Oncogenic Fatty Acid Metabolism Rewires Energy Supply Chain in Gastric Carcinogenesis.

BACKGROUND & AIMS: Gastric carcinogenesis develops within a sequential carcinogenic cascade from precancerous metaplasia to dysplasia and adenocarcinoma, and oncogenic gene activation can drive the process. Metabolic reprogramming is considered a key mechanism for cancer cell growth and proliferation. However, how metabolic changes contribute to the progression of metaplasia to dysplasia remains unclear. We have examined metabolic dynamics during gastric carcinogenesis using a novel mouse model that induces Kras activation in zymogen-secreting chief cells. METHODS: We generated a Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse model that continuously induces active Kras expression in chief cells after doxycycline treatment. Histologic examination and imaging mass spectrometry were performed in the GCK mouse stomachs at 2 to 14 weeks after doxycycline treatment. Mouse and human gastric organoids were used for metabolic enzyme inhibitor treatment. The GCK mice were treated with a stearoyl- coenzyme A desaturase (SCD) inhibitor to inhibit the fatty acid desaturation. Tissue microarrays were used to assess the SCD expression in human gastrointestinal cancers. RESULTS: The GCK mice developed metaplasia and high-grade dysplasia within 4 months. Metabolic reprogramming from glycolysis to fatty acid metabolism occurred during metaplasia progression to dysplasia. Altered fatty acid desaturation through SCD produces a novel eicosenoic acid, which fuels dysplastic cell hyperproliferation and survival. The SCD inhibitor killed both mouse and human dysplastic organoids and selectively targeted dysplastic cells in vivo. SCD was up-regulated during carcinogenesis in human gastrointestinal cancers. CONCLUSIONS: Active Kras expression only in gastric chief cells drives the full spectrum of gastric carcinogenesis. Also, oncogenic metabolic rewiring is an essential adaptation for high-energy demand in dysplastic cells.

Clinicopathologic and molecular characterization of stages II-IV gastric cancer with Claudin 18.2 expression.

BACKGROUND: Claudin 18.2 (CLDN18.2) is a promising target for targeted therapies in gastric cancer (GC). This study investigated the prevalence of CLDN18.2 expression in patients with stages II-IV GC or gastroesophageal junction (GEJ) adenocarcinoma and its correlation with clinicopathologic features and other crucial GC biomarkers. METHODS: We enrolled 1000 patients diagnosed with stages II-IV GC after surgical treatment. Immunohistochemistry for CLDN18 (43-14A clone), PD-L1 (22C3 pharmDx), HER2, and FGFR2 was performed. CLDN18.2 positivity was defined as moderate-to-strong (2+/3+) membranous staining in ≥75% of tumor cells. CLDN18.2 expression was compared with biomarker expression, Epstein-Barr virus (EBV) association and microsatellite instability status, and clinicopathologic features. RESULT: CLDN18.2 was positive in 34.4% of the patients. CLDN18.2 positivity was significantly higher in the middle and upper thirds than in the lower third gastric location (P < .001), but there was no correlation with age, sex, or stage (P > .05). CLDN18.2 positivity was rare (2.8%) in mucinous adenocarcinoma but frequent (90.9%) in a majority of gastric carcinomas with lymphoid stroma. CLDN18.2 positivity was higher in EBV-associated (P < .001) and PD-L1-positive (PD-L1 CPS ≥ 5) GC (P = .014) but lower in HER2 positive GC (P = .005). CLDN18.2 positivity was not significantly associated with overall survival and disease-free survival. CONCLUSION: This study provides a comprehensive evaluation of CLDN18.2 status and its correlation with the clinicopathologic characteristics of patients with stages II-IV GC in Korea and with crucial biomarkers. It may be valuable for guiding future drug development, expanding treatment options, and ultimately improving patient outcomes in GC.

Distinctive Phenotypic and Microenvironmental Characteristics of Neuroendocrine Carcinoma and Adenocarcinoma Components in Gastric Mixed Adenoneuroendocrine Carcinoma.

This study aimed to conduct an in-depth examination of gene expression and microenvironmental profiles of gastric neuroendocrine carcinoma (NEC) and mixed adeno-NEC (MANEC). Tissue microarrays from 55 patients with gastric MANEC (N = 32) or NEC (N = 23) were analyzed using digital spatial profiling (GeoMx DSP, NanoString Technologies). Representative regions of interest were selected from the adenocarcinoma (ADC) portion (ADC-MANEC) and the NEC portion (NEC-MANEC) of the MANEC cores, and pure NEC (pNEC) cores. All regions of interest were separated into epithelial components and stromal components using the masking procedure in the GeoMx platform, followed by transcriptome analysis. Comparison of gene expression between ADC-MANEC and NEC-MANEC/pNEC identified several differentially expressed genes in the epithelial (including PEG10, MAP1B, STMN3, and AKT3) and stromal (FN1, COL1A1, SPARC, and BGN) components. Gene set enrichment analysis revealed that pathways related to the E2F target and G2M checkpoint were more enriched in NEC-MANEC and pNEC than in ADC-MANEC. Deconvolution analysis showed that the microenvironmental profile varied according to histologic differentiation. In ADC-MANEC, intraepithelial infiltrating immune cells were relatively more numerous, whereas fibroblasts in the stroma were more abundant in NEC-MANEC and pNEC. This study confirmed the distinct expression profile of each histologic component of MANEC according to its tumor vs stromal compartment using the DSP platform. Although each component of MANEC shares the same genetic origin, distinctive phenotypes should not be overlooked when managing patients with MANEC. This study provides a useful validation data set for future studies.

Distribution of Perigastric Station 4d Lymph Nodes in Vascularized Gastroepiploic Lymph Node Transfer: An Anatomic Study and Case Series.

BACKGROUND: Vascularized gastroepiploic lymph node transfer (VGLNT) is a well-accepted surgical treatment for restoring physiological function in chronic lymphedema. However, the inclusion of substantial lymph nodes (LNs) in the flap remains uncertain. This study aimed to identify the anatomical basis for reliable flap harvest for VGLNT. PATIENTS AND METHODS: The anatomy of perigastric station 4d LNs was studied in healthy cadavers (n = 15) and patients with early gastric cancer (EGC) (n = 27). The omentum was divided into three segments: proximal, middle, and distal from the origin of the right gastroepiploic vessels. The flap dimension, number, location, size of LNs, and caliber of the vessels were reviewed. Eight patients underwent VGLNT for upper/lower limb lymphedema. RESULTS: The mean numbers of LNs in the proximal, middle, and distal segment were 2.5, 1.4, 0.5 in the cadavers, and 4.9, 2.7, 0.7 in the gastrectomy specimens, respectively. The proximal third included a significantly greater number of LNs than the distal third in the cadaveric (p = 0.024) and ECG (p = 0.016) specimens. A total of 95% of the LNs were located within proximal two-thirds of the flap from the vessel origin both in the cadavers (21.0 × 5.0 cm) and in the gastrectomy specimens (20 × 3.5 cm). In VGLNT, the transferred flap was 25.5 ± 6.9 × 4.1 + 0.7 cm in dimension, containing a mean number of 6.5 ± 1.9 LNs. At postoperative 6 months, the volumetric difference was significantly reduced by 22.8 ± 9.2% (p < 0.001). CONCLUSIONS: This study provides a distinct distribution pattern of station 4d LNs. Inclusion of the proximal two-thirds of the flap, which carries majority of the LNs, is recommended for VGLNT.

Sex-dependent different clinicopathological characterization of Epstein-Barr virus-associated gastric carcinoma: a large-scale study.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) has been reported to account for approximately 5-16% of all GCs with good prognosis compared to EBV-negative GC. We evaluated the clinicopathological characteristics of EBVaGC including survival rate in South Korea. METHODS: A total of 4,587 patients with GC who underwent EBV in situ hybridization (EBV-ISH) were prospectively enrolled at the Seoul National University Bundang Hospital from 2003 to 2021. Age, sex, smoking status, cancer type and stage, tumor size and location, histological type, molecular features and survival information were analyzed. RESULTS: A total of 456 patients with GC (9.9%) were positive for EBV. The EBVaGC group displayed a higher proportion of males (P < 0.001), a predominant presence in the proximal stomach (P < 0.001), a higher proportion of undifferentiated cancer (P < 0.001), and a lower cancer stage (P = 0.004) than the EBV-negative group. Cox multivariate analyses revealed age (hazard ratio [HR] = 1.025, P < 0.001), tumor size (HR = 1.109, P < 0.001), and cancer stage (stage2 HR = 4.761, P < 0.001; stage3 HR = 13.286, P < 0.001; stage4 HR = 42.528, P < 0.001) as significant risk factors for GC-specific mortality, whereas EBV positivity was inversely correlated (HR = 0.620, P = 0.022). Furthermore, the EBVaGC group displayed statistically significant survival advantages over the EBV-negative cancer group in terms of both overall (P = 0.021) and GC-specific survival (P = 0.007) on the Kaplan-Meier survival curve. However, this effect was evident only in males. CONCLUSIONS: EBVaGC patients showed better prognoses despite their association with proximal location and poorly differentiated histology in male, probably due to the difference in immunity between males and females.

AZD8186 in Combination With Paclitaxel in Patients With Advanced Gastric Cancer: Results From a Phase Ib/II Study (KCSG ST18-20).

BACKGROUND: Loss of PTEN function leads to increased PI3Kß signaling. AZD8186, a selective PI3Kß/δ inhibitor, has shown anti-tumor activity in PTEN-deficient preclinical models. Although the combination of AZD8186 and paclitaxel was well tolerated, limited clinical efficacy was observed in advanced gastric cancer with PTEN loss. METHODS: In the phase Ib dose-escalation, subjects with advanced solid tumors received oral AZD8186 (60 mg or 120 mg; twice daily (BID); 5 days on/2 days off) plus intravenous paclitaxel (70 mg/m2 or 80 mg/m2; days 1, 8, and 15) every 4 weeks. In the phase II part, MRGC patients with PTEN loss or PTEN/PIK3CB gene abnormality were enrolled and received recommended phase II dose (RP2D) of AZD8186 plus paclitaxel. Primary endpoints were to determine maximum tolerated dose (MTD) and RP2D in phase Ib and 4-month progression-free survival (PFS) rate in phase II. RESULTS: In phase Ib, both MTD and RP2D were determined at paclitaxel 80 mg/m2 and AZD8186 120 mg BID. In phase II, 18 patients were enrolled [PTEN loss (n = 18) and PIK3CB mutation (n = 1)]. The 4-month PFS rate was 18.8% (3 of 16 evaluable patients) and further enrollment stopped due to futility. CONCLUSION: Although the combination of AZD8186 and paclitaxel was well tolerated, limited clinical efficacy was observed.ClinicalTrials.gov Identifier: NCT04001569.

Dysplastic Stem Cell Plasticity Functions as a Driving Force for Neoplastic Transformation of Precancerous Gastric Mucosa.

BACKGROUND & AIMS: Dysplasia carries a high risk of cancer development; however, the cellular mechanisms for dysplasia evolution to cancer are obscure. We have previously identified 2 putative dysplastic stem cell (DSC) populations, CD44v6neg/CD133+/CD166+ (double positive [DP]) and CD44v6+/CD133+/CD166+ (triple positive [TP]), which may contribute to cellular heterogeneity of gastric dysplasia. Here, we investigated functional roles and cell plasticity of noncancerous Trop2+/CD133+/CD166+ DSCs initially developed in the transition from precancerous metaplasia to dysplasia in the stomach. METHODS: Dysplastic organoids established from active Kras-induced mouse stomachs were used for transcriptome analysis, in vitro differentiation, and in vivo tumorigenicity assessments of DSCs. Cell heterogeneity and genetic alterations during clonal evolution of DSCs were examined by next-generation sequencing. Tissue microarrays were used to identify DSCs in human dysplasia. We additionally evaluated the effect of casein kinase 1 alpha (CK1α) regulation on the DSC activities using both mouse and human dysplastic organoids. RESULTS: We identified a high similarity of molecular profiles between DP- and TP-DSCs, but more dynamic activities of DP-DSCs in differentiation and survival for maintaining dysplastic cell lineages through Wnt ligand-independent CK1α/ß-catenin signaling. Xenograft studies demonstrated that the DP-DSCs clonally evolve toward multiple types of gastric adenocarcinomas and promote cancer cell heterogeneity by acquiring additional genetic mutations and recruiting the tumor microenvironment. Last, growth and survival of both mouse and human dysplastic organoids were controlled by targeting CK1α. CONCLUSIONS: These findings indicate that the DSCs are de novo gastric cancer-initiating cells responsible for neoplastic transformation and a promising target for intervention in early induction of gastric cancer.

Evaluation of an eight marker-panel including long mononucleotide repeat markers to detect microsatellite instability in colorectal, gastric, and endometrial cancers.

BACKGROUND: Accurate determination of microsatellite instability (MSI) status is critical for optimal treatment in cancer patients. Conventional MSI markers can sometimes display subtle shifts that are difficult to interpret, especially in non-colorectal cases. We evaluated an experimental eight marker-panel including long mononucleotide repeat (LMR) markers for detection of MSI. METHODS: The eight marker-panel was comprised of five conventional markers (BAT-25, BAT-26, NR-21, NR-24, and NR-27) and three LMR markers (BAT-52, BAT-59 and BAT-62). MSI testing was performed against 300 specimens of colorectal, gastric, and endometrial cancers through PCR followed by capillary electrophoresis length analysis. RESULTS: The MSI testing with eight marker-panel showed 99.3% (295/297) concordance with IHC analysis excluding 3 MMR-focal deficient cases. The sensitivity of BAT-59 and BAT-62 was higher than or comparable to that of conventional markers in gastric and endometrial cancer. The mean shift size was larger in LMR markers compared to conventional markers for gastric and endometrial cancers. CONCLUSIONS: The MSI testing with eight maker-panel showed comparable performance with IHC analysis. The LMR markers, especially BAT-59 and BAT-62, showed high sensitivity and large shifts which can contribute to increased confidence in MSI classification, especially in gastric and endometrial cancers. Further study is needed with large number of samples for the validation of these LMR markers.

Safety and Efficacy of Intraperitoneal Paclitaxel Plus Intravenous Fluorouracil, Leucovorin, and Oxaliplatin (FOLFOX) for Gastric Cancer with Peritoneal Metastasis.

BACKGROUND: Peritoneal metastasis (PM) remains a major obstacle in the treatment of stage IV gastric cancer. This is a dose-escalation study of intraperitoneal (IP) paclitaxel combined with intravenous (IV) fluorouracil, leucovorin, and oxaliplatin (FOLFOX) to determine the recommended phase II dose in gastric cancer patients. METHODS: Patients with gastric adenocarcinoma and PM were enrolled. The recommended phase II dose of IP paclitaxel was determined using the standard "3 + 3" dose escalation with planned doses ranging from 40 to 100 mg/m2. IV FOLFOX was administered on the same day (oxaliplatin 100 mg/m2 (day 1), leucovorin 100 mg/m2 (day 1), fluorouracil 2,400 mg/m2 over 46 hours (day 1)). Both IP and IV regimens were repeated every 2 weeks. RESULTS: Among the 13 patients, there was no DLT at 40 and 60 mg/m2. Two patients had grade 3 febrile neutropenia at 80 mg/m2, and the recommended phase II dose was 60 mg/m2. Other patients underwent IP paclitaxel and FOLFOX without serious adverse events. Seven patients underwent second-look diagnostic laparoscopy, and the average change in PCI score was -7.0 ± 9.7. Conversion surgery rate was 23.1% (n = 3). The median overall survival was 16.6 months (95% confidence interval, 16.6-N/A), and progression-free survival was 9.6 months (95% confidence interval, 4.7-N/A). All adverse events were tolerable and manageable. CONCLUSIONS: The biweekly regimen of IP paclitaxel and FOLFOX is safe and the recommended dose of IP paclitaxel for a phase II trial is 60 mg/m2.

Resolvin D1 suppresses inflammation-associated tumorigenesis in the colon by inhibiting IL-6-induced mitotic spindle abnormality.

While failure in resolution of inflammation is considered to increase the risk of tumorigenesis, there is paucity of experimental as well as clinical evidence supporting this association. Resolvin D1 (RvD1) is a representative pro-resolving lipid mediator that is endogenously generated from docosahexaenoic acid for the resolution of inflammation. Here, we report a decreased level of RvD1 in the blood from colorectal cancer patients and mice having inflammation-induced colon cancer, suggesting plasma RvD1 as a potential biomarker for monitoring colorectal cancer. Administration of RvD1 attenuated dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM) plus DSS-induced colorectal carcinogenesis by suppressing the production of interleukin-6 (IL-6) and IL-6-mediated chromosomal instability. The protective effect of RvD1 against chromosomal instability is associated with downregulation of IL-6-induced Cyclin D1 expression, which appears to be mediated by blocking the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) axis. RvD1 inhibited the STAT3 signaling pathway by interfering with the binding of IL-6 to its receptor (IL-6R), suggesting the novel function of RvD1 as a putative IL-6R antagonist. Together, our findings suggest that RvD1-mediated blockade of IL-6 signal transmission may contribute to inhibition of chromosomal instability and tumorigenesis.

Effect of Helicobacter pylori infection and its eradication on the expression of tight junction proteins in the gastric epithelium in relation to gastric carcinogenesis.

BACKGROUND: Tight junction proteins (TJPs) play a role in epithelial defense mechanisms. However, the effect of Helicobacter pylori (Hp) on TJPs remains unclear. This study aimed to evaluate the expression of TJPs in relation to Hp infection and eradication in gastric carcinogenesis. METHODS: In total, 510 subjects (284 controls and 226 gastric cancer [GC] patients) were prospectively enrolled in the study. The expression of claudin-1 and -2 (CLDN-1, -2), occludin (OCLN), and tight junction protein 1 (TJP1) was measured based on their Hp infection status in normal corpus mucosa and evaluated following Hp eradication using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC). RESULTS: The expression of TJP1 in Hp+ controls was significantly lower than that in Hp- controls (p = 0.006), whereas it was higher in Hp+ than in Hp- GC patients (p = 0.001). Moreover, the increased expression of TJP1 in Hp+ GC patients was reduced to levels in Hp- within a year after Hp eradication and was maintained for more than 5 years. Furthermore, IHC results for TJP1 were similar to qPCR results. In particular, the higher IHC staining intensity of TJP1 in the cytosol of GC patients (p = 0.019) decreased after Hp eradication (p = 0.040). CONCLUSION: Hp infection affects TJP expression. The high expression of TJP1 in Hp+ GC patients was restored to control levels after Hp eradication, suggesting that TJP1 plays a role in gastric carcinogenesis.

Comprehensive genetic features of gastric mixed adenoneuroendocrine carcinomas and pure neuroendocrine carcinomas.

We aimed to determine the pathogenesis of gastric mixed adenoneuroendocrine carcinoma (MANEC) and pure neuroendocrine carcinoma (NEC), which is largely unknown. Targeted DNA sequencing was performed on 34 tumor samples from 21 patients - 13 adenocarcinoma (ADC)/NEC components from MANECs and eight pure NECs - and 21 matched non-neoplastic gastric tissues. Mutational profiles of MANECs/NECs were compared with those of other tumors using public databases. The majority (64.1%; 59/92) of mutations in MANEC were shared by both ADC and NEC components. TP53 was the most commonly mutated gene in MANEC (69.2%, 9/13) and pure NEC (87.5%, 8/9). All TP53 mutations in MANEC were pathogenic mutations and were shared by both ADC and NEC components. A subset of TP53WT MANECs had a microsatellite-unstable phenotype or amplifications in various oncogenes including ERBB2 and NMYC, and the only TP53WT pure NEC harbored MYC amplification. Compared to NEC in other organs, NECs arising from the stomach had unique features including less frequent RB1 mutations. Differentially altered genes of MANEC ADC components were significantly associated with receptor tyrosine kinase signaling pathways, while differentially altered genes of MANEC NEC components were significantly associated with the NOTCH signaling pathway. Our data provide evidence suggesting a possible clonal origin of ADC and NEC components of MANEC, and we found that gastric MANECs and pure NECs are distinct entities with unique mutational profiles and underlying protein networks. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Combinatory statuses of tumor stromal percentage and tumor infiltrating lymphocytes as prognostic factors in stage III colorectal cancers.

BACKGROUND AND AIM: Tumor stroma and tumor-infiltrating lymphocytes (TILs) are major constituents of the tumor microenvironment, although they have different effects on the prognosis of patients with colorectal cancer (CRC). Combinatory statuses of tumor-stromal percentage (TSP) and TILs are expected to provide more powerful prognostic information but have never been studied in CRCs. METHODS: Stage III CRCs from patients (n = 487) treated with adjuvant chemotherapy were assessed for their TSP and CD3-TIL or CD8-TIL densities using computer-aided methodology. With cut-off values set at median values for intraepithelial TIL (iTIL) and stromal TIL (sTIL) densities, CRCs were sorted into low and high iTIL or sTIL groups. CRCs were classified into five quintile (Q1-Q5) groups according to their TSP and divided into high TSP (Q5) and low TSP (Q1-4) groups. RESULTS: The combination of CD8 iTIL density and TSP was found to be an independent prognostic parameter in multivariate survival analysis in terms of cancer-specific survival and recurrence-free survival. CRCs with low CD8 iTIL density and high TSP showed the worst survival. The combinatory status showed more prognostic power than CD8 iTIL density or TSP alone. Multivariate survival analysis in an independent cohort of stage III CRC validated the prognostic power of the combinatory statuses. CONCLUSIONS: The findings suggest that the combinatory status might serve as a prognostic parameter in stage III CRCs. Further research in a large-scale cohort of patients with stage III CRC is needed to validate the prognostic power of the combinatory status.

The Difference of Endoscopic and Histologic Improvements of Atrophic Gastritis and Intestinal Metaplasia After Helicobacter pylori Eradication.

BACKGROUND/AIMS: Helicobacter pylori (H. pylori) is an important risk factor of atrophic gastritis (AG), intestinal metaplasia (IM), and gastric cancer (GC). However, no report to date has described the endoscopic improvement of AG and IM after H. pylori eradication. Thus, the aim of this study was to evaluate the improvement of AG and IM after H. pylori eradication using endoscopic and histologic analyses. METHODS: A total of 380 subjects were prospectively enrolled for up to 12 years and grouped by their H. pylori infection status: negative, non-eradicated, and eradicated. Endoscopic and histologic analyses of AG and IM were performed in the antrum and the corpus, by annual follow-up endoscopy. RESULTS: Endoscopic AG and IM in the antrum and corpus in the eradicated group improved compared to that in the non-eradicated group (AG, P = 0.002 and P = 0.005; IM, P = 0.038 and P = 0.048, respectively). Histologic AG and IM in the antrum and corpus in the eradicated group also improved compared to that in the non-eradicated group (all P < 0.001). Time taken to the endoscopic improvement of AG and IM after H. pylori eradication was significantly longer than time taken to the histologic improvement in the antrum and corpus (AG in antrum: 3.47 ± 2.60 vs. 2.34 ± 1.71 years, P = 0.004; AG in corpus: 3.19 ± 2.30 vs. 1.87 ± 1.48 years, P = 0.002; IM in antrum: 4.40 ± 2.38 vs. 3.62 ± 2.35 years, P = 0.043; and IM in corpus: 4.82 ± 1.08 vs. 3.61 ± 2.22 years, P = 0.007, respectively). CONCLUSIONS: Both endoscopic and histologic improvements of AG and IM were observed after H. pylori eradication, while endoscopic improvement took significantly longer time than histologic improvement.

Expression of human leukocyte antigen class I and ß2-microglobulin in colorectal cancer and its prognostic impact.

Downregulation of human leukocyte antigen (HLA) class I has been postulated to be a mechanism of adaptive immune escape in various tumors, especially microsatellite instability-high (MSI-H) colorectal cancer (CRC). In this study, we aimed to investigate HLA class I and ß2-microglobulin (ß2M) expression in MSI-H and microsatellite-stable (MSS) CRCs and determine its prognostic impact. The representative areas from the tumor center (TC) and tumor periphery (TP) from 300 CRCs, including 161 MSI-H and 139 MSS cases, were selected to construct a tissue microarray. Immunohistochemistry (IHC) for HLA A/B/C, ß2M, CD3, and CD8 was performed. Reduced HLA A/B/C expression was detected in 113 (70.2%) MSI-H and 54 (38.8%) MSS cases, while reduced ß2M expression was observed in 69 (42.9%) MSI-H and 17 (12.2%) MSS cases. Although reduced ß2M expression was associated with higher pathological tumor (pT) stage in MSI-H CRC with borderline significance, no association was found between HLA A/B/C and ß2M expression and survival. Interestingly, reduced HLA A/B/C expression in MSS was associated with higher stage, and reduced HLA A/B/C and ß2M expression was an independent prognostic factor in multivariate analysis. In conclusion, reduced HLA A/B/C and ß2M expression was frequently observed in immunotherapy-naive MSI-H CRC, suggesting the possibility of primary resistance to immune checkpoint inhibitor. Interestingly, downregulation of HLA A/B/C and ß2M was associated with poor prognosis in MSS cancers. Overall, IHC for HLA A/B/C and ß2M might be a feasible predictive or prognostic tool in CRC.

Immunoscore is a strong predictor of survival in the prognosis of stage II/III gastric cancer patients following 5-FU-based adjuvant chemotherapy.

The prognostic impact of Immunoscore (IS) in gastric cancer (GC) patients treated with adjuvant chemotherapy remains unelucidated. We evaluated the CD3 + , CD8 + , and Foxp3 + T-lymphocyte densities in tumor centers and invasive margin regions of 389 patients with surgically resected stage II/III GC who received 5-FU-based adjuvant chemotherapy and investigated the impact of IS on survival. In univariate analysis, high CD3 + , CD8 + , and Foxp3 + T-lymphocyte densities in the invasive margin were correlated with better prognosis (all P < 0.05). Patients with high IS had significantly longer disease-free survival (DFS; P < 0.001) and overall survival (OS; P < 0.001). In multivariate analysis, IS demonstrated a powerful prognostic impact on patient outcome [DFS, hazard ratio (HR) = 0.465; 95% confidence interval (CI), 0.306-0.707, P < 0.001; OS, HR = 0.478; 95% CI, 0.308-0.743, P = 0.001]. Additionally, although all EBV-positive cases had high IS, IS was similar in both microsatellite instability (MSI)-high and microsatellite stable (MSS)/MSI-low groups (83.3% and 80.5%, respectively). Subgroup analysis according to MSI status revealed that high IS patients had significant DFS and OS benefits in both MSS/MSI-low (DFS, HR = 0.527, 95% CI, 0.341-0.816, P = 0.004; OS, HR = 0.528, 95% CI, 0.334-0.837, P = 0.007) and MSI-high (DFS, HR = 0.166, 95% CI, 0.033-0.826, P = 0.028; OS, HR = 0.177, 95% CI, 0.036-0.883, P = 0.035) groups. Thus, the assessment of immune cell infiltration based on IS may provide a strong indicator of survival in stage II/III GC patients with curative resection following 5-FU-based adjuvant chemotherapy.

Development of a Novel Orthotopic Gastric Cancer Mouse Model.

BACKGROUND: Gastric cancer metastasis is a highly fatal disease with a five-year survival rate of less than 5%. One major obstacle in studying gastric cancer metastasis is the lack of faithful models available. The cancer xenograft mouse models are widely used to elucidate the mechanisms of cancer development and progression. Current procedures for creating cancer xenografts include both heterotopic (i.e., subcutaneous) and orthotopic transplantation methods. Compared to the heterotopic model, the orthotopic model has been shown to be the more clinically relevant design as it enables the development of cancer metastasis. Although there are several methods in use to develop the orthotopic gastric cancer model, there is not a model which uses various types of tumor materials, such as soft tissues, semi-liquid tissues, or culture derivatives, due to the technical challenges. Thus, developing the applicable orthotopic model which can utilize various tumor materials is essential. RESULTS: To overcome the known limitations of the current orthotopic gastric cancer models, such as exposure of tumor fragments to the neighboring organs or only using firm tissues for the orthotopic implantation, we have developed a new method allowing for the complete insertion of soft tissue fragments or homogeneously minced tissues into the stomach submucosa layer of the immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. With this completely-closed transplantation method, tumors with various types of tissue may be used to establish orthotopic gastric cancer models without the risks of exposure to nearby organs or cell leakage. This surgical procedure was highly reproducible in generating forty-eight mouse models with a surgery success rate of 96% and tumor formation of 93%. Among four orthotopic patient-derived xenograft (PDX) models that we generated in this study, we verified that the occurrence of organotropic metastasis in either the liver or peritoneal cavity was the same as that of the donor patients. CONCLUSION: Here we describe a new protocol, step by step, for the establishment of orthotopic xenograft of gastric cancer. This novel technique will be able to increase the use of orthotopic models in broader applications for not only gastric cancer research but also any research related to the stomach microenvironment.

Prognostic significance of natural killer cell-associated markers in gastric cancer: quantitative analysis using multiplex immunohistochemistry.

BACKGROUND: Natural killer (NK) cells mediate the anti-tumoral immune response as an important component of innate immunity. The aim of this study was to investigate the prognostic significance and functional implication of NK cell-associated surface receptors in gastric cancer (GC) by using multiplex immunohistochemistry (mIHC). METHODS: We performed an mIHC on tissue microarray slides, including 55 GC tissue samples. A total of 11 antibodies including CD57, NKG2A, CD16, HLA-E, CD3, CD20, CD45, CD68, CK, SMA, and ki-67 were used. CD45 + CD3-CD57 + cells were considered as CD57 + NK cells. RESULTS: Among CD45 + immune cells, the proportion of CD57 + NK cell was the lowest (3.8%), whereas that of CD57 + and CD57- T cells (65.5%) was the highest, followed by macrophages (25.4%), and B cells (5.3%). CD57 + NK cells constituted 20% of CD45 + CD57 + immune cells while the remaining 80% were CD57 + T cells. The expression of HLA-E in tumor cells correlated with that in tumoral T cells, B cells, and macrophages, but not CD57 + NK cells. The higher density of tumoral CD57 + NK cells and tumoral CD57 + NKG2A + NK cells was associated with inferior survival. CONCLUSIONS: Although the number of CD57 + NK cells was lower than that of other immune cells, CD57 + NK cells and CD57 + NKG2A + NK cells were significantly associated with poor outcomes, suggesting that NK cell subsets play a critical role in GC progression. NK cells and their inhibitory receptor, NKG2A, may be potential targets in GC.

Expression of the immune checkpoint molecule V-set immunoglobulin domain-containing 4 is associated with poor prognosis in patients with advanced gastric cancer.

BACKGROUND: Recent clinical studies on immune checkpoint (IC) inhibitors in the context of advanced gastric cancer (AGC) have failed to show significant survival benefits but have suggested the possible role of IC inhibitors in anti-AGC immunity. Considering the low efficacy of targeted drugs in AGC, there is an urgent need for the discovery of new targets for the development of immunotherapeutics and prognostic markers for patient selection. This study aimed to investigate the expression of a new IC molecule, V-set Ig domain-containing 4 (VSIG4), and its clinical significance in AGC and other major cancers. METHODS: We analyzed the expression of VSIG4 and its correlation with survival in various carcinomas, including 882 surgically resected samples from patients with stage II-III AGC (two academic hospitals). RESULTS: VSIG4 positivity in AGC was significantly associated with overall survival (OS; Hazard ratio (HR) = 2.661, 95% confidence interval [CI] = 2.012-3.519, P < 0.001) and event-free survival (HR = 2.8, 95% CI = 2.18-3.72, P < 0.001). These findings were successfully validated in independent cohorts. VSIG4 expression was also significantly correlated with low intratumoral CD8 + T-cell infiltration (CD8i) (P = 0.029) and high Foxp3 + /CD8i ratio (P = 0.026), which is consistent with the previously reported immunological function of VSIG4. However, VSIG4 expression was not associated with survival in other cancers (colon, P = 0.459; lung, P = 0.275; kidney, P = 0.121; breast, P = 0.147). CONCLUSION: Our results suggest that VSIG4 is an independent prognostic factor in AGC and also implies that VSIG4 is a second-tier IC molecule in AGC, thus, providing an important basis for the development of gastric cancer-specific immunotherapeutics.

Different effects of p53 protein overexpression on the survival of gastric cancer patients according to Lauren histologic classification: a retrospective study.

BACKGROUND: Inactivation of TP53, a tumor suppressor gene, is associated with the development of several malignancies, including gastric cancer (GC). The present study aimed to evaluate the correlation between the overexpression of p53 and survival in different Lauren-type GCs. METHODS: From May 2003 to December 2019, 3608 GC patients treated endoscopically or surgically at the Seoul National University Bundang Hospital were enrolled for the study. Immunohistochemical staining for p53 was performed on all endoscopic and surgical gastric specimens. Clinicopathologic characteristics with Lauren classification, survival rate, and cancer recurrence were analyzed according to p53 overexpression. RESULTS: Among 3608 GC patients, p53 overexpression was seen in 1334 patients (37%). p53 overexpression was associated with lower depth of invasion (P = 0.026) and Early gastric cancer (P = 0.044) in intestinal-type GC, and with advanced TNM stage (P < 0.001) and Advanced gastric cancer (P < 0.001) in diffuse-type GC. The overall survival (OS) and GC-specific survival (GCSS) were significantly lower in p53 overexpression positive patients. This significance was more pronounced and enhanced in the diffuse-type GC and was absent in the intestinal-type GC. In multivariate analyses, p53 overexpression was associated with poor OS in both subtypes of GC and cancer recurrence in diffuse-type GC. (OS in intestinal-type: adjusted hazard ratio [aHR] = 1.423, P = 0.022; OS in diffuse-type: aHR = 1.401 P = 0.035; cancer recurrence in diffuse-type: aHR = 1.502, P = 0.039). CONCLUSION: p53 overexpression was associated with poor prognosis in GC, especially in diffuse-type. In addition, p53 overexpression was associated with early stage disease in intestinal-type GC and with advanced stage disease in diffuse-type GC.