Hereditary Diffuse Gastric Cancer: A 2022 Update

Gastric cancer is ranked fifth among the most commonly diagnosed cancers, and is the fourth leading cause of cancer-related deaths worldwide. The majority of gastric cancers are sporadic, while only a small percentage, less than 1%, are hereditary. Hereditary diffuse gastric cancer (HDGC) is a rare malignancy, characterized by early-onset, highly-penetrant autosomal dominant inheritance mainly of the germline alterations in the E-cadherin gene (CDH1) and β-catenin (CTNNA1). In the present study, we provide an overview on the molecular basis of HDGC and outline the essential elements of genetic counseling and surveillance. We further provide a practical summary of current guidelines on clinical management and treatment of individuals at risk and patients with early disease.


Introduction
Gastric cancer remains an important health issue worldwide, ranking fifth among the most commonly diagnosed malignancies (5.6%) and the fourth leading cause of cancerrelated deaths (7.7%); it was responsible for over one million new incidents and an estimated 769,000 deaths in 2020 worldwide [1]. There are two major subtypes of gastric cancer characterized by distinct molecular, morphological and clinical features, the Lauren intestinal/WHO tubular and Lauren diffuse/WHO poorly cohesive subtype [2]. The majority of gastric cancers are sporadic; however, a small percentage (5-10%) arise within inherited or familial cancer syndromes [3]. Among gastric cancers, only 1% to 3% are hereditary [4], including Hereditary Diffuse Gastric Cancer (HDGC), Familial Intestinal Gastric Cancer (FIGC) and Familial Diffuse Gastric Cancer (FDGC) [5][6][7]. Moreover, several syndromes, such as Lynch, Peutz-Jeghers, Li Fraumeni and Familial Adenomatous Polyposis syndromes, are predisposed to the development of gastric cancer [6]. Hereditary Diffuse Gastric Cancer is an autosomal dominant cancer syndrome characterized by an increased risk of diffuse gastric cancer (DGC) and lobular breast cancer (LBC) [8]. It was first described in 1998 in an extended New Zealand Māori family [9], now the prevalence of HDGC is less than 0.1 per 100,000 in the general population and in less than 1% of patients with gastric cancer [10]. HDGC exhibits high penetrance and invasive disease often manifests before age 40; the median age of HDGC diagnosis is 38 years with the overall risk increasing with age [11,12]. The majority of confirmed cases are caused by inactivating germline mutations in the CDH1 tumor suppressor gene [13]. The lifetime risk of developing gastric cancer in individuals carrying a mutation in CDH1 gene is estimated to be less than 1% at the age of 20, around 4% by the age of 30, increasing to about 20% for men and 45% for women by the age of 50 [14][15][16][17][18][19]. These mutations in penetrance genes widely vary based on tested populations. In fact, original data were in DGC patients with later reports in laboratory-based populations. It has been shown that HDGC may be responsible for a more aggressive disease phenotype compared to sporadic cancer, with a 5-year survival rate of 4% compared to 13% for sporadic cancer [20,21]. Three distinct tumor cell populations are proposed in HDGC, i.e., well-differentiated large cells, well-differentiated small cells and poorly differentiated small cells; the latter group, with aberrant p16 expression, may represent a more aggressive phenotype [22]. Advanced HDGC is composed by poorly differentiated small cells invading the whole thickness of the gastric wall [22]. In this review, we discuss the available literature on the molecular basis of HDGC and outline the essential elements of genetic counseling and surveillance. Moreover, we refer to recent guidelines on clinical management and treatment recommendations.

Molecular Basis of HDGC
HDGC, although rare, is an aggressive cancer inherited in an autosomal dominant fashion [23]. The genetic basis was identified in 1998 by Guilford et al., who reported mutations in the E-cadherin gene (CDH1) [9]. The human CDH1 gene is a 2.6 kb tumor suppressor gene located in chromosome 16q22.1 [24,25]. The CDH1 gene encodes a type I calcium transmembrane glycoprotein expressed on epithelial tissues, responsible for calcium-dependent cell adhesion basolateral membrane in adherent junctions, maintenance of cell differentiation and cell polarity, both during development and in adult life [26][27][28]. The CDH1 120-kDa mature glycoprotein is composed by three domains, an extra-cellular domain containing binding sites for Ca 2+ ions, a transmembrane domain and a highly conserved cytoplasmic domain containing binding sites for β-catenin (CTNNA1) [26,29]. E-cadherin is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis (Table 1) [30]. The binding of β-catenin prevents its translocation to the nucleus, thus leading to the activation of the Wnt signaling pathway, epithelialmesenchymal transition process and malignant transformation [31][32][33][34].
Mutated sites in the CDH1 gene are scattered. There have been over 155 different germline CDH1 mutations identified so far [13], including insertions, deletions and point mutations [12,37]. Moreover, 38% of germline mutations are frameshift mutations, 23% are splice site, 17% are missense and 17% are nonsense mutations affecting the entire coding sequence, resulting in truncated inactive proteins [46]. Almost 90% of the known mutations have been predicted to lead to premature protein truncation or a lack of mRNA expression [47]. Epigenetic factors, such as DNA methylation, have been shown to influence gene expression and serve as the second genetic hit leading to gene inactivation, therefore promoting tumorigenesis [48][49][50]. Oliveira et al. reported that CDH1 epigenetic and genetic alterations were detected in 80% of HDGC families analyzed. Out of 28 HDGC lesions, CDH1 promoter hypermethylation was found in nine (32.1%) cases [50]. Association between promoter hypermethylation of CDH1 and gastric cancer was also confirmed in a meta-analysis by Jing et al. [51]. In this direction, demethylating drugs may emerge as therapeutic options in early CDH1-driven HDGC stages without evidence of metastatic disease [50]. A clinical trial (NCT04253106), currently in the recruiting stage, aims to detect methylation profiles in asymptomatic carriers who refuse gastrectomy and in controls using liquid biopsies of both blood and gastric fluid [52]. Moreover, post-translational machinery was reported by Figueiredo et al. as a new molecular basis for the loss of E-cadherin [53]. Disruption of the highly conserved hydrophobic core of the signal peptide of E-cadherin impairs the interaction of E-cadherin with cellular components crucial for E-cadherin translation and translocation into the endoplasmic reticulum (ER), decreasing protein synthesis, ER import and membrane activity [53]. Pathogenic E-cadherin missense mutant cells fail to form correct cell-cell adhesions and become more invasive in comparison with cells expressing the wild-type (WT) protein [54,55]. Mutations in CDH1 gene have been associated with the development of signet ring cell carcinoma in situ (SRCC-pTis), characterized by tumor cells located between normal foveolar epithelial cells and the basement membrane. SRCC-pTis is considered the precursor lesion of HDGC [56,57]. Penetrance of HDGC in CDH1 mutated carriers is approximately 40% to 67% for men and 63% to 83% for women [12,58]. The cumulative incidence of HDGC by the age of 80 years is 70% (95% CI, 59-80%) for males and 56% (95% CI, 44-69%) for females [13], while the combined risk is estimated at 80% [11,15].  [43][44][45] Another gene implicated in HDGC is CTNNA1 (Table 1). The human CTNNA1 gene is located in chromosome 5q31.2, encoding a member of the catenin family that plays an important role in cell adhesion to the actin filaments inside the cell [59]. The A-catenin family consists of three members, aE-catenin, aN-catenin and aT-catenin expressed in epithelia, neurons and testis-heart, respectively, playing an important role in cell adhesion by connecting to cadherins [59][60][61]. A lack of function of CTNNA1 leads to the destabilization of adherent junctions, making cells less sensitive to cell contact-mediated inhibition, thus facilitating cell migration and invasion [62][63][64]. This correlates with poor prognosis. Pathogenic variants of CTNNA1 have been described in 14 families [38,39]. In the United States, all CTNNA1 variants commercially used in the laboratory are classified as variants of uncertain significance, including CTNNA1 loss-of-function (LOF) variants, thus justifying the low numbers [39]. Thus far, only one frameshift mutation has been detected leading to a germline truncating allele of α-E-catenin in family members with invasive HDGC, and four in patients with intramucosal signet ring cells detected as part of endoscopic surveillance [13,37]. Majewski et al. reported a 2 bp deletion in exon 2, which results in a frameshift mutation after Arg27 and truncated CTNNA1 gene [37]. In all cases, the onset of HDGC occurred after the age of 50 [13,37]. Moreover, rare germline missense mutations in other genes (Table 1) [13] of high and moderate penetrance have been previously reported as pathogenic. Included genes are: insulin receptor (INSR) [40], which has been shown to affect tumor cell invasion by modulating E-cadherin glycosylation [65,66]; F-box protein 24 (FBXO24) [40], which may regulate cell proliferation by mediating ubiquitination and degradation of PRMT6 [67]; DOT1-like histone H3K79 methyltransferase (DOT1L) [40], which is required for proper DNA damage response [68]; mitogen-activated protein 3kinase 6 (MAP3K6) [41], which acts as a tumor suppressor gene [41]; the pathogenic missense variant (F354L) in the highly penetrant Peutz-Jeghers syndrome susceptibility gene, serine/threonine kinase 11 (STK11) (also called LKB1) [43,44]; and the partner and localizer of BRCA2 (PALB2) [43][44][45], which have also been implicated in HDGC occurrence. However, Weren et al. concluded that MAP3K6 should no longer be considered a gastric cancer predisposition gene since, according to their data [42], two missense variants, which have previously been associated with familial gastric cancer, are also frequently observed in the control data set and no significant differences between the two datasets were found.

Genetic Testing and Counseling
The International Gastric Linkage Consortium (IGCLC) set criteria to provide critical information for medical care providers and for those patients and families at risk of HDGC syndrome. The 2020 updated guidelines [8] recommend genetic testing in individuals who have a confirmed cancer diagnosis and meet one of the following family criteria (Table 2): (1) two or more gastric cancer cases, regardless of age, with at least one histologically confirmed diffuse gastric cancer (DGC); (2) one or more cases of DGC at any age, and one or more cases of LBC at an age less than 70 years, in different family members; and (3) two or more cases of LBC in family members less than 50 years of age. Individual criteria include: confirmed DGC at an age less than 50 years; DGC at any age in individuals of Māori ethnicity; DGC at any age in individuals with a personal or family history (first-degree relative) of cleft lip/cleft palate; history of DGC and LBC both diagnosed at an age earlier than 70 years; bilateral LBC diagnosed at an age earlier than 70 years; and in situ gastric signet ring cells or pagetoid spread of signet ring cells in individuals less than 50 years [8,69]. Table 2. Criteria for genetic evaluation for HDGC in affected families according to ACG 2020 updated clinical guidelines [8,69].

Individuals with a Confirmed Cancer Diagnosis Plus One of:
I. ≥2 cases of DGC cases regardless of age, with at least one confirmed histologically for DGC II. ≥1 cases of DGC at any age, and one or more cases of LBC at age less than 70 years, in different family members III. ≥2 cases of LBC in family members less than 50 years Hereditary cancer syndromes presuppose an elevated risk of cancer in family members [3]; therefore, before testing, genetic counseling for HDGC should take place by a team of experts including a gastroenterologist, a geneticist, a surgeon and an oncologist. All possible outcomes of testing and the associated management options should be discussed. It is suggested that individuals who fulfill the criteria for HDGC should be offered genetic testing from the legal age of consent (18 years) [70]; testing should be performed first for CDH1 mutations, and, if there is no variant identified, then they should subsequently be considered for CTNNA1 analysis [71]. However, age of testing should also consider the earliest age of cancer onset in HDGC families from the local population. For example, in New Zealand, CDH1 mutation carriers have developed gastric cancer in their mid-teens; as a consequence, genetic testing begins at 16 years of age, and occasionally 1-2 years before, on a case-by-case basis [15]. Using the criteria for HDGC, the detection rate of CDH1 pathogenic or likely pathogenic variants is detected in 48% of families with multiple cases of gastric cancer [72]. Genetic evaluation should include a three-generation family pedigree, histopathological confirmation of DGC diagnoses or precursor lesions, and a discussion of lifetime risks of DGC and LBC and current CDH1 mutation detection rates [11,12,50]. Analysis for CDH1 and CTNNA1 germline variants must include sequencing of the entire open reading frame (ORF), including intron-exon boundaries [8]. Germline CDH1 point or small frameshift mutations can be identified in 30-50% of HDGC families, while CDH1 large deletions account for less than 5% of pathogenic variants [47,73]. Available variant information can be obtained from the LOVD team [69] in the locus-specific database [74]. Genetic testing should include copy number analysis of exons to detect deletions or duplications of the CDH1 gene [8]. Results interpretation should be performed using the ACMG/AMP guidelines [75,76]. As pathogenic germline CDH1 variants have high prevalence in the New Zealand Māori population, all Māori with confirmed DGC are recommended to undergo CDH1 genetic testing [77]. Moreover, individuals with a personal or family history of cleft lip/palate and DGC or with HDGC precursor lesions are recommended to undergo genetic testing [69,78]. Initially, genetic testing should be performed in an affected patient in order to identify the genetic alteration. Thereafter, first-degree family members may be analyzed for that specific mutation [57,79]. In case no living affected family member exists, formalinfixed paraffin-embedded specimens from the deceased relative may be performed [79]. However, this is not the common method for most laboratories. Nevertheless, it is worth mentioning that no genetic testing should be performed in patients with no family history of cancer and no previous genetic counseling [80].
Concerning molecular genetic analysis approaches, single-gene testing is recommended followed by gene-targeted deletion/duplication analysis if no pathogenic variant is found [81]. A multigene panel testing (MGPT) that includes CDH1 and other genes of interest may also be considered using next generation sequencing techniques searching for mutations [39,[82][83][84]. The MGPT is a type of genetic testing that looks for mutations in several genes at once, and has become a critical component of cancer risk assessment in clinical practice [85]. Other methods used may include quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and a gene-targeted microarray designed to detect single exon deletions or duplications [81]. Using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology updated guidelines, germline sequence variants found in genetic analysis are classified as: (a) pathogenic; (b) likely pathogenic; (c) variant of uncertain significance; (d) likely benign; and (e) benign [75,86,87].
HDGC is associated with LBC which may be the presenting pathology in a patient [88]. The results presented by S. Masciari et al. were not isolated cases, but rather cases of familial LBC. LBC is characterized by small, dyscohesive, diffusely infiltrating epithelial cells which do not form a well-defined mass and have a high predisposition to metastasize to the gastrointestinal and peritoneal surface, as well as to the female reproductive tract [79]. LBC is frequently multicentric and bilateral compared to ductal breast cancer, and bilateral prophylactic mastectomy is usually suggested in asymptomatic individuals with a strong family history of LBC and a CDH1 pathogenic variant [69]. In contrast to ductal breast carcinomas, approximately 90% of invasive LBCs are estimated to display E-cadherin loss [89]. Interestingly, the estimated cumulative risk for LBC in CDH1 mutation female carriers is 39% (95%CI, 12-84%) while the combined risk of gastric cancer and breast cancer in women rises to 90% by age 80 years, indicating that family history of multiple LBCs at a young age should encourage genetic screening for CDH1 mutations [11]. The American College of Gastroenterology also recommends breast cancer screening for women with HDGC through annual mammography and semiannual breast MRI, with breast examinations starting at the age of 35 years [8,90] in concordance with the National Comprehensive Cancer Network (NCCN) guidelines [91]. Bilateral LBC under the age of 50 years or in at least two close relatives under the age of 50 years [92,93], as well as individuals with a personal or family history of cleft lip/cleft palate and DGC [78,94,95], may justify testing for CDH1 mutations.

Clinical Surveillance and Management
Management of patients with symptomatic invasive HDGC is not different to that of sporadic cases [96][97][98][99]. Prognosis in patients with widely invasive DGC is very poor, with less than 10% of them being potentially curable [100]. Furthermore, the 5-year survival rate is still less than 30%, even for the potentially curable DGC [101], which does not differ much from sporadic gastric cancer [102,103]. On the other hand, despite the fact that prophylactic gastrectomy has a post-operative mortality rate of approximately 1% and is a life-changing procedure reducing quality of life (risks of infections, dumping syndrome and weight loss) [58,104], it has a curative result. When gastric cancer is detected early and resected by total gastrectomy, the 5-year survival rate is 90% [81,105]. For patients with metastatic cancer, a systemic treatment is recommended after testing for the expression of human epidermal growth factor receptor-2 (HER-2) in order to check whether trastuzumab should be added to well-established chemotherapeutic regimens [106]. The most commonly used first-line chemotherapy regimens for metastatic disease combine a platinum and a fluoropyrimidine compound, such as FOLFOX, CAPOX, cisplatin/5-fluorouracil (5-FU) or cisplatin/capecitabine [98,107]. These combinations have demonstrated prolongation of survival by approximately 7 months compared to palliative care alone [97]. Recent advances in immunotherapy have revolutionized the treatment of oncological patients [108][109][110][111]. Immunotherapy utilizes monoclonal antibodies to target tumor-produced immune checkpoint receptors, such as Programed cell Death protein-1 (PD-1), Programed cell Death protein Ligand-1 (PD-L1) and Cytotoxic T-Lymphocyte-associated Antigen 4 (CTLA-4) that prevent T cells from recognizing and eliminating cancer cells [112,113]. Recently, concerning the initial treatment of patients with advanced or metastatic gastric cancer, the addition of immunotherapy, namely of PD-1 inhibitor nivolumab to chemotherapy, achieved a survival benefit after more than a decade [114]. Moreover, the anti-PD1 antibody pembrolizumab has been approved for the treatment of patients with advanced, recurrent or metastatic gastric cancer with either microsatellite instability (MSI-H) or DNA mismatch repair deficiency (dMMR) [115]. Importantly, differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 from HDGC patients versus sporadic gastric cancer cells identified increased sensitivity to EGFR inhibitors, including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C) and TOPO2 (Topoisomerase II) inhibitors [116], suggesting that anti-mTOR, anti-PI3K and anti-EGFR therapies should be clinically trialed in HDGC patients. Moreover, Beetham et al. reported the identification of two novel compounds with significant synthetic lethal activity that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC [117].
Moreover, there have been several studies on cell-therapy applications in solid tumors. Chimeric antigen receptor (CAR)-T cell immunotherapy using genetic engineering to harness the anti-tumor activity of T-lymphocytes. T cells from the patients' bloodstream were modified using viral vectors to introduce the CAR to recognize a particular tumor antigen and then were infused back into the patient [118]. Promising results have been shown in preclinical models where engineering of CAR-T cells with integrin αEβ7 results in augmented therapeutic efficacy against E-cadherin positive tumors [119]. In addition, CD24 is an attractive prognostic factor for HDGC [120], and therefore, a potential target for CAR-T cells [121]. Nevertheless, a number of factors result in a reduction in the effectiveness of CAR-T cell therapy in solid tumors, including immunosuppressive tumor microenvironment (TME). TME significantly weakens T-cell function by overexpressing inhibitory receptors and immunosuppressive cells, such as regulatory T-cells (Tregs); and tumor-associated neutrophils (TAN); myeloid-derived suppressor cells (MDSC); and tumorassociated macrophages (TAM), which facilitate tumor immune escape [122]. On the other hand, IL-12 has been proved efficacious in reversing the TME to Th1 anti-tumor phenotype [123]. In addition, E-cadherin also plays a crucial role in negatively regulating Wnt signaling [124] which in turn is correlated with the inhibition of migration and invasion of GC cells [125]. Moreover, down-regulation of Wnt/β-catenin is correlated with an increased sensitivity of GC cells to PD-1 antibody [126] providing new options also for HDGC therapeutic interventions in the future.

Conclusions
Despite the fact that HDGC is a rare cancer syndrome, it is highly penetrant and difficult to diagnose and manage. HDGC requires the successful collaboration of a team of experts, including a geneticist, a gastroenterologist, a surgeon and an oncologist for the best decision making at each stage of disease, in order to achieve optimal long-term results. The IGCLC 2010 criteria have evolved to help clinicians make the most effective decisions. Genetic counseling should be performed before testing, while it is crucial to identify a mutation and to further determine whether unaffected relatives are at risk for cancer. There has also been progress in endoscopic techniques for the surveillance of patients who wish to postpone surgery or for those whose risk is not well defined. Nevertheless, endoscopy does not appear to offer a reliable reduction of risk in individuals with a proven pathogenic germline CDH1 mutation. Therefore, prophylactic total gastrectomy remains the cornerstone of HDGC management. However, the impact of gastrectomy should not be underestimated, since it may have severe consequences on a metabolic and nutritional level and, therefore, patients' quality of life. Importantly, significant progress has been made in recent years in the identification of CDH1 mutations. Future efforts should be directed towards the development of improved genetic screening modalities and the identification of new genes, especially in CDH1-negative families, aiming at the early identification of asymptomatic carriers.

Conflicts of Interest:
The authors declare no conflict of interest.