Gastric cancer (GC) remains one of the most common deadly malignancies worldwide. Recently, several targeted therapeutics for treating unresectable or metastatic GC have been developed. Comprehensive characterization of the molecular profile and of the tumor immune microenvironment of GC has allowed researchers to explore promising biomarkers for GC treatment and has enabled a new paradigm in precision-targeted immunotherapy. In this article, we review established and promising new biomarkers relevant in GC, with a focus on their clinical implications, diagnostic methods, and the efficacy of targeted agents.

Inactivation of phosphatase and tensin homolog (PTEN) is caused by multiple mechanisms, and loss of PTEN activity is related to the progression of various cancers. In gastric cancer (GC), the relationship between the loss of PTEN protein expression and various genetic alterations remains unclear. The effects of microsatellite instability (MSI), Epstein–Barr virus (EBV), HER2 overexpression, and PD-L1 expression on PTEN mutation have not been fully explored. We performed comprehensive cancer panel tests with a cohort of 322 tumor samples from patients with advanced GC. Immunohistochemistry for PTEN protein was performed in all cases, and the loss of protein expression was defined as a complete absence of nuclear staining. In total, 34 cases (10.6%) had pathogenic PTEN mutations, of which 19 (55.9%) showed PTEN protein loss. The most common PTEN variants associated with protein loss were p.R130 (n = 4) followed by p.R335, p.L265fs, and deletions (n = 2). All the ten nonsense mutations identified in the samples resulted in PTEN inactivation. In the remaining 288 GC cases with wild-type PTEN, protein loss was found in 35 cases (12.2%). Thus, PTEN mutations were significantly associated with PTEN protein loss (p = 5.232 × 10⁻¹⁰), high MSI (p = 3.936 × 10⁻⁸), and EBV-positivity (p = 0.0071). In conclusion, our results demonstrate that loss-of-function mutations in PTEN are a frequent genetic mechanism of PTEN inactivation in GC.

Although immune checkpoint inhibitors can induce durable responses in patients with multiple types of advanced cancer, only a limited number of patients have a known reliable biomarker. This study aimed to validate the IMmunotherapy Against GastrIc Cancer (IMAGiC) model, which was developed based on a previous study of four-gene and PD-L1 level, to predict immunotherapy response. We developed a clinical assay for formalin-fixed paraffin-embedded samples using quantitative real-time polymerase chain reaction to measure the expression level of the previously published four-gene set. The predictive performance was validated in a cohort of 89 patients with several advanced tumor types. The IMAGiC score was derived from tumor samples of 89 patients consisting of eight cancer types, and 73 out of 89 patients available for clinical response were analyzed with clinicopathological factors. The IMAGiC group (responder vs. non-responder) was determined with a specific value of the IMAGiC score as a cutoff, which was set by log-rank statistics for progression-free survival (PFS) divided the patients into 56 (76.7%) non-responders and 17 (23.3%) responders. Clinical responders (complete response/partial response) were higher in the IMAGiC responder group than in the non-responder group (70.6 vs. 21.4%). The median PFS of the IMAGiC responder group and non-responder was 20.8 months (95% CI 9.1-not reached) and 6.7 months (95% CI 4.9–11.1, p = 0.007), respectively. Among the 17 IMAGiC responders, 11 patients had tumor mutation burden-low and microsatellite-stable tumors. This study validated a predictive model based on a four-gene expression signature. Along with conventional biomarkers, our model could be useful for predicting response to immunotherapy in patients with advanced cancer.

BRAF mutation is an important diagnostic and prognostic marker in patients with papillary thyroid carcinoma (PTC). To be applicable in clinical laboratories with limited equipment, diverse testing methods are required to detect BRAF mutation. A shifted termination assay (STA) fragment analysis was used to detect common V600 BRAF mutations in 159 PTCs with DNAs extracted from formalin-fixed paraffin-embedded tumor tissue. The results of STA fragment analysis were compared to those of direct sequencing. Serial dilutions of BRAF mutant cell line (SNU-790) were used to calculate limit of detection (LOD). BRAF mutations were detected in 119 (74.8%) PTCs by STA fragment analysis. In direct sequencing, BRAF mutations were observed in 118 (74.2%) cases. The results of STA fragment analysis had high correlation with those of direct sequencing (p < 0.00001, κ = 0.98). The LOD of STA fragment analysis and direct sequencing was 6% and 12.5%, respectively. In PTCs with pT3/T4 stages, BRAF mutation was observed in 83.8% of cases. In pT1/T2 carcinomas, BRAF mutation was detected in 65.9% and this difference was statistically significant (p = 0.007). Moreover, BRAF mutation was more frequent in PTCs with extrathyroidal invasion than tumors without extrathyroidal invasion (84.7% versus 62.2%, p = 0.001). To prepare and run the reactions, direct sequencing required 450 minutes while STA fragment analysis needed 290 minutes. STA fragment analysis is a simple and sensitive method to detect BRAF V600 mutations in formalin-fixed paraffin-embedded clinical samples. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5684057089135749

Fibroblast growth factor receptor-2 (FGFR2) gene alterations have been identified in solid tumors. FGFR2 amplification is found in 2–9% of gastric carcinomas. We hypothesized that FGFR2 could be associated with peritoneal seeding and studied 360 advanced gastric carcinoma patients; 222 (61.7%) were male, 246 (73.7%) had poorly differentiated histology, and 175 (48.6%) presented with peritoneal seeding. High tumor mutation burden (TMB) was observed in 44 (12.2%) patients, high microsatellite instability (MSI) was observed in 12 (3.33%) patients, ERBB2 amplification was observed in 44 (12.2%) patients, EBV positivity was observed in 10 (10/278; 3.6%) patients, and PD-L1 positivity was observed in 186 (186/264; 70.5%) cases. We found FGFR2 amplification in 26 (7.2%) patients, of which 12 (46.2%) were female and 22 (84.6%) had poorly differentiated histology. In these 26 cases, the copy number of FGFR2 amplification ranged from 3.7 to 274. Eighteen of them showed seeding, and this association was statistically significant (18/26, 69.2%; 157/334, 47%; p = 0.023). In addition, high TMB was significantly associated with seeding (p = 0.028; OR = 1.83). Poorly differentiated histology was significantly associated with seeding (p = 0.04) but not with FGFR2 amplification (p > 0.1). Seeding was frequent in gastric carcinoma patients with FGFR2 amplification, in patients with high TMB, or in those who were female. The subgroup of patients with FGFR2 amplification could be potential candidates for targeted therapeutic agents.

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.