**3. Molecular classification of gastric cancers by "The Cancer Genome Atlas (TCGA)" project**

The goals of "The Cancer Genome Atlas (TCGA)" project were to develop a molecular classification of gastric cancer with clinical impact and to detect the major dysregulated pathways of distinct subtypes of gastric cancer [31].

The researchers have analyzed fresh frozen gastric adenocarcinoma primary tumor tissue from 295 patients with no prior chemo- or radiotherapy, using six genomic and molecular platforms including genome/exome/methylome DNA sequencing, RNA sequencing, and protein arrays. Germline DNA from blood/nonmalignant gastric mucosa was used as a control for detecting somatic alterations. Nonmalignant gastric samples were also collected for DNA methylation (*n* = 527) and expression (*n* = 529) assessment.

Tumors were first subgrouped by EBV-positivity (9%), then by MSI-high status (named MSI; 22%), and the remaining tumors were classified by degree of aneuploidy into genomically stable cancers (20%) or those exhibiting chromosomal instability (CIN; 50%).

This project revealed that the vast majority of the diffuse histological subtype belongs to the genomically stable group. CIN tumors were mostly located in the gastroesophageal junction/ cardia, whereas most of the EBV-positive tumors were located in the gastric fundus or body. Genomically, stable tumors were diagnosed at an earlier age compared to MSI tumors; most EBV-positive cases were male (81%), in concordance with previous results [32].

As previously reported [33], all EBV-positive tumors exhibited extreme CIMP, distinct from that in the MSI subtype [10] (CDKN2A promoter hypermethylation versus MLH1 hypermethylation) [34]. Furthermore, in concordance with prior data [35, 36], the study revealed a strong predilection for PIK3CA mutation in EBV positive tumors, with nonsilent PIK3CA mutations found in 80% of this subgroup (P < 0.001), that could be targeted using PI(3)-kinase inhibition.

By assessing 63 hypermutated tumors, there were identified 37 significantly mutated genes including TP53, KRAS, ARID1A, PIK3CA, ERBB3, PTEN, and HLA-B. The analysis of genes mutated within MSI subgroup of gastric cancers revealed alterations in major histocompatibility complex class I genes, a beneficial event for hypermutated tumors by dysregulating antigen presentation to the immune system.

Through the analysis of the 215 nonhypermutated cancers, there were identified 25 significantly mutated genes, including TP53, ARID1A, KRAS, PIK3CA, and RNF43, genes involved in the β-catenin pathway, the TGF-b pathway, RASA1, and ERBB2 (therapeutic target).

In addition to PIK3CA mutations, EBV-positive tumors had frequent ARID1A and BCOR (encoding an anti-apoptotic protein) gene mutations. TP53 mutations were detected in 71% of CIN tumors. The genomically stable subtype presented a high frequency of CDH1somatic mutations and inactivating ARID1A mutations. RHOA mutations were detected almost exclusively in genomically stable tumors. In its activated form, RHOA controls cellular motility [37, 38] and activates STAT3 to initiate tumorigenesis [39, 40]. It seems that the activation of RHOA-driven pathways contribute to the invasive phenotype of diffuse gastric cancer.

Oncogenic signaling pathways, including candidate therapeutic targets such as receptor tyrosine kinases (RTKs), RAS, and PI(3)-kinase signaling were characterized. EBV-positive tumors contained PIK3CA mutations and recurrent JAK2 and ERBB2 amplifications. MSI cases presented some mutations in PIK3CA, ERBB3, ERBB2, and EGFR [41]. The genomically stable subtype expressed recurrent RHOA and CLDN 18 events. CIN tumors showed genomic amplifications of RTKs that may be therapeutically blocked. Recurrent amplification of the gene encoding ligand VEGFA and frequent amplifications of cell cycle mediators highlight the role of the VEGFR2 targeting antibody (the already approved agent ramucirumab) [42] and suggest the possible efficacy of cyclin-dependent kinases.

All the subtypes (to a lesser degree the genomically stable tumors) showed increased expression of components involved in the mitotic process, such as AURKA/B and E2F, DNA damage response pathways, targets of MYC activation, and FOXM1 and PLK1 signaling pathways. In addition, the genomically stable subtype exhibited elevated expression of cell adhesion pathways, the B1/B3 integrins, syndecan-1 mediated signaling, and angiogenesis-related pathways, suggesting more potential therapeutic targets, including the aurora kinases (AURKA/B) and polo-like (PLK) family members. The elevated IL-12-mediated signaling expression, along with evidence of PD-L1/2 overexpression in EBV-positive tumors, suggests the importance of immune checkpoint inhibitors evaluation in this subtype of gastric cancer [31].

including genome/exome/methylome DNA sequencing, RNA sequencing, and protein arrays. Germline DNA from blood/nonmalignant gastric mucosa was used as a control for detecting somatic alterations. Nonmalignant gastric samples were also collected for DNA methylation

Tumors were first subgrouped by EBV-positivity (9%), then by MSI-high status (named MSI; 22%), and the remaining tumors were classified by degree of aneuploidy into genomically

This project revealed that the vast majority of the diffuse histological subtype belongs to the genomically stable group. CIN tumors were mostly located in the gastroesophageal junction/ cardia, whereas most of the EBV-positive tumors were located in the gastric fundus or body. Genomically, stable tumors were diagnosed at an earlier age compared to MSI tumors; most

As previously reported [33], all EBV-positive tumors exhibited extreme CIMP, distinct from that in the MSI subtype [10] (CDKN2A promoter hypermethylation versus MLH1 hypermethylation) [34]. Furthermore, in concordance with prior data [35, 36], the study revealed a strong predilection for PIK3CA mutation in EBV positive tumors, with nonsilent PIK3CA mutations found in 80% of this subgroup (P < 0.001), that could be targeted using PI(3)-kinase inhibition. By assessing 63 hypermutated tumors, there were identified 37 significantly mutated genes including TP53, KRAS, ARID1A, PIK3CA, ERBB3, PTEN, and HLA-B. The analysis of genes mutated within MSI subgroup of gastric cancers revealed alterations in major histocompatibility complex class I genes, a beneficial event for hypermutated tumors by dysregulating

Through the analysis of the 215 nonhypermutated cancers, there were identified 25 significantly mutated genes, including TP53, ARID1A, KRAS, PIK3CA, and RNF43, genes involved in the β-catenin pathway, the TGF-b pathway, RASA1, and ERBB2 (therapeutic target).

In addition to PIK3CA mutations, EBV-positive tumors had frequent ARID1A and BCOR (encoding an anti-apoptotic protein) gene mutations. TP53 mutations were detected in 71% of CIN tumors. The genomically stable subtype presented a high frequency of CDH1somatic mutations and inactivating ARID1A mutations. RHOA mutations were detected almost exclusively in genomically stable tumors. In its activated form, RHOA controls cellular motility [37, 38] and activates STAT3 to initiate tumorigenesis [39, 40]. It seems that the activation of RHOA-driven pathways contribute to the invasive phenotype of diffuse gastric cancer.

Oncogenic signaling pathways, including candidate therapeutic targets such as receptor tyrosine kinases (RTKs), RAS, and PI(3)-kinase signaling were characterized. EBV-positive tumors contained PIK3CA mutations and recurrent JAK2 and ERBB2 amplifications. MSI cases presented some mutations in PIK3CA, ERBB3, ERBB2, and EGFR [41]. The genomically stable subtype expressed recurrent RHOA and CLDN 18 events. CIN tumors showed genomic amplifications of RTKs that may be therapeutically blocked. Recurrent amplification of the gene encoding ligand VEGFA and frequent amplifications of cell cycle mediators highlight the role of the VEGFR2 targeting antibody (the already approved agent ramucirumab)

[42] and suggest the possible efficacy of cyclin-dependent kinases.

stable cancers (20%) or those exhibiting chromosomal instability (CIN; 50%).

EBV-positive cases were male (81%), in concordance with previous results [32].

(*n* = 527) and expression (*n* = 529) assessment.

112 Gastric Cancer

antigen presentation to the immune system.

Therefore, the four major genomic subtypes defined by "The Cancer Genome Atlas (TCGA)" may provide a guide to molecular targeted agents that should be assessed in clinical trials for distinct populations of gastric cancer patients.
