of the bacterial mutS and S.cerevisiae MSH genes, and concluded that mutations in this gene were likely to be causative of LS. Using positional cloning techniques, Leach etal.48 mapped MSH2 to the first LS-linked 2p21 region and identi-fied deleterious mutations within MSH2 that

Box 1 | Amsterdam I and Amsterdam II Criteria, and Bethesda Guidelines

Amsterdam I CriteriaFor a diagnosis of Lynch syndrome (LS), the Amsterdam I Criteria23 require at least three relatives with histologically verified colorectal cancer (CRC):1. One is a first-degree relative of the other two;2. At least two successive generations are affected;3.AtleastoneoftherelativeswithCRCisdiagnosedat

segregated with cancer in LS families. In 1994, Papadopoulos etal.49 and Bronner etal.50 cloned and mapped the human homologue of the yeast MUTL MMR gene, MLH1, to the second linked 3p2123 region, and identified deleterious mutations in several LS families. In 1994, Nicolaides etal.51 identified PMS1 and PMS2 on 2q3133 and 7p22, respectively, on the basis of their sequence conservation with the yeast homologues and described two LS patients with a germline mutation in either gene. However, subsequent studies argued against a role for PMS1 in LS51,52. In 1997, Miyaki etal.53 reported a deleterious muta-tion of MSH6 in an LS family with multiple affected members, although this family did not meet the Amsterdam I Criteria owing to the predominance of extracolonic cancers and an age of first cancer onset above 50years. Miyaki etal.53 at that time proposed pheno-typic heterogeneity in LS according to which of the MMR genes contained the causative mutation, a concept that was subsequently substantiated through collective epidemiolog-ical studies (TABLE1). In 2000, a further MMR gene, MLH3 on 14q24, was identified through biochemical analyses of the MMR system54. However, few carriers of MLH3 mutations have been identified, and either these have had no family history or their cancers do not exhibit MSI, so there currently is no defini-tive evidence for the role of MLH3 mutations inLS5558.

MMR mutations. The rate and nature of mutations detected in LS have reflected tech-nological advances over the years. Mutation detection initially involved sequence analyses of exons in genomic DNA, and intronexon boundaries were subsequently incorporated. Numerous point mutations have been iden-tified, including frameshift, nonsense and splicing mutations that result in nonsense-mediated mRNA decay of the transcripts and/or truncated or altered protein structure. The loss of MMR protein expression in LS-associated tumours has led to the rou-tine use of immunohistochemistry (IHC) to determine the status of MMR proteins both as a biomarker to identify potential LS-associated cancers and as a guide to uncover the gene that is most likely to con-tain the germline mutation (see below). Numerous missense variants that result in amino acid substitutions have also been identified. A proportion have been shown to be pathogenic through a combination of functional studies demonstrating that they alter the MMR capability of the encoded variant protein59 and that they segregate with the cancer phenotype in families and

epidemiology studies. However, for mis-sense variants, which are collectively termed variants of uncertain significance (VUS), the pathogenic importance still remains in ques-tion. Occasional sequence analysis of cDNAs led to the detection of large exonic deletions within MLH1 and MSH2 (REFS60,61). In 1998, Wijnen etal.62 showed, by Southern blotting, that genomic rearrangements of MSH2 in particular including interstitial deletions and duplications that result in loss of an intact protein were a frequent cause of LS. In 2000, Yan etal.63 described the technique of conversion of diploidy to haploidy, in which a patients chromosome homologues could be separated from one another. This technique enabled genetic analyses to be performed on the two genetic alleles individually and led to the discovery of previously unidentified cryptic point mutations and large genomic rearrangements, including the identification of the MSH2 c.646-3TG mutation within the splice acceptor site of exon 4 (REF.63), which is responsible for LS in FamilyG6.

With the advent of PCR-based dosage analysis that evolved into multiplex ligation-dependent probe amplification (MLPA) that is currently in use, large copy number variants, primarily within MSH2 and MLH1, were found to be common in LS64,65. Most of these variants were attributable to mal-recombination events between Alu repeats, which are present at especially high density across MSH2. A number of founder mutations have also been identified among LS families within particular populations6668. Current genetic screening for germline MMR

mutations involves sequence analysis of all coding exons and exonintron boundaries and MLPA, given the broad distribution of mutations throughout these genes. In the case of PMS2, highly conserved pseudogenes complicate mutation detection, but the use of long-range PCR, cDNA sequencing and novel MLPA strategies circumvents this issue6972. Many laboratories are adopting diagnostic testing strategies based on targeted next-generation sequencing73.

In 2004, a database of all known cancer-causing mutations in LS was assem-bled, which is curated and continuously updated by the International Society for Gastrointestinal and Hereditary Tumours (InSiGHT)66. In 2012, the database showed LS-associated mutation contributions of 42% for MLH1, 33% for MSH2, 18% for MSH6 and 7.5% for PMS2 (REF.66).

Alternative causes of LS. Despite the improvements in genetic screening tech-nologies, germ line mutations of the MMR genes remained undetected in up to 30% of families with a clinical suspicion of LS. In 2002, Gazzoli etal.74 identified a case with the epi genetic defect now referred to as a constitutional epimutation of MLH1, which is characterized by monoallelic methylation and transcriptional loss of expression throughout normal somatic tissues, thereby serving as an alternative cause for LS in MMR gene muta-tion-negative cases75. Hitchins etal.76 showed that MLH1 epimutations tended to arise denovo, which provided an explanation for the lack of a family history in most carriers.

Table 1 | Phenotypic heterogeneity by germline MMR gene defect

Causative mechanism Associated phenotypic heterogeneity

Heterozygous MLH1 mutation

LS: CRC predominance; extracolonic cancers less frequent than with MSH2 mutations

Heterozygous MSH2 mutation

LS: greater frequency of extracolonic cancers

Heterozygous MSH6 mutation

LS: predominance of endometrial cancer; tumours sometimes exhibit lowlevel MSI

Heterozygous PMS2 mutation

LS: may contain excess colonic polyps; lower frequency of cancer

Heterozygous EPCAM deletion

LS: silences MSH2 expression; often lower risk of extracolonic cancers, although if the deletion is close to the MSH2 gene, risk for endometrial cancer increases

Monoallelic MLH1 epimutation

LS: phenotypic expression seems to be similar to that of MLH1 mutation carriers; a proportion of cases with MLH1 epimutation are inherited but, more commonly, this epigenetic defect arises denovo

Biallelic mutation in any of the four MMR genes

CMMRD syndrome: very earlyonset (paediatric) haematological, colorectal, urinary tract and brain (glioblastoma) cancers, and neurofibromatosis

CMMRD, constitutional mismatch repair deficiency; CRC, colorectal cancer, EPCAM, epithelial cell adhesion molecule; LS, Lynch syndrome; MLH1, mutL homologue 1; MMR, mismatch repair; MSH, mutS homologue; MSI, microsatellite instability; PMS2, postmeiotic segregation increased 2.





GGGGCCCC

GGGGGGGGGGCCCCCCCCCC

CCCCCG

G

G

G

G

GGG

C10

GGCCCCCCCCCCC10

GGCCCCCCCCCCC10

C10

G10

GGGGGGGGGGCCCCCCCCCCC10

G11

GGGGCCCCCCCCCCC10

GGGGGGGGGGGCCCCCCCCCCC C11

G11

GGGGGGGGGGGCCCCCCCCCCC C11

G11

G11

MSS MSI

Stranddenaturation

Strandmisalignment

Replicationproceeds

PCNA

GGGGGGGGGGGCCC

Pol

GGGGGGGGGGGCCCCCCCCCCC10

G11

GG

MMR-proficient

MMR-deficient

Misalignedstrandexcised

Nextround ofreplication

EXO1

MLH1

PMS2

MSH6MSH2

The likelihood of vertical transmission of epimutations to the next generation was hotly debated for the next few years. In 2007, Hitchins etal.199 reported the verti-cal transmission of an MLH1 epimutation from mother to son that occurred in a

non-Mendelian pattern. In 2011, Hitchins etal.77 and Morak etal.78 reported families with autosomal dominant inheritance of MLH1 epimutation due to the presence of an underlying genetic alteration in the vicinity of the MLH1 gene, including a private promoter sequence variant and a large duplication encompassing MLH1 and neighbouring genes. An ancestral haplo-type underlying a dominant form of MLH1 epimutation has now been described in a collection of European families79. Thus, epi-mutations ofMLH1 have been shown to be heritable but with distinct Mendelian and non-Mendelian patterns depending on the underlying mechanism. MLH1 epimutation has been estimated to account for up to 10% of MMR gene mutation-negative cases of LS with MLH1deficient tumours and for a simi-lar proportion of incidental CRC cases with MLH1-hypermethylated tumours in patients below 60years ofage75.

In 2006, Chan etal.80 reported an auto-somal dominant LS family with a heritable MSH2 epimutation characterized by methyl-ation of the MSH2 promoter within normal tissues. In 2009, Ligtenberg etal.81 demon-strated that MSH2 epimutations in some LS families resulted from germline deletions of the terminal end of the adjacent gene, epithelial cell adhesion molecule (EPCAM; also known as TACSTD1). EPCAM deletions encompass the transcription termination signal, resulting in abnormal transcriptional elongation from EPCAM into MSH2. As EPCAM is expressed exclusively in epithelial tissues, the downstream methylation and silencing of MSH2 manifests only within epithelial tissues. This has clinical ramifica-tions in terms of the relative risk of cancer development. Screening for EPCAM dele-tions that are associated with MSH2 epimu-tation by MLPA has now been incorporated into routine molecular genetic testing forLS.

MMR function in normal cells. While knowledge of the MMR pathway in lower organisms aided the discovery of the human MMR genes, several biochemical experi-ments carried out in the decades since most notably, the complete reconstitution of the MMR reaction invitro82,83 have further expanded our understanding of this pathway. The MMR pathway repairs single-base-pair mismatches and small insertion or deletion loops (IDLs) that form when the polymerase attempts to replicate small repeat sequences84,85. MSH2 and MSH6 form hetero-dimers that recognize both of these types of errors, whereas an MSH2MSH3 hetero-dimer recognizes larger IDLs86. In addition to

providing lesion recognition specificity, the formation of the two MSH heterodimers pro-vides some functional redundancy87,88. This redundancy may underlie the distribution of MMR gene mutations identified in LS, in which MSH2 mutations are much more prevalent than MSH6 mutations66. Upon recognition of a single-base-pair mismatch in the presence of ATP, the MSH2MSH6 heterodimer undergoes a conformational change to form a sliding clamp that can dif-fuse along DNA89,90 and recruits a second heterodimer comprised of MLH1 and PMS2 (REFS28,91,92). MLH1 also pairs with MLH3, which again may provide some functional redundancy that could provide an explana-tion for the reduced frequency of mutations within PMS2 in LS compared with MLH1 (REF.93). Together, the MSH2MSH6 and MLH1PMS2 complexes regulate the down-stream steps of repair. The DNA processiv-ity factor proliferating cell nuclear antigen (PCNA) is also loaded near the site of the mismatch and can stimulate a latent endo-nuclease activity of PMS2, which introduces single-strand nicks into the daughter DNA strand94,95. These nicks facilitate the removal of a segment of the errant daughter strand that encompasses the sequence error, either by exonuclease1 (EXO1)-dependent exci-sion96,97,82 or by other mechanisms that may include polymerase-induced strand displacement synthesis98.

The mutator phenotype and cancer develop-ment. Hemminki etal.99 showed that loss of the remaining wild-type allele of the mutated MMR gene was a feature of LS tumours, which is consistent with Knudsons two-hit model of carcinogenesis100. The loss of function of both alleles in somatic cells results in loss of MMR function and the establishment of a mutator pheno-type101. The idea of a mutator phenotype was first proposed in 1974 by Loeb etal.102, who argued that defects in DNA replica-tion or repair would enhance the mutation frequency in cancer cells and increase the chances of a mutation in an important oncogene or tumour suppressor gene. Loss of MMR would result in DNA polymerase errors remaining intact. Upon the next round of DNA replication, an incorrectly inserted base or an extra repeat sequence bulge would be replicated and permanently fixed in the genome (FIG.2). This phenom-enon underlies the presence of MSI and the several hundred-fold increase in mutation frequency observed in MMR-deficient cells85. The recognition that microsatellite sequences were particularly susceptible

Figure 2 | Molecular mechanism of MSI. During replication of tandem repeat sequences (C10), DNA strand denaturation may occur, resulting in strands reannealing out of register (that is, becoming misaligned). This may lead to the addition (or subtraction) of one or more nucleotides during replication (G11). The extra nucleotide bulge is recognized by the mismatch repair (MMR) heterodimer mutS homologue 2 (MSH2)MSH6 which, together with MLH1 postmeiotic segregation increased 2 (PMS2), promotes excision of this portion of the errant daughter strand. However, in the absence of MMR activity, the extra nucleotide remains. During the next round of DNA replication, the G11 strand becomes the template strand. Successful replication of this errant strand results in permanent fixation of the additional nucleotide and the generation of a new allele (C11). EXO1, exonuclease 1; MSI, microsatellite instability; MSS, microsatellite stability; PCNA, proliferating cell nuclear antigen; Pol, DNA polymerase.





a Cellular phenotype

b Parallel intracellular molecular events

MMRhaploinsufficiency?

Somatic drivermutation?

MMR function intact(normal by IHC)

MSS

MMR usuallynormal by IHC

MSS

Heterozygous germlinemutation encodes defectiveprotein and predisposesto cancer

Wild-type allele encodesnormal protein

MMR protein functionintact

Carcinoma withmutator phenotype

Somatic loss of wild-typeallele with consequent MMRloss results in replicationerrors being unrepaired

Normal colonic epithelium

Accelerated polyp-to-carinoma sequence

Polyp

MMR genotypephenotype heterogeneity. The widespread implementation of MSI and genetic testing has resulted in a better under-standing of the disease phenotype among MMR mutation carriers. While heterozygous MMR gene mutations, by definition, cause LS, a more severe cancer phenotype known as constitutional mismatch repair deficiency (CMMR-D) syndrome is caused by biallelic mutations within the MMR genes. In 1999, Ricciardone etal.111 and Wang etal.112 concur-rently reported the paediatric onset of can-cers, including haematological malignancy and neurofibromatosis in two sets of siblings. Both were from consanguineous families and found to be homozygous for an MLH1 muta-tion. Subsequent case reports of individuals with biallelic germline MMR gene mutations have shown manifestations of CRC, haema-tological malignancies, features of neuro-fibromatosis, glioblastoma and urinary tract tumours, with age of onset as early as 6years. Non-consanguineous cases of CMMR-D syndrome have also been described with compound hetero zygous MMR gene muta-tions within MSH6 and PMS2, including point mutations and complex deletions113.

The complete inability to repair sequence errors introduced during DNA replication in individuals with CMMR-D syndrome explains the high penetrance and early onset of cancers. By contrast, patients with LS, who are carriers of heterozygous MMR gene muta-tions, are afforded some protection by the presence of one wild-type allele. The parents of patients with CMMR-D syndrome are also at risk of developing LS-associated can-cers, given that they are obligate carriers of a hetero zygous MMR mutation.

Disease expression in LS is variable, although particular phenotypic traits have been correlated with the MMR gene affected by a germline mutation (TABLE1). Cases with a germline mutation within MLH1 or MSH2 typically develop a classic LS phenotype that fulfils the Amsterdam I Criteria with a mean age of CRC onset of 4346years and with tumours exhibiting MSI, although cases with MSH2 mutations tend to develop more extra-colonic tumours114116. MSH2 mutations pre-dominate in the MuirTorre variant ofLS117.

By contrast, LS cases with germline muta-tions within MSH6 and PMS2 tend to develop an atypical LS phenotype. MSH6 mutation

carriers were found to be at the highest risk of endometrial cancer, with a mean age of onset above 50years118,119. Furthermore, the tumours did not consistently display MSI120,121, although instability specifically at mono-nucleotide repeat sequences was consistently observed122. PMS2 mutation carriers typically develop CRC with MSI but sometimes in the absence of a family history or at a later age of onset123126. The higher penetrance and earlier mean age of cancer onset associated with MLH1 and MSH2 mutations than with MSH6 and PMS2 mutations may be partly explained by the functional redundancy with MSH3 and MLH3 noted above, whereas loss of MLH1 or MSH2 expression results in the destabilization of their respective interaction partners. LS patients with EPCAM deletions were found by Kempers etal.127 to be at a similar risk of developing CRC as their counterparts with mutations within MSH2; however, they had a significantly reduced risk of endo metrial can-cer. This phenomenon was subsequently con-firmed by Lynch etal.128. Only patients with large EPCAM deletions that extended as far as the MSH2 promoter had an increased risk of endometrial cancer.

Variation in the LS phenotype has been observed even among cases with the same germline mutation, which suggests that this is influenced by additional factors. For example, Peltomki etal.116 reported a variable age of cancer onset among Finnish families carry-ing the same founder mutations of MLH1. Halvarsson etal.129 studied the pathological features of CRCs and adenomas from 12 members of 2 LS families, and found that the tumour morphology and IHC expression of -catenin (the nuclear localization of which is a hallmark of cancers driven by chronic activation of the WNT signalling pathway, for example, owing to APC mutation) varied extensively within families and even between synchronous or metachronous CRCs from the same individual. Proximal CRCs more frequently showed poor tumour differentia-tion, expanding growth pattern and tumour-infiltrating lymphocytes, whereas distal CRCs often lacked distinct LS-associated morpho-logical features. Interestingly, glioblastoma and kidney cancers in patients with either LS or CMMR-D syndrome are more likely to be microsatellite stable, despite loss of the relevant MMR protein as determined by IHC and the finding of MSI in the CRC and other tumours from the same individuals, suggest-ing these LS tumours develop through dis-tinct pathways130132. The nature of the genes in which somatic mutations accumulate as a consequence of the mutator phenotype is likely to contribute to some of the variation in

Box 2 | Screening and management

Prediction of cancer risk is partially defined by clinical pathology findings in an extended pedigree, coupled with the presence or absence of a mismatch repair (MMR) gene mutation.

Colorectal cancer (CRC) prevention requires colonoscopy, which is initiated at age 18 and repeated biennially through age 40 and then annually thereafter. Jrvinen etal.172 evaluated the efficacy of colonoscopic surveillance in a 15-year controlled study, which investigated 133 at-risk family members who underwent colonoscopy at 3-year intervals and compared them with 119 at-risk family members who declined surveillance. Those who underwent colonoscopy had a 62% 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.

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 and bilateral salpingo-oophorectomy is performed at approximately age 35 (or when pregnancy is no longer an option) in germline MMR gene mutation carriers. This was first recommended by Lynch as early as 1978 (REF.173). In 2006, Schmeler etal.174 reported strong evidence supporting the role of prophylactic hysterectomy and bilateral salpingo-oophorectomy in women with Lynch syndrome (LS)-associated germline mutations. Ketabi etal.175 indicated that based on the study of 19,334 women years of endometrial cancer surveillance in LS patients, such surveillance should be targeted at MMR gene mutation carriers.

Upper urological cancer involving urine cytology (which requires a highly experienced pathologist) is coupled with ultrasound scans annually starting at age 30.

Owing to evidence of increased synchronous and metachronous CRCs in LS in which the risk is approximately2030%formetachronouscancer10yearsafterinitialCRCiflessthanasubtotalcolectomy is performed in 1996 Church176 and Lynch177 suggested a role for prophylactic colectomy in LS patients. This must include genetic counselling and referral to one or more colorectal surgeons for their opinions; most importantly, this must involve LS-confirmed patients who have declined further colonoscopy. Scaife and Rodriguez-Bigas178 gave further support for both gynaecological and colorectal prophylactic surgeries in 2003. In 2010, Natarajan etal.179 provided data that showed the importance of extended colectomy versus limited resection in LS patients with CRC.

It is clear that in order for a clinical and prophylactic surgical management programme to be successful, there must be full patient compliance.

Identification of a deleterious germline MMR gene mutation provides a definitive diagnosis.




tumour pathology. However, the reasons that certain tissues show a propensity for cancer in the context of both LS and CMMR-D syndrome remain poorly understood.

Diagnosis and managementDifferential diagnosis. Diagnosis of LS is now based on a combination of clinical phe-notype, routine tumour pathology and/or genetic screening practices. The Revised Bethesda Guidelines (BOX1) remain the cur-rent clinical criteria for the identification of patients with an increased likelihood of hav-ing LS27. However, there is now much evi-dence for the efficacy and cost effectiveness of universal reflex testing for MSI and/or IHC for loss of MMR protein expression of all CRCs133135. The US Multi-Society Task Force on Colorectal Cancer has issued guidelines that call for the use of reflex testing135 while noting the difficulty of implementation in the clinical setting.

The identification of MSI in LS-associated tumours and the use of IHC to detect the expression of MMR proteins have revolution-ized the diagnosis of LS. Combinatorial IHC testing of all four MMR proteins can provide an indication of the specific MMR gene that is most likely to contain a pathogenic germ-line mutation135. For instance, specific loss of PMS2 or MSH6 protein expression impli-cates a germline mutation within the PMS2 or MSH6 genes, respectively. However, dual loss of MLH1 and PMS2 protein expression would suggest a germline mutation within MLH1, as the PMS2 protein is destabilized in the absence of MLH1. Similarly, IHC loss of both MSH2 and MSH6 staining implies a germline mutation within MSH2, as MSH6 is unstable in the absence of MSH2. A move towards more population-based testing of incident cancers came with the revision of the Bethesda Guidelines in 2004. In 2008, Hampel etal.133 studied 500 consecutive patients with CRC and showed that 18 (3.5%) harboured MMR gene mutations, which accounted for 1 in 35 patients with CRC. The demonstration that LS was more prevalent than previously assumed provided a strong rationale for the implementation of more widespread reflex testing of every incidental case of CRC for the possibility ofLS.

Although MSI is a characteristic feature of LS-associated cancers, approximately 12% of sporadic CRCs also exhibit MSI, which poses an additional challenge to identifying LS-associated cancers3436. These sporadic tumours share similar clinicohistopatho-logical features with cases of LS-associated CRC, including a tendency to develop in the proximal colon, and poorly differentiated and

mucinous histology136. MSI+ sporadic CRCs were found to be caused primarily by somati-cally acquired hypermethylation of both alleles of the MLH1 promoter, with resultant loss of MLH1 protein expression137139, which was closely associated with the presence of the oncogenic BRAF-V600E mutation140. Given the comparative rarity of these two molecular events in LS tumours, BRAF-V600E mutation or MLH1 hypermethylation testing has been used in reflex testing of CRC to differentiate LS-associated CRC from the more common sporadic MSI+ counterparts141,142. However, MLH1 methylation testing in tumours will not distinguish LS cases with a constitutional

MLH1 epimutation or methylation as a sec-ond hit during tumorigenesis from sporadic MSI+cases.

A definitive diagnosis of LS is provided by the identification of a deleterious germ-line mutation or epimutation affecting one of the associated MMR genes. LS screening and management recommendations are discussed in BOX2. Molecular genetics-based diagnosis has also become crucial in providing a differ-ential diagnosis of LS, as there are additional cancer syndromes with clinically overlap-ping features, including FAP, mutY homo-logue (MYH)-associated polyposis (MAP) and familial CRC type X (BOX3). LS, FAP

Box 3 | Other syndromes involved in differential diagnosis

MYH-associated polyposis (MAP)In 2002, Al-Tassan etal.180 reported biallelic germline mutations of the mutY homologue (MYH) base excision repair gene in a family with multiple colorectal adenomas and colorectal cancer (CRC) 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 study, Morak etal.181 found a great deal of variability in the clinical phenotype of monoallelic and biallelic MUTYH mutation carriers. It is estimated that monoallelic MUTYH mutation carriers have an approximately 2.5-fold increased risk of CRC compared with the general population182,183.

Morak etal.184 noted that the highly variable phenotype of MAP may overlap with the Lynch syndrome (LS) phenotype. As an illustration of this, they described one patient with biallelic MUTYH mutations who manifested CRC, urothelial carcinoma and sebaceous gland carcinoma. LS had been suspected because of a positive family history of CRC, as well as high levels of microsatellite instability (MSI) and immunohistochemistry (IHC) results that showed mutS homologue 2 (MSH2) 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.

Familial adenomatous polyposis (FAP)Variants include: Gardners syndrome that contains extracolonic phenotypic aspects;

Turcots syndrome that is associated with medulloblastoma (as opposed to the LS variant of Turcots syndrome, which is associated with glioblastoma);

Attenuated FAP (AFAP), which can pose considerable diagnostic problems because its phenotype overlaps with that of LS, as colonic adenomas are fewer, more frequently flat and more likely to 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.

Familial colorectal cancer type X (FCC-X)FCC-X comprises families that meet the Amsterdam Criteria for diagnosis of LS but that lack evidence of mismatch repair (MMR) defects.

Clinical differences between LS and FCC-X are as follows. Lindor etal.186 found only a twofold increased risk for CRC with an older average age of CRC onset(61years)inFCCXcomparedwithLS(49years).

In a German study187, two-thirds of the tumours in FCC-X were left-sided, which is the reverse of CRC in LS. There were fewer synchronous and metachronous CRCs than in LS, and there was a greater adenoma:carcinoma ratio and a tendency towards more adenomas, which suggests slower adenomacarcinoma progression.

In a 2005 study of 100 individuals from 25 families fulfilling the Amsterdam Criteria188, 40% showed normal DNA MMR and, in these individuals, 89% of the tumours were left-sided. None showed tumour-infiltrating lymphocytes, whereas half of the LS-associated CRCs showed this pathology phenomenon.

In a 2007 study189, FCC-X cases were found to be less likely to have mucinous tumours.The recent discovery by Nieminen etal. of the role of germline mutations within the bone

morphogeneticproteinreceptortype1A(BMPR1A) gene190 and the ribosomal protein S20 (RPS20) gene191 in FCC-X families has provided new evidence of the genetic heterogeneity among FCC-X families and the distinction of FCC-X from LS.

A summary of studies on FCC-X was published by Lindor192 in 2009.





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knowledge that she was positive for the family MLH1 mutation (exon2; 1-bp inser-tion mutation at codon64 that results in frameshift and stop codon at position91) enabled her to work closely with her physi-cians to develop a cancer screening and prevention plan. Conversely, individualIII-5 was found to be negative for the MLH1 mutation, meaning that all of his descend-ants are automatically negative for this mutation; consequently, they are at general population risk for LS-associated cancers and do not require intensive screening.

Some individuals choose not to be tested for the family mutation and, therefore, their cancer risk remains defined by the family history. For example, individualIV-18 is at a 50% risk of inheriting the mutation (FIG.4) but has chosen not to be tested. Studies have shown that individuals who choose not to know their genetic result have a higher rate of anxiety than those who tested positive147. Genetic counselling implications for LS are discussed by Eliezer etal.148 and Aktan-Collan etal.149; considerations regarding genetic counselling and testing are presented in BOX4.

Pharmacogenetics and chemopreventionGenetic factors have recently been shown to affect cancer therapeutics150. The relatively new field of hereditary cancer therapeu-tics has potential to influence cancer care through the elucidation of relationships between a mutation and response to specific chemotherapy and/or chemoprevention.

Continued pharmacogenetics research might yield a more tailored treatment for patients with LS based on a better under-standing of the impact of a patients germline mutation on therapeutic outcome. Basic science research has shown that the MMR pathway is also involved in a global response to certain forms of DNA damage that ulti-mately result in the activation of cell cycle checkpoints and cell death151,152. Karran and Marinus153 first showed that, in addition to hypermutability, MMR-defective E.coli also display an increased resistance to the cytotoxic effects of the DNA alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG). Kat etal.154 later determined that an isolated lymphoblastoid cell line that survived the effects of MNNG lacked MMR activity, whereas the MNNG-sensitive parental cell line maintained MMR func-tion. Numerous studies have shown that the MMR proteins recognize lesions created by MNNG and other DNA damaging agents; however, in the attempt to repair these lesions, secondary DNA damage, including

double-strand breaks, is generated, which results in checkpoint activation and, ultimately, cell death151,152,155.

The relevance of this DNA damage response function in tumorigenesis is unclear; however, it is likely to be an impor-tant consideration for how MMR-deficient tumours respond to chemotherapy. For example, 5-fluorouracil treatment may be less effective in MMR-deficient CRC156,157, although how this relates specifically to LS-associated CRC is not yet known158. Preclinical studies have shown that treat-ment of cells with 5-fluorouracil leads to the incorporation of 5-fluoro-2-deoxyuridine into DNA, which can mispair with gua-nine and be recognized by MSH2MSH6 (REF.159). In addition, 5-fluorouracil also decreases thymidine nucleotide pools, leading to increased incorporation of deoxyuridine into DNA. This event leads to the production of UG mispairs, which also stimulate an MMR-dependent DNA damage response. However, prolonged exposure to 5-fluorouracil may also lead to lesions that are repaired by the base excision repair (BER) pathway which, particularly in combination with agents that block down-stream steps of BER, can result in MMR-independent cytotoxicity160. Although MSI cancers may be less susceptible to 5-fluoro-uracil, some studies have indicated an improved response in tumours with MSI to another key CRC chemotherapeutic agent, irinotecan161163. An improved understand-ing of the role of the MMR pathway in response to topo isomerase stabilizers such as irinotecan will be beneficial, as the selec-tive benefit of this treatment is not entirely clear164,165. More recent studies suggest syn-thetically lethal approaches that capitalize

on MMR deficiency in LS-associated tumours by treating these cancers with inhibitors of oxidative DNA damage repair166,167. Martinetal.166,167 showed that MSH2-deficient cancer cells were differen-tially susceptible to RNA interference-based knockdown of DNA polymerase-, which is involved in the repair of 8-oxoguanine. Similarly, they showed that MLH1-deficient cells were sensitive to loss of DNA polymerase-, an enzyme involved in the repair of these same lesions in mitochon-dria. These studies suggest that MMR has a back-up role in the repair of oxidative dam-age and that loss of both repair pathways leads to an overload of unrepaired lesions that affects cell viability.

An initial study by Burn etal.168 found aspirin to be ineffective in preventing colonic adenomas and CRC in LS. However, when follow-up time was increased, the chemopreventive effect on polyps and CRC began to emerge169. These findings are consistent with observations in mice, which showed that aspirin and nitric oxide-donating aspirin (an aspirin derivative) increase lifespan and delay tumour onset in an intestinal tumour model withMSI170.

ConclusionWe have catalogued the salient clinical, pathological and molecular aspects of LS. The story of LS is exemplary in illustrating how the combination of clinical observa-tions and basic scientific discoveries was integral to its evolution. The predisposi-tion of patients with LS to develop multiple primary cancers in a single family and the genotypic heterogeneity of LS have, and will continue to, advance the elucidation of carcinogenictheory.

Box 4 | Genetic counselling and testing

Detailed cancer family history with pathology documentation is required whenever possible.

Involvement of a knowledgeable physician, trained genetic counsellor or centre of medical genetics expertise is needed.

Family history should be extended to four generations whenever possible and should be inclusive of both parents, progeny, maternal and paternal aunts and uncles, grandparents and their siblings and descendants and, whenever possible, great-grandparents.

Trust, compassion and confidentiality are prerequisites to success.

Recognition of a hereditary cancer syndrome in a family poses a challenge in reaching and communicating with relatives. In most genetic counselling settings, information is limited and forthcoming only for the probands nuclear family. More-distant relatives are rarely evaluated, which is unfortunate because DNA testing and surveillance screening could be lifesaving in patients with Lynch syndrome (LS) and other hereditary cancer syndromes. Lynch etal.193 summarized the type of information technology that can be effectively used for the dissemination of susceptibility information and cancer risk knowledge to members of extended families. This involves an effective use of telephone and video counselling, which has been found to be useful in contacting and counselling distant relatives.

A detailed educational programme for the entire family membership is crucial.




Henry T.Lynch, Carrie L.Snyder and Trudy G.Shaw are at the Department of Preventive Medicine and Public Health, Creighton University, 2500 California Plaza,

Omaha, Nebraska 68178, USA.

Christopher D.Heinen is at the Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut

060303101, USA.

Megan P.Hitchins is at the Department of Medicine (Oncology), Stanford Cancer Institute,

Stanford University, Grant Building S169, 1291 Welch Road, Stanford, California 94305, USA.

Correspondence to H.T.L. e-mail: [email protected]

doi:10.1038/nrc3878 Published online 12 February 2015

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2. Mendel,G. Versuche ber Pflanzen-Hybriden. Verh. Naturforsch. Ver. Brnn 4, 347 (1866); English translation available in J.R.Hortic. Soc. 26, 132 (1901).

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5. Lynch,H.T. & Krush,A.J. Cancer family G revisited: 18951970. Cancer 27, 15051511 (1971).

6. Douglas,J.A. etal. History and molecular genetics of Lynch syndrome in Family G: a century later. JAMA 294, 21952202 (2005).

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8. Boland,C.R. & Troncale,F.J. Familial colonic cancer without antecedent polyposis. Ann. Intern. Med. 100, 700701 (1984).This was the first paper to suggest the term LS for CFS.

9. Tempero,M.A., Jacobs,M.M., Lynch,H.T., Graham,C.L. & Blotcky,A.J. Serum and hair selenium levels in hereditary nonpolyposis colorectal cancer. Biol. Trace Element Res. 6, 5155 (1984).

10. Kalady,M.F., Kravochuck,S., LaGuardia,L., OMalley,M. & Church,J.M. Adenomas in Lynch syndrome: dont be fooled by the non in hereditary nonpolyposis colorectal cancer. Fam Cancer 12 (Suppl. 2), 53 (2013).This is a recent study demonstrating an excess of adenomas in LS.

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14. Lanspa,S.J. etal. Surveillance in Lynch syndrome: how aggressive? Am. J.Gastroenterol. 89, 19781980 (1994).This paper calls for complete colonoscopy in surveillance for LS-associated CRCs.

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19. Kastrinos,F. etal. Risk of pancreatic cancer in families with Lynch syndrome. JAMA 302, 17901795 (2009).

20. Win,A.K. etal. Colorectal and other cancer risks for carriers and noncarriers from families with a DNA mismatch repair gene mutation: a prospective cohort study. J.Clin. Oncol. 30, 958964 (2012).

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24. Vasen,H.F.A., Watson,P., Mecklin,J.-P. & Lynch,H.T. & ICG-HNPCC. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC. Gastroenterology 116, 14531456 (1999).This is the source of the revised Amsterdam II Criteria.

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29. Farabaugh,P.J., Schmeissner,U., Hofer,M. & Miller, J.H. Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli. J.Mol. Biol. 126, 847857 (1978).

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31. Levinson,G. & Gutman,G.A. High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12. Nucleic Acids Res. 15, 53235338 (1987).

32. Modrich,P. Methyl-directed DNA mismatch correction. J.Biol. Chem. 264, 65976600 (1989).

33. Strand,M., Prolla,T.A., Liskay,R.M. & Petes,T.D. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365, 274276 (1993).

34. Aaltonen,L.A. etal. Clues to the pathogenesis of familial colorectal cancer. Science 260, 812816 (1993).This was the first study to link MSI with LS.

35. Ionov,Y.M., Peinado,M.A., Malkhosyan,S., Shibata,D. & Perucho,M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363, 558561 (1993).

36. Thibodeau,S.N., Bren,G. & Schaid,D. Microsatellite instability in cancer of the proximal colon. Science 260, 816819 (1993).

37. Parsons,R. etal. Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75, 12271236 (1993).This is an early description of hypermutability in MSI.

38. Jass,J.R. Natural history of hereditary non-polyposis colorectal cancer. J.Tumor Marker Oncol. 10, 6571 (1995).

39. Lengauer,C., Kinzler,K.W. & Vogelstein,B. Genetic instabilities in human cancers. Nature 396, 643649 (1998).

40. Jass,J.R. etal. Pathology of hereditary non-polyposis colorectal cancer. Anticancer Res. 14, 16311634 (1994).This was the first comprehensive listing of pathological features found in LS-associated CRCs.

41. Watson,P. etal. Colorectal carcinoma survival among hereditary nonpolyposis colorectal cancer family members. Cancer 83, 259266 (1998).

This study documents the survival advantage of LS-associated CRC compared with sporadic CRC.

42. Deschoolmeester,V. etal. Tumor infiltrating lymphocytes: an intriguing player in the survival of colorectal cancer patients. BMC Immunol. 11, 19 (2010).

43. Hamilton,S.R. in Cancers of the Colon and Rectum: A Multidisciplinary Approach to Diagnosis and Management Ch. 6 (eds Benson, A. B, Chakravarthy, A., Hamilton, S. R., Sigurdson, E. & Thomas, C. Jr) 115128 (Demos Medical Publishing, 2014).This book chapter gives an up-to-date understanding of pathological features in CRC.

44. Bartley,A.N. etal. Colorectal adenoma stem-like cell populations: associations with adenoma characteristics and metachronous colorectal neoplasia. Cancer Prev. Res. 6, 11621170 (2013).

45. Peltomki,P. etal. Genetic mapping of a locus predisposing to human colorectal cancer. Science 260, 810812 (1993).This was the first study to show a germline mutation as a cause of LS.

46. Lindblom,A., Tannergard,P., Werelius,B. & Nordenskjold,M. Genetic mapping of a second locus predisposing to hereditary nonpolyposis colorectal cancer. Nature Genet. 5, 279282 (1993).This study links LS to a second locus.

47. Fishel,R. etal. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75, 10271038 (1993).

48. Leach,F.S. etal. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 75, 12151225 (1993).References 47 and 48 document the discovery of the connection between MSH2 and LS.

49. Papadopoulos,N. etal. Mutation of a mutL homolog in hereditary colon cancer. Science 263, 16251629 (1994).

50. Bronner,C.E. etal. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary nonpolyposis colon cancer. Nature 368, 258261 (1994).References 49 and 50 document the discovery of the connection between MLH1 and LS.

51. Nicolaides,N.C. etal. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371, 7580 (1994).This study was the first to link the PMS genes to LS.

52. Liu,T. etal. The role of hPMS1 and hPMS2 in predisposing to colorectal cancer. Cancer Res. 61, 77987802 (2001).

53. Miyaki,M. etal. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nature Genet. 17, 271272 (1997).This paper shows the involvement of MSH6 in LS.

54. Lipkin,S.M. etal. MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability. Nature Genet. 24, 2734 (2000).This paper connects MLH3 with MSI.

55. Wu,Y. etal. A role for MLH3 in hereditary nonpolyposis colorectal cancer. Nature Genet. 29, 137138 (2001).

56. Liu,H.X. etal. The role of hMLH3 in familial colorectal cancer. Cancer Res. 63, 18941899 (2003).

57. Korhonen,M.K., Vuorenmaa,E. & Nystrm,M. The first functional study of MLH3 mutations found in cancer patients. Genes Chromosomes Cancer 47, 803809 (2008).

58. Ou,J. etal. Biochemical characterization of MLH3 missense mutations does not reveal an apparent role of MLH3 in Lynch syndrome. Genes Chromosomes Cancer 48, 340350 (2009).

59. Rasmussen,L.J. etal. Pathological assessment of mismatch repair gene variants in Lynch syndrome. Hum. Mutat. 33, 16171625 (2012).

60. Nystrom-Lahti,M. etal. Founding mutations and Alu-mediated recombination in hereditary colon cancer. Nature Med. 1, 12031206 (1995).

61. Marshall,B., Isidro,G. & Boavida,M.G. Insertion of a short Alu sequence into the hMSH2 gene following a double cross over next to sequences with chi homology. Gene 174, 175179 (1996).

62. Wijnen,J. etal. MSH2 genomic deletions are a frequent cause of HNPCC. Nature Genet. 20, 326328 (1998).This study shows the use of Southern blotting to identify genomic rearrangements in MSH2.

63. Yan,H. etal. Conversion of diploidy to haploidy: individuals susceptible to multigene disorders may now be spotted more easily. Nature 403, 723724 (2000).

64. Charbonnier,F. etal. MSH2 in contrast to MLH1 and MSH6 is frequently inactivated by exonic and promoter rearrangements in hereditary nonpolyposis colorectal cancer. Cancer Res. 62, 848853 (2002).

65. Gille,J.J. etal. Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br. J.Cancer 87, 892897 (2002).




66. Plazzer,J.P. etal. The InSiGHT database: utilizing 100years of insights into Lynch syndrome. Fam. Cancer 12, 175180 (2013).

67. Wagner,A. etal. Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene. Am. J.Hum. Genet. 72, 10881100 (2003).

68. Lynch,H.T. etal. A founder mutation of the MSH2 gene and hereditary nonpolyposis colorectal cancer in the United States. JAMA 291, 718724 (2004).This paper describes the discovery of a widespread founder mutation in the United States.

69. Clendenning,M. etal. Long-range PCR facilitates the identification of PMS2-specific mutations. Hum. Mutat. 27, 490495 (2006).

70. Vaughn,C.P. etal. Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes. Hum. Mutat. 31, 588593 (2010).

71. Vaughn,C.P., Baker,C.L., Samowitz,W.S. & Swensen,J.J. The frequency of previously undetectable deletions involving 3 exons of the PMS2 gene. Genes Chromosomes Cancer 52, 107112 (2013).

72. Wimmer,K. & Wernstedt,A. PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference. Methods Mol. Biol. 1167, 289302 (2014).

73. Pritchard,C.C. etal. ColoSeq provides comprehensive Lynch and polyposis syndrome mutational analysis using massively parallel sequencing. J.Mol. Diagn. 14, 357366 (2012).

74. Gazzoli,I., Loda,M., Garber,J., Syngal,S. & Kolodner, R.D. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor. Cancer Res. 62, 39253928 (2002).

75. Hitchins,M.P. The role of epigenetics in Lynch syndrome. Fam. Cancer 12, 189205 (2013).This is a recent review of the role of epigenetics in LS.

76. Hitchins,M. etal. MLH1 germline epimutations as a factor in hereditary nonpolyposis colorectal cancer. Gastroenterology 129, 13921399 (2005).This was the first description of MLH1 epimutations in LS.

77. Hitchins,M. etal. Dominantly inherited constitutional epigenetic silencing of MLH1 in a cancer-affected family is linked to a single nucleotide variant within the 5UTR. Cancer Cell 20, 200213 (2011).This paper describes a dominantly inherited epimutation in MLH1.

78. Morak,M. etal. Biallelic MLH1 SNP cDNA expression or constitutional promoter methylation can hide genomic rearrangements causing Lynch syndrome. J.Med. Genet. 48, 513519 (2011).

79. Kwok,C.T. etal. The MLH1 c.-27C>A and c.85G>T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype. Eur. J.Hum. Genet. 22, 617624 (2014).

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81. Ligtenberg,M.J.L. etal. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3 exons of TACSTD1. Nature Genet. 41, 112117 (2009).This paper describes the effect of EPCAM deletions on MSH2.

82. Zhang,Y. etal. Reconstitution of 5-directed human mismatch repair in a purified system. Cell 122, 693705 (2005).

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87. de Wind,N. etal. HNPCC-like cancer predisposition in mice through simultaneous loss of Hsh3 and Msh6 mismatch-repair protein functions. Nature Genet. 23, 359362 (1999).

88. Edelmann,W. etal. The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression. Cancer Res. 60, 803807 (2000).

89. Gradia,S., Acharya,S. & Fishel,R. The human mismatch recognition complex hMSH2hMSH6

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Milestones of Lynch Syndrome

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