Review

Familial gastric cancer: genetic susceptibility, pathology, and implications for management

Carla Oliveira*, Hugo Pinheiro*, Joana Figueiredo, Raquel Seruca, Fátima Carneiro

Familial gastric cancer comprises at least three major syndromes: hereditary diff use gastric cancer, gastric Lancet Oncol 2015; 16: e60–70 adenocarcinoma and proximal polyposis of the stomach, and familial intestinal gastric cancer. The risk of development *These authors contributed of gastric cancer is high in families aff ected b-y these syndromes, but only hereditary diff use gastric cancer is equally genetically explained (caused by germline alterations of CDH1, which encodes E-cadherin). Gastric cancer is also Ipatimub-Institute of associated with a range of several cancer-associated syndromes with known genetic causes, such as Lynch, Li- Molecular Pathology and Immunology & Instituto Fraumeni, Peutz-Jeghers, hereditary breast–ovarian cancer syndromes, familial adenomatous polyposis, and juvenile Instituto de Investigação e polyposis. We present contemporary knowledge on the genetics, pathogenesis, and clinical features of familial gastric Inovação em Saúde, cancer, and discuss research and technological developments, which together are expected to open avenues for new (C Oliveira PhD, H Pinheiro PhD, genetic testing approaches and novel therapeutic strategies. J Figueiredo PhD, R Seruca MD, Prof F Carneiro MD), and Department of Pathology and Introduction expected to improve the identifi cation of novel causative Oncology, Faculty of Medicine Gastric cancer aff ects nearly 1 million individuals every genetic events, which will aff ect genetic testing and the (C Oliveira, R Seruca, F Carneiro), year, 70–85% of whom die within 5 years of diagnosis, management of families with a high frequency of gastric University of Porto, Porto, Portugal; and Centro 1 making it the third most lethal cancer worldwide. The cancers. Hospitalar S João, Porto, high mortality associated with gastric cancer (nearly HDGC was the fi rst of the hereditary gastric cancer Portugal (F Carneiro) 800 000 deaths per year) is mainly a result of late syndromes to be recognised, initiated by inherited Correspondence to: diagnosis, and limited therapeutic options. The two causative mutations in the E-cadherin gene (CDH1).5 Prof Fátima Carneiro, Institute of major subtypes—diff use and intestinal2—are Mutations in the alpha-E-catenin gene (CTNNA1) have Molecular Pathology and Immunology, University of characterised by distinct epidemiological, morphological, been identifi ed as an additional genetic cause of HDGC Porto, Rua Dr Roberto Frias s/n, 6 and molecular features. Although most gastric cancers (table 1). Several patients with early-onset diff use-type 4200-465, Porto, Portugal are sporadic, aggregation within families occurs in gastric cancers with no apparent family history have [email protected] roughly 10% of cases. In regions where the incidence of been found to be carriers of CDH1 germline mutations, gastric cancer is low, most familial cases are probably which supports the role of CDH1 germline defi ciency in due to heritable pathogenic mutations that increase risk disease initiation and helped to identify de-novo HDGC from birth.3,4 Truly hereditary cases are thought to families.7,8 In HDGC families, lobular breast cancer is the account for 1–3% of the global burden of gastric cancer second most frequent type of neoplasia.9 Although and comprise at least three main syndromes: hereditary colorectal cancer also arises as part of the tumour diff use gastric cancer (HDGC), gastric adenocarcinoma spectrum, whether the risk in HDGC families is higher and proximal polyposis of the stomach (GAPPS), and than that in the general population remains unclear.9 familial intestinal gastric cancer (FIGC). A genetic basis Congenital malformations, such as cleft lip or cleft palate, has been found in only around 40% of families aff ected occur in some CDH1-mutation-driven HDGC families,10 by HDGC. The identifi cation of inherited factors among but are not a defi ning clinical characteristic because of individuals with family histories of gastric cancer is their rarity. However, clinicians should collect infor- therefore a crucial step for early diagnosis and disease mation about them when counselling at-risk families.11 management. According to published data, the penetrance of diff use In this Review, we discuss the available knowledge on gastric cancer in mutation carriers reaches more than hereditary gastric cancer and cancer-associated 80% in both men and women by 80 years of age, and the syndromes from which gastric cancer can arise, with the probability of women developing lobular breast cancer is aim of clarifying questions relevant for the translation of 60%.9 The combined risk of gastric cancer and breast basic research into clinical practice. cancer in women has been calculated to be 90% at 80 years.12 Eff orts are being made to calculate the Hereditary gastric cancer syndromes penetrance of gastric cancer in a larger number of HDGC The recognition of familial aggregation (eg, high CDH1 mutant families. occurrence in siblings or off spring) is the fi rst step GAPPS was identifi ed in 2012, and is characterised by towards the identifi cation of a disease with a genetic the autosomal dominant transmission of fundic gland component and is clinically useful. Family history, polyposis (including dysplastic lesions or intestinal-type histological classifi cation, and age at onset of disease gastric adenocarcinoma, or both) that are restricted to the (<45 years) are used to guide genetic testing and clinical proximal stomach with no evidence of colorectal or surveillance in the context of a particular gastric cancer duodenal polyposis or other hereditary gastrointestinal hereditary syndrome. The advent of new, fast, and cancer syndromes (table 1).13 It is characterised by incom- inexpensive massive parallel sequencing technologies is plete penetrance, and some elderly obligate carriers have www.thelancet.com/oncology Vol 16 February 2015 e60 Review

Clinical criteria Genetic screening Alterations described Hereditary diff use Two or more cases of gastric cancer, one confi rmed case of diff use gastric Sequencing of CDH1 coding Mutations throughout the gastric cancer cancer in someone younger than 50 years; sequences; CDH1 gene and deletions Three or more confi rmed diff use gastric cancer cases in fi rst-degree or Multiplex ligation-dependent mainly implicating fl anking second-degree relatives, independent of age of onset; probe amplifi cation (large CDH1 untranslated regions; Diff use gastric cancer before age 40 years without a family history; rearrangements); Personal or family history of diff use gastric cancer and lobular breast Sequencing of CTNNA1 coding One germline truncating cancer, one of which must be diagnosed before age 50 years sequences mutation in CTNNA1 Gastric Gastric polyps restricted to the body and fundus with no evidence of No screening available No inherited inherited adenocarcinoma and colorectal or duodenal polyposis; mutations so far proximal polyposis More than 100 polyps carpeting the proximal stomach in the index case or of the stomach more than 30 polyps in a fi rst-degree relative of another case; Mainly fundic gastric polyps, some with regions of dysplasia (or a family member with either dysplastic fundic gastric polyps or gastric adenocarcinoma); Autosomal dominant pattern of inheritance; Exclusions include other heritable gastric polyposis syndromes and use of proton-pump inhibitors* Familial intestinal Two or more cases of gastric cancer in fi rst-degree or second-degree No screening available No inherited inherited gastric cancer relatives, with at least one confi rmed case of intestinal histology in mutations so far someone younger than 50 years; Three or more confi rmed cases of intestinal gastric cancer in fi rst-degree or second-degree relatives, independent of age

*Proton-pump inhibitors can induce a phenotype similar to that of gastric adenocarcinoma and proximal polyposis of the stomach. Patients taking these drugs should undergo a repeat endoscopy off -therapy to confi rm diagnosis of gastric adenocarcinoma and proximal polyposis of the stomach.

Table 1: Clinical criteria, recommended screening, and inherited alterations of familial gastric cancer syndromes

normal endoscopies. The genetic cause has yet to be Li-Fraumeni syndrome encompasses several tumour identifi ed.13,14 types that develop generally before 45 years of age because FIGC is characterised mainly by intestinal gastric of inherited TP53 mutations,19,20 including early-onset cancer (table 1). An autosomal dominant inheritance gastric cancer. The frequency of gastric cancer in families pattern has been noted in many families with intestinal- carrying TP53 mutations ranges from 1·8% to 4·9%.21,22 type gastric cancer.15 In practical terms, FIGC should be 40% of families with TP53 mutations present with at least thought of as a potential diagnosis when histopathological one gastric cancer (mean age 43 years, median age 36; age reports refer to intestinal-type adenocarcinoma range 24–74 years), with an excess of early onset gastric segregating within families without gastric polyposis. cancers.22 These data support periodic screening with The genetic cause is unknown, and only a few oesophagogastroduodenoscopy in young carriers of TP53 recommendations have been advanced for the clinical germline mutations, and particularly in Li-Fraumeni management of patients at risk of FIGC.16 syndrome families in which at least one case of gastric cancer has been reported.22 Data for the histological type Associations with other hereditary cancer of gastric cancer are insuffi cient to associate a particular syndromes histotype with Li-Fraumeni syndrome. Importantly, Gastric cancer has been identifi ed as part of the tumour gastric cancer can occur in Li-Fraumeni syndrome spectrum in several other hereditary cancer syndromes, families without TP53 mutations (two of 73 families and thus the risk of it should be taken into account in studied), suggesting that incidence could be independent patients with these syndromes. Lynch syndrome is a from the presence of germline TP53 mutation.21 highly penetrant colorectal cancer syndrome bearing a Familial adenomatous polyposis is caused by APC molecular phenotype of microsatellite instability that is germline mutations and is characterised by the caused by mutations in one of the mismatch repair development of more than 100 colonic and rectal genes MLH1, MSH2, MSH6, PMS1, PMS2, or EPCAM.17 adenomas and early development of colorectal cancer.23,24 The frequency of gastric cancer in carriers of Lynch Roughly 8% of patients have attenuated disease, in syndrome mutations has been estimated at 1·6%, and which fewer adenomas are present and disease onset is intestinal-type disease is the main histotype.18 The risk of later.24 Adenomas also develop in the upper development of gastric cancer was 4·8% in patients with gastrointestinal tract, especially in the duodenum, and, germline defects of MLH1 and 9% in those with if untreated, can progress to malignant disease in germline defects of MSH2. Surveillance with oesophago- roughly 5% of cases.24 A grading system was developed gastroduodenoscopy is necessary in patients with Lynch for duodenal polyps in familial adenomatous polyposis syndrome who carry mutations in mismatch repair for assessment of disease severity.25 In the stomach, genes.18 gastric fundic gland polyps and adenomas in the antrum e61 www.thelancet.com/oncology Vol 16 February 2015 Review

can also occur.24,26 Fundic gland polyps are reported in in BRCA1 or BRCA2 carriers is 1·69 (95% CI 1·21–2·38), around 88% of people in familial adenomatous polyposis which is higher than the relative risk for pancreatic, families,27 and occur more frequently in those with prostate, and colorectal cancer. No details of pathological attenuated disease.28 Gastric adenocarcinomas in familial changes were provided in the studies included in the adenomatous polyposis are generally thought to arise meta-analysis, and whether a histological subtype of from the adenomas, and evidence suggests that fundic gastric cancer predominates in BRCA-associated gland polyps can transform into adenocarcinomas.26,28 families is unknown.38 Despite these concerns about fundic gland polyps, in western countries, the risk of gastric adenocarcinoma Diagnostic issues does not seem appreciably higher in patients with The search for a specifi c inherited germline mutation in familial adenomatous polyposis than in patients with clinically selected families with aggregation of gastric sporadic fundic gland polyps.26,27 Carriers of APC cancer or early onset gastric cancer, or both, relies on mutations undergo prophylactic colectomies because the information from pathology reports from at least the probability of malignant transformation of colorectal proband in the family. Studying pathological changes polyps is 100% with this mutation. In patients who have establishes whether a family is classifi ed as HDGC, undergone this operation, the main cause of death is GAPPS, or FIGC, and determines which diagnostic upper gastrointestinal malignancy, justifying surveillance genetic test—if any—should be done. In most molecular with oesophagogastroduodenoscopy.29 Prophylactic diagnostic laboratories, conventional genetic testing is gastrectomy has been discussed for patients with familial generally off ered on the basis of one gene for one adenomatous polyposis (including those with attenuated syndrome (or, infrequently, several genes for one disease) displaying diff use fundic gland polyps, and syndrome—eg, MLH1, MSH2, MSH6, PMS1, PMS2, high-grade dysplasia or large polyps.30 EPCAM for Lynch syndrome). In hereditary gastric Peutz-Jeghers syndrome is caused by mutations in cancer, only families fulfi lling the criteria for HDGC STK11, and is characterised by the association of proposed by the International Gastric Cancer Linkage hamartomatous gastrointestinal polyps with muco- Consortium9 are tested for CDH1. Endoscopic cutaneous pigmentation and increased cancer risk, surveillance is off ered periodically to individuals at risk particularly for gastrointestinal and breast cancers, which in non-HDGC families, because no germline causative develop at younger ages than in the general population.31,32 mutations have been described to guide genetic testing.9 The frequency of gastric cancer in Peutz-Jeghers Dependence on clinical and pathological data to guide syndrome families has been reported in two cohorts—it appropriate genetic testing can delay or prevent the was 2·1% in one32 (mean age 40 years) and 3·0% in the fi nding of causative genetic mutations in individuals at other33 (mean age 41·5 years). Age at diagnosis ranged risk. To circumvent some of these shortcomings, genetic from 20 years to 61 years.32,33 A meta-analysis32 suggested a diagnosis with multigene sequencing panels, which has cumulative risk of gastric cancer of 29% by age 65 years. proved eff ective in several hereditary disorders, could be Based on the increased cancer risk, surveillance used. Genes causing both hereditary gastric cancer recommendations for patients with Peutz-Jeghers syndromes and cancer-associated syndromes in which syndrome include oesophagogastroduodenoscopy from gastric cancers are implicated are obvious candidates for age 20 years (repeated every 2–5 years depending on such panels. The identifi cation of more susceptibility endoscopic and histological fi ndings).32 genes will improve informed genetic testing, prevention, Juvenile polyposis syndrome is a hereditary cancer early detection, and management of individuals at high syndrome characterised by numerous juvenile polyps risk of gastric cancer. An increased attentiveness for developing in the colon or stomach, or both, and is caused these issues amongst medical doctor and genetic by SMAD4 or BMPR1A mutations (at similar counsellor communities is therefore required. frequencies).34,35 Gastric cancer has been noted in 21% of patients with juvenile polyposis syndrome who are HDGC aff ected by gastric polyps.35 Surveillance recommendations Genetic susceptibility encompass monitoring of gastrointestinal symptoms The genetic susceptibility to, and the molecular basis for, from infancy and upper endoscopy at age 15 years or when HDGC were fi rst identifi ed in Maori families in work symptoms are present, in addition to surveillance for published in 1998,5 which was shortly followed by colorectal cancer. Endoscopy should be repeated every studies39–41 reinforcing the role of CDH1 in susceptibility 1–3 years, depending on the load of gastric polyps.35 to HDGC in other populations.39–41 Since then, several Hereditary breast or ovarian cancer syndrome is hundred probands (aff ected individuals through whom a caused by germline BRCA1 and BRCA2 mutations, family with a genetic disorder is ascertained) meeting the which increase the risk of gastric cancer development. 2010 International Gastric Cancer Linkage Consortium Gastric tumours are included in the tumour spectrum of criteria for HDGC have been tested for CDH1 mutations, this syndrome.36 A meta-analysis37 of more than roughly 40% of whom carry CDH1 germline alterations. 30 studies showed that the relative risk of gastric cancer So far 104 diff erent CDH1 deleterious structural changes www.thelancet.com/oncology Vol 16 February 2015 e62 Review

causing germline fi rst-hit inactivation mechanisms have mutations.45,47 In-silico methods allow the prediction of been reported in HDGC probands (table 2). A third of the eff ect of missense variants in E-cadherin native-state these changes have been detected in two or more stability and in cryptic splicing.45,46 However, they cannot families. Notably, germline CDH1 changes aff ect the be used to establish the clinical implications of missense entire coding sequence and all the protein’s functional mutations or to derive informed reports for clinicians domains. 90 of the 104 known mutations are predicted to and patients. In vitro functional and biochemical analysis lead to premature protein truncation or lack of mRNA of cell lines expressing wild-type CDH1 and pathogenic expression, and enclose small frameshifts, splice-site and missense mutants were implemented to address these nonsense mutations, and large rearrangements (table 2). issues.44,47 When compared with wild-type-expressing The identifi cation of large deletions (from 357 bp to cells, CDH1 missense mutants are thought to be 275 kb in length) led to the addition of multiplex ligation- deleterious because of the following eff ects: disruption of dependent probe amplifi cation to the screening strategy cell adhesion, incorrect binding of E-cadherin to in HDGC families who test negative for CDH1 point fundamental adhesion-complex regulators, impairment mutations.9,42 of E-cadherin stability at the plasma membrane, and The genotype–phenotype correlation is straightforward induction of cell migration or invasion.43,45–47 CDH1 for truncating mutations, but the same is not true for so- missense mutations should be functionally analysed in called pure missense mutations. 14 pure missense reference laboratories. Novel bioimaging technologies mutations and four missense mutations that activate that are based on immunofl uorescence and detect cryptic splicing have been reported and classifi ed as changes in E-cadherin protein expression and abnormal potentially pathogenic, accounting for 18 (17%) of the localisation are expected to improve further the mutations described so far (table 2). Seven of these interpretation of functional consequences of CDH1 18 missense mutations recur in unrelated HDGC families. missense mutations.48,49 Many other missense alterations have been described, Once a germline missense CDH1 mutation has been although their pathogenicity remains to be fully classifi ed as deleterious, clinicians and genetic established, raising a substantial problem for genetic counsellors might suggest that mutant carriers enter a counselling and clinical management. Clinical and surveillance programme similar to that off ered to carriers genetic assessments of the pathogenicity of missense of truncating CDH1 mutations. Microscopic foci of variants should take into account their allelic frequency invasive cancer have been detected in carriers of in healthy controls, cosegregation within families and missense mutations carriers who were submitted to recurrence in unrelated HDGC families.43,44 This prophylactic gastrectomy, showing the relevance of this analytical pipeline has clear limitations because of the type of mutation in the clinical setting.50 low number of aff ected individuals and small pedigrees Roughly 60% of HDGC families do not have mutations that prevent segregation analysis.44 Furthermore, the in CDH1 coding regions, but present with a germline absence of mutation hotspots precludes the establishment phenotype of monoallelic CDH1 downregulation which of a correlation between the mutation site and its can be detected with allele-specifi c expression analysis.51,52 functional consequence.43–45 More than 90% of patients with such a phenotype show In-silico and in-vitro approaches have been developed loss or low expression of E-cadherin in tumours.53,54 Three to predict the pathogenicity of CDH1 missense families who have germline monoallelic CDH1 downregulation with deleterious large deletions encompassing CDH1 non-coding regulatory regions Frequency of mutation Recurrence have been described, supporting allele-specifi c expression CDH1 analysis as a promising prescreening strategy.51 Frameshift 39/104 (38%) 10/39 (26%) The only gene other than CDH1 that has been Splice site 24*/104 (23%) 10/24 (42%) implicated in HDGC is CTNNA1. So far, only one Missense† 18‡/104 (17%) 7/18 (39%) CTNNA1 germline truncating mutation has been Nonsense 18/104 (17%) 7/18 (39%) detected, in a large HDGC pedigree.6 In this family, the Large rearrangements 9/104 (9%) 4/9 (44%) onset of HDGC occurred relatively late in life (after age CTNNA1 50 years), which could be a feature of CTNNA1-driven Frameshift 1/1 NA disease. Furthermore, penetrance was incomplete,

Data are n/N (%), where N=total number of diff erent pathogenic mutations similar to what happens in CDH1-driven HDGC. reported to date. NA=not applicable. *Includes four mutations that activate Importantly, reduced expression of E-cadherin was noted cryptic splicing because of a missense mutation. †Only missense mutations with in addition to complete loss of CTNNA1 expression in proven pathogenicity based on recurrence, segregation, or in vitro functional the tumours.6 The defi nitive proof of CTNNA1 mutations analysis (or any combination thereof), and that have been reported as such, are shown. ‡Includes four missense mutations that activate cryptic splicing. as drivers of HDGC depends on the identifi cation of additional families harbouring inactivated alleles. Table 2: Type, frequency, and recurrence of pathogenic germline defects Data published in 2014 point to other possible in hereditary diff use gastric cancer families susceptibility genes for hereditary gastric cancer. In one e63 www.thelancet.com/oncology Vol 16 February 2015 Review

study, MAP3K6 was suggested as a novel predisposing mutation, or combinations of diff erent second hits factor because the investigators detected fi ve germline occurred only rarely. In lymph node metastases, loss of mutations in the gene in fi ve unrelated individuals.55 The heterozygosity was the most prevalent second-hit event authors of another study56 claim to have identifi ed several (fi ve occurrences), followed by combination of loss of candidate genes for early-onset and familial gastric heterozygosity with promoter hypermethylation (fi gure 1; cancer, and the full description of their results are keenly table 3). However, second hits associated with CDH1 awaited. Discovery and characterisation of novel inactivation were not identifi ed in 16 primary tumours susceptibilities will broaden knowledge of clinical and and four nodal metastases. pathological aspects of familial gastric cancer, generate These results are especially relevant because HDGC data for penetrance analysis and tumour spectrum is a disease characterised by multiple tumour foci assessment, and improve genetic screening, risk scattered in the stomach of mutation carriers.60 The stratifi cation, and management of patients. heterogeneity of second hits in neoplastic lesions from Molecular somatic events in HDGC lead to inactivation the same patient, the diff erence between type of second of the remaining wild-type CDH1 allele through a hit in primary and metastatic lesions, and the plasticity second-hit mechanism, causing aberrant E-cadherin of hypermethylated promoters during tumour expression and initiation of gastric signet ring cell development, suggest that drugs targeting only carcinoma (SRCC).53,57–59 Initial reports addressing the epigenetic changes might be less eff ective than was type and frequency of CDH1 second hits in HDGC initially anticipated, particularly in patients with tumours suggested that promoter hypermethylation is metastatic HDGC. the most common mechanism leading to CDH1 biallelic C-Src, fi bronectin, P-Fak, and P-Stat3, all of which have inactivation (table 3).57,59 A second mutation or deletion roles in the c-Src kinase pathway are strongly expressed (loss of heterozygosity or intragenic deletions) has been in large intramucosal HDGC lesions with poorly described less frequently.57–59 In the most comprehensive diff erentiated cells and in cells invading the muscularis study53 of CDH1 second-hit mechanisms in HDGC, mucosae, by contrast with small intramucosal cancer several primary and metastatic HDGC lesions were foci, in which the expression is low or absent.61 These analysed (table 3).53 This work showed for the fi rst time fi ndings start to shed light into the pathways implicated that diff erent neoplastic lesions from the same patient in cancers in which E-cadherin function is lost, and can present distinct second hits, and that the same warrant further study. neoplastic lesion can present diff erent second-hit In CDH1-driven HDGC, no somatic mutations have mechanisms (table 3).53 been searched for in tumours, apart from those in Overall, studies of HDGC cases analysed for CDH1 CDH1. By contrast, in the single CTNNA1-associated second hits encompass 50 lesions: 38 primary tumours, HDGC family reported, several genes have been and 12 lymph node metastases.53,57,59 Promoter identifi ed to be somatically mutated in a single tumour hypermethylation was the most frequent second hit in sample analysed, including LMTK3, MCTP2, MED12, HDGC primary tumours (occurring in 13 primary PIK3CA, ARID1A, and other genes recently shown to tumours); isolated loss of heterozygosity, a second be mutated in sporadic gastric cancer exomes.6

Lesions Primary tumours (n=38) Lymph node metastases (n=12) Promoter Loss of Mutation Promoter hyper- Promoter Loss of No Promoter Loss of Promoter No hyper- hetero- methylation and hyper- hetero- alteration hyper- hetero- hyper- alteration methylation zygosity loss of methylation zygosity and methylation zygosity methylation heterozygosity and mutation mutation and loss of hetero zygosity Grady et al,57 Six lesions from 3/6 0/6 2/4 0/6 0/4 0/4 1/6 NA NA NA NA 2000 six people in two families Barber et al,59 16 lesions from 2/16 0/5 0/16 0/5 2/16 0/5 12/16 NA NA NA NA 2008 16 people in nine families Oliveira et al,53 28 lesions from 8/16 2/16 0/16 3/16 0/16 0/16 3/16 1/12 5/12 2/12 4/12 2009 17 people in 15 families Combined ·· 13/38 2/27 2/36 3/27 2/36 0/25 16/38 1/12 5/12 2/12 4/12 frequency of second hits*

The numbers in this table are descriptive and represent the summary of mechanisms reported in three published works. *In some lesions there was a combination of diff erent second hits.

Table 3: CDH1 somatic mechanisms acting as second hits in tumours of patients with hereditary diff use gastric cancer in multi-family studies

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Macroscopic and microscopic features pale areas are visible on standard white light endoscopy,64 Macroscopic features in the stomachs of asymptomatic which, after formalin fi xation, show up as white patches CDH1 mutation carriers submitted to prophylactic under close inspection, suggesting intramucosal diff use gastrectomy diff er from those in index cases with HDGC. carcinoma or SRCC. Most index cases with HDGC Stomachs of asymptomatic carriers nearly always seem present with advanced cancers that are indistinguishable normal to the naked eye because of the absence of mass from sporadic diff use gastric cancer. They often have lesion, and slicing shows normal mucosal thickness.62,63 features of linitis plastica, which can implicate all However, in some normal-seeming stomachs, subtle topographical regions in the stomach. Microscopic features of HDGC were initially assessed Primary tumour in the stomachs of CDH1 mutation carriers who Promoter hypermethylation underwent total gastrectomies. Multiple foci of intramucosal (T1a) SRCC were noted in most cases.59,60,62,63,65 The individual foci are small, ranging from 0·1 mm to 10 mm (fi gure 2). The neoplastic cells are small at the neck-zone level and usually enlarge towards the surface of the gastric mucosa, displaying the Loss of heterozygosity distinctive SRCC phenotype (fi gure 2). In North American and European families, microscopic foci of intramucosal carcinoma are not restricted to any topographical region in the stomach: foci were identifi ed from cardia to the prepyloric region, without antral clustering.60,65 In New Zealand Maori families, most early Loss of heterozygosity and promoter hypermethylation invasive carcinomas developed in the distal stomach and Nodal metastases the body-antral transitional zone.62 Background genetics

Loss of heterozygosity Somatic mutation and environmental factors are putative contributing Loss of heterozygosity and promoter hypermethylation factors to the diff erent anatomical localisations of the Promoter hypermethylation cancer foci. Somatic mutation and promoter hypermethylation Two distinct types of intraepithelial lesions have been Figure 1: Schematic of advanced hereditary diff use gastric cancer displaying features of linitis plastica, and identifi ed as precursors of invasive cancers in CDH1 summary of CDH1 second-hit inactivating mechanisms identifi ed in primary tumours and nodal metastases mutation carriers:60 in-situ SRCC corresponds to the

Pagetoid spread of signet ring cells

Intramucosal signet ring cell carcinoma

In-situ carcinoma

Figure 2: Macroscopic appearance of a prophylactic gastrectomy displaying lesions identifi ed by histology e65 www.thelancet.com/oncology Vol 16 February 2015 Review

presence of signet ring cells, generally with hyper- In situations in which gastrectomy is not appropriate, chromatic and depolarised nuclei, within the basal yearly surveillance with endoscopy and biopsies is membrane (fi gure 2), and pagetoid spread of signet ring recommended.9,66 Surveillance is crucial to guide the cells below the preserved epithelium of glands or foveolae clinical management of individuals from families with (fi gure 2). CDH1 mutations who refuse genetic testing or prefer to Confi rmation of carcinoma in situ and pagetoid spread delay gastrectomy, in patients carrying CDH1 mutations of signet ring cells by an independent pathologist with of uncertain clinical signifi cance (eg, missense experience in this area is strongly recommended. Strict mutations), and in those from families without CDH1 adherence to the criteria below for the identifi cation of mutations.9,66 The authors of a prospective cohort study66 these precursor lesions will diminish the risk of over- of the outcomes of HDGC families undergoing diagnosis of non-specifi c changes, and will help endoscopic surveillance concluded that careful white pathologists to distinguish legitimate precursor lesions light examination with targeted and random biopsies and tiny foci of early intramucosal carcinoma from combined with detailed histopathology can identify mimics of signet ring cells (eg, telescoped normal glands, early lesions and help to inform decision making about globoid cells in hyperplastic lesions, xanthomatous cells, gastrectomy. In this study, autofl uorescence and neuroendocrine cell nests, and clear or glassy cell change narrow-band imaging were of limited use. Endoscopic of mucous glands).7 E-cadherin is aberrantly expressed biopsies should be assessed by pathologists with (dotted in membrane and cytoplasm), reduced, or absent expertise in the disease to maximise surveillance in precursor lesions and early invasive gastric carcinomas output.9 (T1a); expression should be normal in adjacent non- Because of the high lifetime risk of developing lobular neoplastic mucosal membranes.7 breast cancer, women carrying CDH1 mutations are Advanced HDGC presents as a poorly diff erentiated recommended to undergo yearly mammography and diff use carcinoma, sometimes with a few signet ring breast MRI from age 35 years onwards.9 Prophylactic cells, that invades widely the whole thickness of the mastectomy might be an option for some, although data gastric wall, which becomes rigid (linitis plastica). It also are insuffi cient to advise prophylactic breast surgery over presents as undiff erentiated or mixed subtypes with surveillance.9,67 Detailed information about the clinical mucinous and occasionally tubular components. These management of patients with HDGC is available in the advanced gastric carcinomas of CDH1 mutation carriers International Gastric Cancer Linkage Consortium 2010 do not have any characteristics that can be used to guidelines9 and the Clinical Utility Gene Card for diff erentiate them from sporadic gastric cancers. Mild HDGC.67 chronic gastritis has been identifi ed as a background Analysis of pathological changes in HDGC prophylactic change in the gastric mucosa of patients with HDGC; gastrectomy specimens includes a thorough microscopic but intestinal metaplasia and infection with Helicobacter assessment of the complete stomach with haematoxylin pylori have been rarely reported in the HDGC setting in and eosin and a mucin stain, such as periodic acid- families from North America and Europe.60 Schiff .9 Periodic acid-Schiff is a primary stain, and, as such, increases the detection rate of small invasive SRCC Clinical management foci and reduces screening time. The Swiss roll Clinically accepted approaches in HDGC include technique, consisting of rolling up the gastrectomy prophylactic total gastrectomy and endoscopic specimen lengthwise, can be used to include the screening. Prophylactic gastrectomy is the only complete mucosa. The pathology report should mention therapeutic option for carriers of CDH1 pathogenic all gastric abnormalities and localisation. Histological mutations, and is off ered even when carriers are confi rmation of resection margins consisting of proximal asymptomatic in view of the high penetrance of HDGC, oesophageal and distal duodenal mucosa is essential, the limitations of endoscopic surveillance (which often because new gastric cancer can develop in remaining does not detect microscopic disease), and the dismal gastric mucosa.9,60 prognosis of HDGC at advanced stages.9,66,67 Patients considering prophylactic gastrectomy should take into GAPPS account the mortality of the procedure (<1%) and the Genetic susceptibility short-term and long-term adverse events associated Other heritable polyposis syndromes should be excluded with it, which have eff ects on quality of life.66 A before considering GAPPS as a diagnosis. Familial multidisciplinary approach to preoperative counselling adenomatous polyposis, attenuated familial adeno- is essential, and should include input from a gastro- matous polyposis, and MUTYH-associated polyposis enterologist, surgeon, dietitian, genetic counsellor, and (which has autosomal recessive inheritance) are defi ned specialist nurse.9,67,68 For patients with symptomatic by their colorectal phenotype. Patients with Peutz- invasive HDGC, non-surgical treatment regimens used Jeghers syndrome might also have fundic gastric polyps in sporadic diff use gastric cancer are often the only and intestinal gastric cancer, but the polyp distribution therapeutic option. diff ers from that in GAPPS. After the initial description www.thelancet.com/oncology Vol 16 February 2015 e66 Review

of a GAPPS family, two Japanese families with similar so far occurred in someone aged 33 years in one of the clinicopathological features were reported.14 families studied.13 Recommended diagnostic criteria for GAPPS are listed in table 1.13 Mutations in APC, MUTYH, CDH1, SMAD4, Clinical management BMPR1A, STK11, and PTEN have been excluded in fi ve Clinically accepted approaches to the management of GAPPS families by sequence analysis of exons and GAPPS families include endoscopic surveillance, and, fl anking regions and assays for deletion or duplication eventually, prophylactic gastrectomy. The limitations of of exons.13,14 endoscopic surveillance, the patient-specifi c risk of morbidity associated with prophylactic surgery, and the Macroscopic and microscopic features risk of gastric cancer within the specifi c family need to be Macroscopic features of GAPPS include fl orid gastric balanced. All fi rst-degree relatives of aff ected patients polyposis, mainly with gastric polyps with diameters of should be advised to undergo am oesophago gastro- less than 10 mm. More than 100 polyps carpet the gastric duodenoscopy and a colonoscopy.13 body and fundus, with relative sparing along the lesser curve of the stomach.13,14 The oesophagus, gastric antrum, FIGC pylorus and duodenum are usually unaff ected. Genetic susceptibility Microscopy shows mainly fundic gastric polyposis and The genetic cause of FIGC is currently unknown. The regions of dysplasia (fi gure 3). Occasional hyperplastic diagnosis is considered when there is a family history of and pure adenomatous polyps and some mixed polyps gastric cancer, intestinal-type, in families without gastric containing discrete areas of fundic-gastric-polyposis-like, polyposis. adenomatous, and hyperplastic features can be detected amid the fundic gastric polyps. Gastric cancers that Macroscopic and microscopic features develop in patients with GAPPS display the features of The stomach cancers display the common macroscopic intestinal-type gastric cancer. The earliest case reported features observed in the sporadic setting of gastric cancer. By histology, the tumours show the features of A B adenocarcinoma, intestinal-type.

Clinical management Few recommendations have been suggested for the management of patients at risk of FIGC.16

Future directions in research and treatment of HDGC In readthrough therapies, which are also called nonsense suppression therapies, low-molecular-weight com- pounds are used to induce the translation machinery to recode a nonsense codon into a sense codon.69 Aminoglycosides and PTC124, for instance, partly restore expression of proteins with normal function from C D nonsense-mutated mRNA.69 Roughly 50% of CDH1 germline mutations are either nonsense or small insertions or deletions that cause premature truncation of E-cadherin nascent proteins. Amino glycosides and PTC124 have not been tested in the context of HDGC, mainly because of the high likelihood of generation of deleterious missense mutant proteins during correction. Nevertheless, when suppressor tRNA that replaces a specifi c premature stop codon by a cognate aminoacid was used, E-cadherin readthrough was induced and full- length wild-type proteins were generated in an HDGC cell line.70 The objective of such a strategy is to reverse the eff ect of germline CDH1-truncating mutations, thereby restoring expression of full-length normal E-cadherin Figure 3: Gastric adenocarcinoma and proximal polyposis of the stomach molecules, although further research is needed.70 (A) Florid fundic gastric polyposis (haematoxilyn and eosin, 40× magnifi cation), (B) fundic gland polyp without Chemical chaperones (eg, dimethyl sulfoxide) can dysplasia (haematoxilyn and eosin, 400× magnifi cation), (C) fundic gland polyp with high-grade dysplasia (haematoxilyn and eosin, 400× magnifi cation), and (D) invasive intestinal-type adenocarcinoma (haematoxilyn restore E-cadherin folding, expression, and function in and eosin, 200× magnifi cation). vitro, because they are modulators of traffi cking-related e67 www.thelancet.com/oncology Vol 16 February 2015 Review

signalling pathways.47,71 About a fi fth of HDGC-causing mutations are missense, and often generate E-cadherin Search strategy and selection criteria molecules that are incapable of proper folding, which get We searched PubMed with the terms “hereditary diff use gastric destabilised and degraded.71 Therefore, natural, chemical, cancer (HDGC)”, “early onset gastric cancer”, “familial gastric or pharmacological chaperones have been advanced as cancer”, “gastric adenocarcinoma and proximal polyposis of the promising drugs for use in carriers of CDH1 germline stomach (GAPPS)”, and “E-cadherin germline mutations” for missense mutations.46,71 articles in English published between Jan 1, 1998, and 30 June, Drugs that induce DNA demethylation and the re- 2014. We also searched our fi les, particularly for papers about expression of specifi c target genes have been tested for gastric-cancer-associated syndromes and the pathology of use in gastric cancer. Vorinostat, a histone deacetylase HDGC. We generated the fi nal reference list on the basis of inhibitor, has shown particular benefi t when combined originality and relevance to the broad scope of this Review. with a taxane or capecitabine and cisplatin.72,73 Given the high frequency of hypermethylation of the CDH1 promoter, demethylating drugs are emerging as on the basis of available information, personalised therapeutic options in early CDH1-driven HDGC stages management of HDGC families, specifi cally with regard without evidence of metastatic disease.53 to targeted therapies, is not foreseeable in the near future. E-cadherin signalling has a crucial role in cell behaviour. It modulates the expression and function of several Animal models of HDGC molecules, such as growth factor receptors, signalling Three mouse models of E-cadherin inactivation exist.80–82 eff ectors, cytoskeletal regulators, catenins, integrins, and CDH1 heterozygous knockout animals, derived from a metalloproteinases.74,75 As an example, EGFR becomes previous homozygous knockout model,83 were used to activated when E-cadherin is mutated, and in the absence establish the fi rst murine model of diff use gastric cancer.80 of E-cadherin expression.76 In-vitro analysis with E-cadherin Wild-type and heterozygous knockout mice were treated mutant cells showed that the inhibition of EGFR prevents with N-methyl-N-nitrosourea to promote gastric carcino- cell motility and invasion.76 Anti-EGFR therapy might still genesis. Heterozygous knockout mice developed intra- have a role in the treatment of patients with HDGC, mucosal SRCC that closely mimicked human disease with although results from clinical trials are not encouraging.77 an 11-times higher frequency than did wild-type mice. All C-Src is overexpressed in large intramucosal HDGC lesions displayed decreased E-cadherin expression and low lesions and cells invading the gastric wall, which suggests proliferative activity.80 Knockout of CDH1 has also been that inhibition of c-Src activation could be a therapeutic specifi cally induced in the parietal cell lineage, leading to strategy for patients with early HDGC.61 MMP-targeting loss of E-cadherin and disturbance of epithelial cell polarity, agents, Hedgehog (Hh) signalling inhibition, and growth, and diff erentiation, but was not suffi cient to induce inhibition of RhoA activity also improve E-cadherin- tumour development.81 The main limitation of the these dependent cellular activities in vitro, and therefore could two models is their inability to spontaneously develop be molecules to investigate when designing targeted cancer, which is probably a result of the mice’s short therapies for HDGC.78 lifespan. Notch and TFF1 have been advanced as molecules A double conditional knockout mouse line in which specifi cally associated with the transcriptional signature CDH1 and TP53 were specifi cally inactivated has been of healthy gastric epithelia.79 These molecules seem to genetically engineered.82 Diff use gastric cancer developed endow gastric epithelial cells with increased resistance to in all mice within 12 months (100% penetrance); and apoptosis in the event of E-cadherin downregulation, in a cancers were mainly composed of poorly diff erentiated tissue-specifi c manner.79 If the hypothesis that normal cells and SRCCs, as in human advanced HDGC.82 gastric epithelial cells survive complete loss of E-cadherin, Furthermore, gene expression patterns of diff use gastric as opposed to other epithelial cells, is correct, it might cancer in double knockout mice showed increased explain why carriers of CDH1 germline mutations have a expression of mesenchymal markers and epithelial- high risk of diff use gastric cancer.79 Gastric tissue-specifi c mesenchymal-transition-related genes, resembling the proteins, including Notch and TFF1, could then become molecular phenotype of the human disease.82 This model targets for therapy in E-cadherin-defi cient gastric cancers mimics more closely human disease but, because it (eg, HDGC). depends on knockout of an additional gene, its use in the HDCG is a rare cancer syndrome, and thus the planning study of the natural history of CDH1-related HDGC of clinical trials to compare compounds that restrore could be limited. E-cadherin functions with prophylactic surgery is quite Animals develop diff use gastric cancer rapidly, and could diffi cult. Alternatively, if the safety and effi cacy of any of be very useful in several ways. They have the potential to these compounds is proved in sporadic diff use gastric clarify the mechanism underlying the formation of diff use cancer, which is far more common, the compound could gastric cancers and they can be used as disease models for potentially be used in patients with HDGC—in particular, preclinical intervention studies to test preventive measures those who refuse or delay prophylactic surgery. However, and potential HDGC-targeted therapies. www.thelancet.com/oncology Vol 16 February 2015 e68 Review

Conclusions 15 Caldas C, Carneiro F, Lynch HT, et al. Familial gastric cancer: The genetic and pathogenic determinants of hereditary overview and guidelines for management. J Med Genet 1999; 36: 873–80. gastric cancer syndromes are not yet fully recognised. 16 Corso G, Roncalli F, Marrelli D, Carneiro F, Roviello F. History, For GAPPS and FIGC, eff orts need to be made to identify pathogenesis, and management of familial gastric cancer: original genetic causes that may guide patients’ genetic testing study of John XXIII’s family. Biomed Res Int 2013; 2013: 385132. 17 Rahner N, Steinke V, Schlegelberger B, Eisinger F, Hutter P, and clinical management. Much more is currently Olschwang S. Clinical utility gene card for: Lynch syndrome known about clinical aspects, genetics, and pathogenesis (MLH1, MSH2, MSH6, PMS2, EPCAM)—update 2012. of HDGC; anticipated future technological developments Eur J Hum Genet 2013; 21. should open avenues for new genetic testing approaches 18 Capelle LG, Van Grieken NC, Lingsma HF, et al. Risk and epidemiological time trends of gastric cancer in Lynch syndrome and novel therapeutic strategies. carriers in the Netherlands. Gastroenterology 2010; 138: 487–92. Contributors 19 Li FP, Fraumeni JF Jr. Rhabdomyosarcoma in children: FC and CO contributed to the conceptual design, supervision, and epidemiologic study and identifi cation of a familial cancer syndrome. J Natl Cancer Inst 1969; 43: 1365–73. preparation of the Review. CO, HP, JF, RS, and FC analysed and interpreted data. HP compiled all relevant references and tabulated data. 20 Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. All authors drafted the paper and read, commented on, and approved the Science 1990; 250: 1233–38. fi nal version before submission. 21 Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P. Declaration of interests The IARC TP53 database: new online mutation analysis and We declare that we have no competing interests. recommendations to users. Hum Mutat 2002; 19: 607–14. 22 Masciari S, Dewanwala A, Stoff el EM, et al. Gastric cancer in Acknowledgments individuals with Li-Fraumeni syndrome. Genet Med 2011; We thank the Portuguese Foundation for Science and Technology (FCT), 13: 651–57. the Programa Operacional Temático Factores de Competitividade 23 Lipton L, Tomlinson I. The genetics of FAP and FAP-like (COMPETE), and fundo Comunitário Europeu FEDER for funding the syndromes. 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