Gastric cancer (GC) pathogenesis is complex and heterogeneous, reflecting morphological, molecular, and genetic diversity. Diffuse gastric cancer (DGC) and intestinal gastric cancer (IGC) are the major histological types. GC may be sporadic or hereditary; sporadic GC is related to environmental and genetic low‐risk factors and hereditary GC is caused by inherited high‐risk mutations, so far identified only for the diffuse histotype. DGC phenotypic heterogeneity challenges the current understanding of molecular mechanisms underlying carcinogenesis. The definition of a DGC‐specific mutational profile remains controversial, possibly reflecting the heterogeneity of DGC‐related histological subtypes (Signet‐Ring Cell Carcinoma (SRCC) and Poorly Cohesive Carcinoma not otherwise specified (PCC‐NOS)). Indeed, DGC and DGC‐related subtypes may present specific mutational profiles underlying the particularly aggressive behaviour and dismal prognosis of DGC vs IGC and PCC‐NOS vs SRCC. In this systematic review, we revised the histological presentations, molecular classifications, and approved therapies for gastric cancer, with a focus on DGC. We then analysed results from the most relevant studies, reporting mutational analysis data specifying mutational frequencies, and their relationship with DGC and IGC histological types, and with specific DGC subtypes (SRCC and PCC‐NOS). We aimed at identifying histology‐associated mutational profiles with an emphasis in DGC and its subtypes (DGC vs IGC; sporadic vs hereditary DGC; and SRCC vs PCC‐NOS). We further used these mutational profiles to identify the most commonly affected molecular pathways and biological functions, and explored the clinical trials directed specifically to patients with DGC. This systematic analysis is expected to expose a DGC‐specific molecular profile and shed light into potential targets for therapeutic intervention, which are currently missing.

E-cadherin is a key player in gastric cancer (GC) and germline alterations of CDH1, its encoding gene, are responsible for Hereditary Diffuse Gastric Cancer (HDGC) syndrome. This study aimed at elucidating the role of genetic variants and DNA methylation of CDH1 promoter and enhancers in the regulation of gene expression. For this purpose, we analyzed genetic variants of the CDH1 gene through Next-Generation Sequencing (NGS) in a series of GC cell lines (NCI-N87, KATO-III, SNU-1, SNU-5, GK2, AKG, KKP) and the corresponding CDH1 expression levels. By bisulfite genomic sequencing, we analyzed the methylation status of CDH1 regulatory regions in 8 GC cell lines, in a series of 13 sporadic GC tissues and in a group of 20 HDGC CDH1-negative patients and 6 healthy controls. The NGS analysis on CDH1 coding and regulatory regions detected genetic alterations in 3 out of 5 GC cell lines lacking functional E-cadherin. CDH1 regulatory regions showed different methylation patterns in patients and controls, GC cell lines and GC tissues, expressing different E-cadherin levels. Our results showed that alterations in terms of genetic variants and DNA methylation patterns of both promoter and enhancers are associated with CDH1 expression levels and have a role in its regulation.

Hereditary diffuse gastric cancer (HDGC) caused by CDH1 variants predisposes to early-onset diffuse gastric (DGC) and lobular breast cancer (LBC). In Northern Portugal, the unusually high number of HDGC cases in unrelated families carrying the c.1901C>T variant (formerly known as p.A634V) suggested this as a CDH1-founder variant. We aimed to demonstrate that c.1901C>T is a bona fide truncating variant inducing cryptic splicing, to calculate the timing of a potential founder effect, and to characterize tumour spectrum and age of onset in carrying families. The impact in splicing was proven by using carriers’ RNA for PCR-cloning sequencing and allelic expression imbalance analysis with SNaPshot. Carriers and noncarriers were haplotyped for 12 polymorphic markers, and the decay of haplotype sharing (DHS) method was used to estimate the time to the most common ancestor of c.1901C>T. Clinical information from 58 carriers was collected and analysed. We validated the cryptic splice site within CDH1-exon 12, which was preferred over the canonical one in 100% of sequenced clones. Cryptic splicing induced an out-of-frame 37bp deletion in exon 12, premature truncation (p.Ala634ProfsTer7), and consequently RNA mediated decay. The haplotypes carrying the c.1901C>T variant were found to share a common ancestral estimated at 490 years (95% Confidence Interval 445–10,900). Among 58 carriers (27 males (M)–31 females (F); 13–83 years), DGC occurred in 11 (18.9%; 4M–7F; average age 33 ± 12) and LBC in 6 females (19.4%; average age 50 ± 8). Herein, we demonstrated that the c.1901C>T variant is a loss-of-function splice-site variant that underlies the first CDH1-founder effect in Portugal. Knowledge on this founder effect will drive genetic testing of this specific variant in HDGC families in this geographical region and allow intrafamilial penetrance analysis and better estimation of variant-associated tumour risks, disease age of onset, and spectrum.

CRISPR/Cas9-mediated homology-directed repair (HDR) allows precise gene editing, but its efficiency remains low for certain cell types, such as human induced pluripotent stem cells (hiPSCs). In this study, we aimed to introduce the CTNNA1: c.2023C>T (p.Q675*) genetic alteration, which is associated with Hereditary Diffuse Gastric Cancer, into hiPSCs using CRISPR/Cas9. We designed a single-guide RNA targeting the alteration site and a single-stranded oligonucleotide donor DNA template for HDR-based repair. Herein, we report the successful introduction of the CTNNA1: c.2023C>T homozygous alteration in one hiPSC line, which resulted in severe phenotypic changes, including impaired colony formation and cell proliferation. Additionally, we established a straightforward protocol to assess hiPSCs karyotype integrity, ensuring the chromosomal stability required for the gene-editing process. This protocol involves routine G-banding analysis that is required for regular quality controls during handling of hiPSCs. This study demonstrates an efficient approach to precisely edit hiPSCs by CRISPR/Cas9 and highlights the essential role of CTNNA1 expression in maintaining hiPSC viability. Our methodology provides a valuable framework for modeling disease-associated alterations in human-derived cellular models that can be reproduced for other genes and other types of cell lines.