Diffuse-type Gastric Cancer Patients Research Articles

Senescent Fibroblasts Potentiate Peritoneal Metastasis of Diffuse-type Gastric Cancer Cells via IL-8-mediated Crosstalk.

Diffuse-type gastric cancer (DGC) often forms peritoneal metastases, leading to poor prognosis. However, the underlying mechanism of DGC-mediated peritoneal metastasis is poorly understood. DGC is characterized by desmoplastic stroma, in which heterogeneous cancer-associated fibroblasts (CAFs), including myofibroblastic CAFs (myCAFs) and senescent CAFs (sCAFs), play a crucial role during tumor progression. This study investigated the CAF subtypes induced by GC cells and the role of sCAFs in peritoneal metastasis of DGC cells. Conditioned medium of human DGC cells (KATOIII, NUGC-4) and human intestinal-type GC (IGC) cells (MKN-7, N87) was used to induce CAFs. CAF subtypes were evaluated by analyzing the expression of α-smooth muscle actin (α-SMA), senescence-associated β-galactosidase (SA-β-gal), and p16 in human normal fibroblasts (GF, FEF-3). A cytokine array was used to explore the underlying mechanism of GC-induced CAF subtype development. The role of sCAFs in peritoneal metastasis of DGC cells was analyzed using a peritoneally metastatic DGC tumor model. The relationships between GC subtypes and CAF-related markers were evaluated using publicly available datasets. IGC cells significantly induced α-SMA+ myCAFs by secreting transforming growth factor-β, whereas DGC cells induced SA-β-gal+/p16+ sCAFs by secreting interleukin (IL)-8. sCAFs further secreted IL-8 to promote DGC cell migration. In vivo experiments demonstrated that co-inoculation of sCAFs significantly enhanced peritoneal metastasis of NUGC-4 cells, which was attenuated by administration of the IL-8 receptor antagonist navarixin. p16 and IL-8 expression was significantly associated with poor prognosis of DGC patients. sCAFs promote peritoneal metastasis of DGC via IL-8-mediated crosstalk.

Tubulointerstitial nephritis antigen-like 1 from cancer-associated fibroblasts contribute to the progression of diffuse-type gastric cancers through the interaction with integrin β1

BackgroundTumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression.MethodsWe conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH).ResultsCAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC.ConclusionsThese results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC.

E-cadherin is a biomarker for ferroptosis sensitivity in diffuse gastric cancer.

Gastric cancer is the third most common cause of cancer-related death worldwide. Diffuse-type gastric cancer (DGC) is a particularly aggressive subtype that is both difficult to detect and treat. DGC is distinguished by weak cell-cell cohesion, most often due to loss of the cell adhesion protein E-cadherin, a common occurrence in highly invasive, metastatic cancer cells. In this study, we demonstrate that loss-of-function mutation of E-cadherin in DGC cells results in their increased sensitivity to the non-apoptotic, iron-dependent form of cell death, ferroptosis. Homophilic contacts between E-cadherin molecules on adjacent cells suppress ferroptosis through activation of the Hippo pathway. Furthermore, single nucleotide mutations observed in DGC patients that ablate the homophilic binding capacity of E-cadherin reverse the ability of E-cadherin to suppress ferroptosis in both cell culture and xenograft models. Importantly, although E-cadherin loss in cancer cells is considered an essential event for epithelial-mesenchymal transition and subsequent metastasis, we found that circulating DGC cells lacking E-cadherin expression possess lower metastatic ability, due to their increased susceptibility to ferroptosis. Together, this study suggests that E-cadherin is a biomarker predicting the sensitivity to ferroptosis of DGC cells, both in primary tumor tissue and in circulation, thus guiding the usage of future ferroptosis-inducing therapeutics for the treatment of DGC.

IL-1β derived from mixed-polarized macrophages activates fibroblasts and synergistically forms a cancer-promoting microenvironment.

Remodeling the tumor microenvironment (TME) to benefit cancer cells is crucial for tumor progression. Although diffuse-type gastric cancer (DGC) preferentially interacts with the TME, the precise mechanism of the complicated network remains unknown. This study aimed to investigate the mutual activation mechanism underlying DGC progression. Mass cytometry analysis of co-cultured macrophages, noncancerous fibroblasts (NFs), and DGC cells was performed. RNA sequencing was applied to examine gene expression in fibroblasts. DGC cells were treated with cytokines to examine their effect on characteristic changes. The TCGA and Kumamoto University cohorts were used to evaluate the clinical relevance of the in vitro findings. Cohort analysis revealed that DGC patients had a poor prognosis. The fibroblasts and macrophages interacted with DGC cells to form a cell cluster in the invasive front of DGC tissue. The original 3D triple co-culture system determined the promotional effects of nonmalignant cells on DGC invasive growth. We notably identified a mixed-polarized macrophage cell type with M1/M2 cell surface markers in a triple co-culture system. IL-1β from mixed-polarized macrophages induced the conversion of NFs to cancer-associated fibroblast-like (CAF-like) cells, promoting the malignant phenotype of DGC cells by inducing the secretion of IL-6, IL-24, and leukemia inhibitory factor (LIF). Moreover, IL-6 and colony stimulating factor 2 (GM-CSF) cooperated to maintain the stable state of mixed-polarized macrophages. Finally, we found that mixed-polarized macrophages were frequently detected in DGC tissues. These findings demonstrated that mixed-polarized macrophages exist as a novel subtype through the reciprocal interaction between DGC cells and nonmalignant cells.

A combined analysis of bulk and single-cell sequencing data reveals that depleted extracellular matrix and enhanced immune processes co-contribute to fluorouracil beneficial responses in gastric cancer.

Fluorouracil, also known as 5-FU, is one of the most commonly used chemotherapy drugs in the treatment of advanced gastric cancer (GC). Whereas, the presence of innate or acquired resistance largely limits its survival benefit in GC patients. Although accumulated studies have demonstrated the involvement of tumor microenvironments (TMEs) in chemo-resistance induction, so far little is known about the relevance of GC TMEs in 5-FU resistance. To this end, in this study, we investigated the relationship between TME features and 5-FU responses in GC patients using a combined analysis involving both bulk sequencing data from the TCGA database and single-cell RNA sequencing data from the GEO database. We found that depleted extracellular matrix (ECM) components such as capillary/stroma cells and enhanced immune processes such as increased number of M1 polarized macrophages/Memory T cells/Natural Killer T cells/B cells and decreased number of regulatory T cells are two important features relating to 5-FU beneficial responses in GC patients, especially in diffuse-type patients. We further validated these two features in the tumor tissues of 5-FU-benefit GC patients using immunofluorescence staining experiments. Based on this finding, we also established a Pro (63 genes) and Con (199 genes) gene cohort that could predict 5-FU responses in GC with an AUC (area under curve) score of 0.90 in diffuse-type GC patients, and further proved the partial applicability of this gene panel pan-cancer-wide. Moreover, we identified possible communications mediated by heparanase and galectin-1 which could regulate ECM remodeling and tumor immune microenvironment (TIME) reshaping. Altogether, these findings deciphered the relationship between GC TMEs and 5-FU resistance for the first time, as well as provided potential therapeutic targets and predicting rationale to overcome this chemo-resistance, which could shed some light on developing novel precision treatment strategies in clinical practice.

Tumorigenic mechanisms of estrogen and Helicobacter pylori cytotoxin-associated gene A in estrogen receptor α-positive diffuse-type gastric adenocarcinoma.

Diffuse-type gastric cancer (DGC), for which Helicobacter pylori infection is a causal factor, is associated with poor prognosis among young women, possibly due to female hormones such as estrogen. We aimed to identify the carcinogenesis induced by estrogen and H. pylori in DGC. We screened and selected estrogen receptor alpha (ERα)-positive (MKN45) and ERα-negative (SNU5) DGC cell lines. H. pylori strain 60190 and its isogenic mutant strain lacking cytotoxin-associated gene A (60190ΔCagA) were used to infect MKN45 cells. And the cytotoxin-related gene A (CagA) cDNA which was cloned into pSP65-SR-HA (cagA-pSP65SRa) vector was used to transfect MKN45 cells. Tumor samples were used for DGC organoid culture. In MKN45 cells, we found that estradiol promotes epithelial-mesenchymal transition (EMT) and stemness phenotypes via HOTAIR expression. These effects were further enhanced by the addition of CagA secreted by H. pylori but were reversed by co-treatment with fulvestrant (ICI 182,780), a selective ER degrader. We also validated the effect of estrogen on DGC organoids. ERα expression was associated with tumor invasion and HOTAIR expression in DGC patients with overt H. pylori infection. These findings may explain the rapid DGC progression in young women with physiologically high levels of estrogen and suggest that fulvestrant with ovarian function suppression could serve as a tumor-suppressive agent in premenopausal patients with DGC.

The dawn of precision medicine in diffuse-type gastric cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. The histology- and morphology-based Lauren classification of GC has been widely used for over 50 years in clinical practice. The Lauren classification divides GC into intestinal and diffuse types, which have distinct etiology, molecular profiles, and clinicopathological features. Diffuse-type GC (DGC) accounts for approximately 30% of GCs. Tumor cells lack adhesion and infiltrate the stroma as single cells or small subgroups, leading to easy dissemination in the abdominal cavity. Clinically, DGC has aggressive traits with a high risk of recurrence and metastasis, which results in unfavorable prognosis. Although systemic chemotherapy is the main therapeutic approach for recurrent or metastatic GC patients, clinical benefits are limited for patients with DGC. Therefore, it is urgent to develop effective therapeutic strategies for DGC patients. Considerable research studies have characterized the molecular and genomic landscape of DGC, of which tight junction protein claudin-18 isoform 2 (CLDN18.2) and fibroblast growing factors receptor-2 isoform IIIb (FGFR2-IIIb) are the most attractive targets because of their close association with DGC. Recently, the impressive results of two phase II FAST and FIGHT trials demonstrate proof-of-concept, suggesting that anti-CLDN18.2 antibody (zolbetuximab) and FGFR2-IIIb antibody (bemarituzumab) are promising approaches for patients with CLDN18.2-positive and FGFR2-IIIb-positive GC, respectively. In this review, we summarize the clinicopathological features and molecular profiles of DGC and highlight a potential therapeutic target based on the findings of pivotal clinical trials.

Abstract 654: Discovery and validation of a gene expression signature for recurrence prediction in high-risk diffuse-type gastric cancer

Abstract Background: Diffuse type gastric cancer (DGC), represented by low sensitivity to 5-fluorouracil based chemotherapy and poor prognosis, is a heterogenous malignancy in which patient subsets exhibit diverse oncological risk-profiles. The current risk stratification criteria based on the TNM staging system are inadequate in predicting the prognosis and in guiding optimal treatment in patients with DGC. Our study aimed to develop molecular biomarkers for prognostic risk-stratification in patients with DGC. Methods: We undertook a systematic and comprehensive discovery and validation effort to identify recurrence prediction biomarkers by analyzing genome-wide transcriptomic expression profiling data from 157 patients with DGC (GSE62254, GSE13861, and TCGA-STAD). Rigorous bioinformatic approaches were used to identify biomarker panels, followed by evaluation of their performance in two independent clinical cohorts (a training cohort, n=180; a validation cohort, n=74) using qRT-PCR assays. Furthermore, using Cox proportional hazard and Kaplan-Meier analyses, the clinical significance of the biomarkers was evaluated for their ability to predict recurrence individually, and when analyzed in combination with key prognostic factors. Results: Genome-wide transcriptomic profiling identified a 7-gene panel for robust prediction of recurrence in DGC patients (AUC=0.91; 95% CI: 0.83-0.96), which was successfully validated in an independent dataset (AUC=0.86; 95% CI: 0.66-0.96). Examination of 180 tissue specimens from a training cohort allowed us to establish a risk prediction model (AUC=0.78; 95% CI: 0.71-0.84), which was subsequently validated in an independent cohort of 74 GC patients (AUC=0.83; 95% CI: 0.72-0.90). The Kaplan-Meier analyses exhibited a consistently superior performance of our 7-gene risk-prediction model in the identification of high and low-risk patient subgroups. Interestingly, the performance of our gene panel to predict recurrence was significantly improved when combined with the tumor stage, in both clinical cohorts (AUC=0.83; 95% CI: 0.76-0.88 - training cohort; AUC=0.89; 95% CI: 0.80-0.95 - validation cohort). Finally, for an easier clinical translation, we established a nomogram that comprised of the 7-gene panel and the tumor stage, which robustly predicted prognosis in patients with DGC. Conclusions: We developed a novel gene expression-based risk-stratification model for predicting recurrence and the identification of high-risk patients, which could facilitate individualized treatments and improving the survival outcomes in patients suffering from DGC. Citation Format: Inseob Lee, Divya Sahu, Hoon Hur, Jeong-Hwan Yook, Byung-Sik Kim, Ajay Goel. Discovery and validation of a gene expression signature for recurrence prediction in high-risk diffuse-type gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 654.

Discovery and validation of an expression signature for recurrence prediction in high-risk diffuse-type gastric cancer.

Diffuse type gastric cancer (DGC), represented by low sensitivity to chemotherapy and poor prognosis, is a heterogenous malignancy in which patient subsets exhibit diverse oncological risk-profiles. This study aimed to develop molecular biomarkers for robust prognostic risk-stratification and improve survival outcomes in patients with diffuse type gastric cancer (DGC). We undertook a systematic and comprehensive discovery and validation effort to identify recurrence prediction biomarkers by analyzing genome-wide transcriptomic profiling data from 157 patients with DGC, followed by their validation in 254 patients from 2 clinical cohorts. Genome-wide transcriptomic profiling identified a 7-gene panel for robust prediction of recurrence in DGC patients (AUC = 0.91), which was successfully validated in an independent dataset (AUC = 0.86). Examination of 180 specimens from a training cohort allowed us to establish a gene-based risk prediction model (AUC = 0.78; 95% CI 0.71-0.84), which was subsequently validated in an independent cohort of 74 GC patients (AUC = 0.83; 95% CI 0.72-0.90). The Kaplan-Meier analyses exhibited a consistently superior performance of our risk-prediction model in the identification of high- and low-risk patient subgroups, which was significantly improved when we combined our gene signature with the tumor stage in both clinical cohorts (AUC of 0.83 in the training cohort and 0.89 in the validation cohort). Finally, for an easier clinical translation, we established a nomogram that robustly predicted prognosis in patients with DGC. Our novel transcriptomic signature for risk-stratification and identification of high-risk patients with recurrence could serve as an important clinical decision-making tool in patients with DGC.

Intratumoral and peritumoral radiomics analysis for preoperative Lauren classification in gastric cancer

BackgroundPreoperative prediction of the Lauren classification in gastric cancer (GC) is very important to the choice of therapy, the evaluation of prognosis, and the improvement of quality of life. However, there is not yet radiomics analysis concerning the prediction of Lauren classification straightly. In this study, a radiomic nomogram was developed to preoperatively differentiate Lauren diffuse type from intestinal type in GC.MethodsA total of 539 GC patients were enrolled in this study and later randomly allocated to two cohorts at a 7:3 ratio for training and validation. Two sets of radiomic features were derived from tumor regions and peritumor regions on venous phase computed tomography (CT) images, respectively. With the least absolute shrinkage and selection operator logistic regression, a combined radiomic signature was constructed. Also, a tumor-based model and a peripheral ring-based model were built for comparison. Afterwards, a radiomic nomogram integrating the combined radiomic signature and clinical characteristics was developed. All the models were evaluated regarding classification ability and clinical usefulness.ResultsThe combined radiomic signature achieved an area under receiver operating characteristic curve (AUC) of 0.715 (95% confidence interval [CI], 0.663–0.767) in the training cohort and 0.714 (95% CI, 0.636–0.792) in the validation cohort. The radiomic nomogram incorporating the combined radiomic signature, age, CT T stage, and CT N stage outperformed the other models with a training AUC of 0.745 (95% CI, 0.696–0.795) and a validation AUC of 0.758 (95% CI, 0.685–0.831). The significantly improved sensitivity of radiomic nomogram (0.765 and 0.793) indicated better identification of diffuse type GC patients. Further, calibration curves and decision curves demonstrated its great model fitness and clinical usefulness.ConclusionsThe radiomic nomogram involving the combined radiomic signature and clinical characteristics holds potential in differentiating Lauren diffuse type from intestinal type for reasonable clinical treatment strategy.

Fusobacterium nucleatum is associated with worse prognosis in Lauren\u2019s diffuse type gastric cancer patients

Fusobacterium nucleatum (F. nucleatum) is frequently detected in primary colorectal cancer (CRC) and matching metastasis, and has been linked to a worse prognosis. We investigated the presence of F. nucleatum in gastric cancer (GC) and gastric preneoplastic conditions of the stomach, and its potential prognostic value in GC patients. Fusobacterium spp. and F. nucleatum were quantified in various specimens from gastrointestinal tract including paired CRC and GC tissues using probe-based qPCR. Fusobacterium spp. and F. nucleatum were more frequently found in tumorous tissue of CRC and GC compared to non-tumorous tissues. The frequency and bacterial load were higher in CRC compared to GC patients. F. nucleatum positivity showed no association to chronic gastritis or preneoplastic conditions such as intestinal metaplasia. F. nucleatum-positivity was associated with significantly worse overall survival in patients with Lauren’s diffuse type, but not with intestinal type GC. There was no association with gender, Helicobacter pylori-status, tumor stage or tumor localization. However, F. nucleatum was positively associated with patient’s age and a trend for a lower global long interspersed element-1 DNA methylation. In conclusion, our work provides novel evidence for clinical relevance of F. nucleatum in GC by showing an association between F. nucleatum positivity with worse prognosis of patients with Laurens’s diffuse type gastric cancer. Further studies are necessary to explore related mechanistic insights and potential therapeutic benefit of targeted antibiotic treatment in GC patients.

Overexpressing PLOD family genes predict poor prognosis in gastric cancer

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) are a set of enzymes involved in the hydroxylation of lysine and stabilization of collagen by crosslinks. Previous studies have highlighted that overexpressing PLOD genes were related to the progression, migration and progression of different human cancers. However, the diverse expression patterns and prognostic values of PLOD genes remain to be elucidated in gastric cancer (GC). In this study, we mined the expression and survival data in GC patients through ONCOMINE, UALCAN and Kaplan-Meier Plotter database. STRING portal couple with DAVID was used to establish a functional protein interaction network of PLOD family genes and analyze the GO and KEGG enriched pathways. Differential gene expression correlated with PLOD family genes was identified with LinkedOmics. We found that PLOD1, 2 and 3 were up-regulated in GC patients compared with normal tissues. High expression levels of PLOD1 and PLOD3 were associated with shorter overall survival (OS), first progression (FP) and post progression survival (PPS) while high expression level of PLOD2 was only associated with shorter FP in all GC patients. Specifically, only high PLOD2 expression had significant correlation with shorter OS, FP and PPS in the diffuse type GC patients. Furthermore, combinatorial use of expressions of all PLOD genes was a superior prognostic indicator for GC patients. Pathway analysis confirmed that PLOD family genes mainly participate in regulating the collagen metabolism and extracellular matrix constitution, and the cellular adaptor protein SHC1, which helps to transduce an extracellular signal into an intracellular signal, could be the regulatory module mediating PLOD's effect on GC. Therefore, we propose that individual PLOD genes or PLOD family genes as a whole could be potential prognostic biomarkers for GC.

155P - Identification of distinct subtypes revealing prognostic and therapeutic relevance in diffuse type gastric cancer

BackgroundAlthough recent advances in high-throughput technology have provided many insights into gastric cancer (GC), few reliable biomarkers for handling diffuse type GC are identified. Here, we aim to identify a prognostic and predictive signature predicting heterogeneous clinical courses of diffuse type GC.Table: 155PUnivariate and multivariate Cox regression analysis of overall survival in diffuse type gastric cancerTable: 155PVariablesUnivariateMultivariatenHR (95% CI)P-valuenHR (95% CI)P-valueAge4021.013 (1.001 - 1.025)0.0374021.02 (1.007 - 1.032)0.003Gender (Male or Female)4021.074 (0.805 - 1.433)0.625AJCC Stage (I, II, III or IV)4022.516 (2.088 - 3.032)<0.0012.67 (2.204 - 3.235)<0.001Tumour site (cardia, body, antrum or whole)4020.985 (0.777 - 1.248)0.9COD-signature (INT or COD subtypes)4021.675 (1.257 - 2.234)<0.0012.058 (1.53 - 2.766)<0.001Abbreviations: HR, hazard ratio; CI, confidence interval; INT, intestinal-like; COD, core diffuse type. MethodsWe analysed RNA-seq based transcriptome data to identify a molecular signature in 150 gastric tissue samples including 107 diffuse type GCs. The predictive value of the signature was verified using other diffuse type GCs in three independent cohorts (n=466). Various statistical methods, including log-rank and Cox regression analyses, were used to estimate an association between the signature and prognosis. The signature was also characterized by somatic variant assessments and tissue microarray analysis between diffuse type GC subtypes. ResultsTranscriptomic profiling revealed two distinct subtypes of diffuse type GC including intestinal-like (INT) and core diffuse type (COD) subgroups. We generated a signature, namely COD-signature, reflecting the best characteristics of subtypes. When estimating prognostic value in other cohorts, COD-signature showed a strong predictability and an independent clinical utility in diffuse type GC prognosis (hazard ratio = 2.058, 95% confidence interval = 1.53-2.77, P<0.001; Table). Integrative mutation and gene expression analyses demonstrated that COD subtype was responsive to chemotherapy, whereas INT subtype showed responsiveness to immunotherapy with immune-check point inhibitor (ICI). Tissue microarray analysis showed practical utility of IGF1 and NXPE2 proteins for predicting diffuse type GC’s heterogeneity. ConclusionsThe COD-signature represents a promising diagnostic tool for the identification of diffuse type GC patients who would display different clinical behaviours as well as response to chemotherapy or ICI treatment. Legal entity responsible for the studyKorea Research Institute of Bioscience and Biotechnology Chungnam National University, College of Medicine Seoul National University, Faculty of Medicine. FundingKorea Research Institute of Bioscience and Biotechnology. DisclosureAll authors have declared no conflicts of interest.

Potential of Helicobacter pylori-uninfected signet ring cell carcinoma to invade the submucosal layer.

The typical histology of Helicobacter pylori-uninfected gastric cancer is signet ring cell carcinoma (SRCC) localized in the mucosal layer, but the potential of these SRCCs to invade the submucosal layer is unclear. This study aimed to investigate the clinicopathological characteristics of SRCC in H.pylori-uninfected patient and its prevalence in diffuse-type gastric cancer (DGC) within Japan. We enrolled consecutive pure DGC patients diagnosed with the disease either localized in the mucosal layer or with submucosal invasion. H.pylori infection was investigated, and the patients were divided into three groups according to histological types: pure SRCC, SRCC with poorly differentiated adenocarcinoma (PDA), and pure PDA. Of the 345 pure DGC patients, 132 (38%), 127 (37%), and 86 (25%) had pure SRCC, SRCC with PDA, and pure PDA histologies, respectively. The prevalence of H.pylori infection and the SM ratio were significantly lower in the pure SRCC group than other groups (P<0.01). Twenty-two (6.4%) patients, including two with submucosal invasion, were negative for H.pylori and had mucosal SRCC component in the cancer lesions. Of the 259 SRCC cases (pure SRCC or SRCC+PDA), H.pylori-uninfected cases had different clinicopathological characteristics compared with H.pylori-positive cases. Particularly, the ratio of patients with submucosal invasive SRCC was significantly lower in the H.pylori-uninfected gastric cancer group than in those with H.pylori infection. Helicobacter pylori-uninfected gastric cancer is not rare among pure DGC patients in Japan. SRCC in patients without H.pylori infection is less likely to be invasive.

Protein Classification of Diffuse-Type Gastric Cancer Using Formalin-Fixed Paraffin-Embedded Samples

Background: Gastric cancer (GC) is the third leading cause of cancer deaths in the world. It is highly heterogeneous. Many molecular therapies for GC have entered clinical trials but, apart from trastuzumab, apatinib and ramucirumab, all have failed. One important reason is that insufficient attention is paid to the underlying subtypes and characteristics of GC, especially the diffuse-type gastric cancer (DGC) according to the Lauren classification with worst clinical outcomes. Aim: Here we firstly investigated formalin-fixed paraffin-embedded (FFPE) samples of DGC to establish clinically relevant molecular classification based on proteomics analysis. Also, we tried to generate a suitable classifier of DGC that can guide patient therapy. Methods: We screened a total of 2548 cases retrospectively, who underwent GC resection at Beijing Cancer Hospital from October 2006 to December 2011. We used a fast mass spectrometry workflow for proteome profiling. Finally we carried out proteome profiling of 99 DGC paired tumor-nearby tissues from FFPE sections. Median overall survival of the whole population was 55.0 months. Proteome profiling data from these samples were used to develop a subtype prediction model. We used consensus clustering to identify molecular subtypes based on differentially expressed proteins. The pathway enrichment was performed by GSEA, and the prediction classifier was generated by elastic-net machine learning. Kaplan-Meier survival analysis and Cox regression multivariate analysis were used. Results: A total of 8201 gene products were identified in this study, and 1249 differential expressed proteins between tumor and nearby-normal tissue was detected (FDR q-value &lt; 0.01 by SAM). Tumor upregulated proteins mostly enriched into pathways including RNA processing, epithelial-mesenchymal transition (EMT), immune response and inflammation related pathways. Tumor downregulated proteins mostly enriched into metabolic pathways such as oxidative phosphorylation pathway. Based on proteome profiling alone, DGC can be subtyped into 3 major classes (PX1-3) that exhibit distinct proteome features and correlate with distinct clinical outcomes. PX1 (31 patients) exhibits RNA processing proteins and associates with the best prognosis; PX2 (26 patients) exhibits highly expressed cell cycle features, and the patients have poorer prognosis than those with cluster1 but better prognosis than those with cluster3; PX3 (42 patients) features EMT and the worst prognosis. We built a classifier of 12 marker proteins that can stratify DGC patients into these 3 subtypes, opening a door for protein classification in clinical application and intervention. Conclusion: Our study demonstrated that proteome profiling alone from FFPE samples was able to subtype DGC into 3 protein subtypes that were linked to distinct patterns of molecular alterations and prognosis. The prediction model need to be further verified in more clinical cohorts.

A proteomic landscape of diffuse-type gastric cancer

The diffuse-type gastric cancer (DGC) is a subtype of gastric cancer with the worst prognosis and few treatment options. Here we present a dataset from 84 DGC patients, composed of a proteome of 11,340 gene products and mutation information of 274 cancer driver genes covering paired tumor and nearby tissue. DGC can be classified into three subtypes (PX1–3) based on the altered proteome alone. PX1 and PX2 exhibit dysregulation in the cell cycle and PX2 features an additional EMT process; PX3 is enriched in immune response proteins, has the worst survival, and is insensitive to chemotherapy. Data analysis revealed four major vulnerabilities in DGC that may be targeted for treatment, and allowed the nomination of potential immunotherapy targets for DGC patients, particularly for those in PX3. This dataset provides a rich resource for information and knowledge mining toward altered signaling pathways in DGC and demonstrates the benefit of proteomic analysis in cancer molecular subtyping.

Abstract 2204: A proteomic landscape of diffuse-type gastric cancer

Abstract Gastric cancer is a heterogeneous disease characterized by poor clinical outcomes and limited targeted treatment options. Among them, diffuse-type gastric cancer (DGC) is the subtype with worst prognosis. Here we describe the first proteomic landscape of DGC. We carried out proteome profiling and targeted exome DNA sequencing of 84 DGC samples. We analyzed the 1,008 (168 x 6) raw files together for uniformed quality control and protein identification with 1% global protein false discovery rate (FDR), which resulted in the identification of 11,340 gene products (GPs). A SAM (significance analysis of microarray) analysis identified 1,641 proteins as differentially expressed between T (tumor) and N (nearby) with statistical significance (FDR q value&amp;lt;0.01 by SAM and differential expression ratio&amp;gt;0.5/ &amp;lt; -0.5), including 1,211 up-regulated and 430 down-regulated GPs. Gene Ontology annotation indicated that tumor proteomes were significantly enriched in cell cycle, DNA replication, checkpoint, E2F, WNT, p53 signaling, epithelial mesenchymal transition (EMT), and inflammation/cytokine-receptor interaction pathways, and the proteomes of the nearby tissues are enriched in metabolism pathways, such as fatty acid metabolism, oxidative phosphorylation, and amino acid metabolism. Notably, many gastric makers (ANXA10, VSIG1, CLDN18, CTSE, TFF2, MUC5AC and MUC6) and signature proteins for stomach functions, including digestion, absorption, secretion, and stomach acid generation (PGC, GIF, GAST, and ATP4A), were lost in tumors. Based on proteome profiling alone, DGC can be subtyped into 3 major classes (PX1-3) that exhibit distinct proteome features and correlate with distinct clinical outcomes (Gehan-Breslow-Wilcoxon P = 0.024). PX1 exhibits proteome stability and the best overall survival; PX2 exhibits dysregulation in DNA replication and cell cycle, and is most sensitive to chemotherapy; PX3 features hyper-activated immune response and is not responsive to chemotherapy. We identified seven-marker proteins that can stratify DGC patients into these three subtypes, opening a door for proteome subtyping in clinical application and intervention. Furthermore, we nominated drug target candidates taking into consideration both the altered DGC proteome and association data with patients’ overall survival. This study revealed the altered signaling pathways in DGC and demonstrated the advantage of proteomic approach in molecular subtyping of cancer. Citation Format: Sai Ge, Xia Xia, Chen Ding, Bei Zhen, Quan Zhou, Jinwen Feng, Jiajia Yuan, Rui Chen, Yumei Li, Zhongqi Ge, Jiafu Ji, Lianhai Zhang, Jiayuan Wang, Zhongwu Li, Yumei Lai, Ying Hu, Yanyan Li, Yilin Li, Jing Gao, Lin Chen, Jianming Xu, Chunchao Zhang, Sung Yun Jung, Mingwei Liu, Lei Song, Wanlin Liu, Gaigai Guo, Tongqing Gong, Yin Huang, Yang Qiu, Tieliu Shi, Weimin Zhu, Yi Wang, Fuchu He, Lin Shen, Jun Qin, CNHPP. A proteomic landscape of diffuse-type gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2204. doi:10.1158/1538-7445.AM2017-2204

Abstract 139: Novel gastric cancer cell lines established from diffuse type gastric cancer patients for potential subgroup-specific therapy

Abstract In vitro culture system is a long-standing useful tool in the study of cell biology for developing new therapeutic modalities and discovering new anti-cancer drugs. Therefore establishment and characterization of cell lines are very important in vitro study. However, establishment of GC patients derived cell lines are very challenging, especially with diffuse type which has different biology and worse prognosis from intestinal type. Yonsei Cancer Center (YCC) has been consistently worked to build human cancer cell lines, and resulting in over 30 novel cancer cell lines from metastatic gastric cancer patients. Most of YCC GC cell lines were established by primary culture of peritoneal fluid from metastatic gastric cancer patients. Interestingly, YCC-16 cells were isolated from the peripheral blood of gastric cancer patients. 20 cells were established from diffuse type GC, and 21 cells were from signet ring cell type. In this study, we evaluated 29 YCC GC cell lines and 23 GC cell lines from other sources (ATCC, KCLB, and JCRB). For molecular characterization of 52 GC cell lines, we analyzed copy number variant (CNV), single number variant (SNV) and gene expression using whole-exome sequencing (WES) and RNA sequencing. The 7 cell lines (YCC-17, 18, 19, 20, 29, 34, 36) were compared with their germline variants of paired PBMC. Also, these cell lines were investigated expression of proteins related to cancer progression and the sensitivity of chemotherapies and diverse molecular targeted drugs/antibodies. We evaluated the tumorigenesis with soft-agar assay and invasiveness by transwell assay. In addition to previous novel EBV infected cell line (YCCEL1/YCC-10, J Gen Virol. 2013), we observed amplification and overexpression of receptor tyrosine kinase (RTK) including HER2 (YCC-19, 32, 33, 38, 42), EGFR (YCC-11, 21), Met (YCC-31, 42), and FGFR2 (YCC-28, 30); confirming that protein was overexpression by Western blot in 20/52 (38.5%). Interestingly, amplification of RTKs was detected mutually exclusive pattern with other RTK amplification. Furthermore, RTK amplified cell lines were shown to be sensitive to target specific small molecules such as BGJ398. Also, we identified cell line specific genetic variations including ARID1A which might be the potential new targets in diffuse type GC cell lines. Then we could subgroup the cell lines based on 4 subtypes of TCGA. In conclusion, our GC cell line bank with genomic and biological characteristics based on the clinical information, would be useful in subgroup specific target selection, drug screening and mechanism evaluation for improving GC treatment. Citation Format: Tae Soo Kim, Kyu Hyun Park, Woo Sun Kwon, Won Suk Lee, In Hye Jeong, Sun Kyoung Kang, Hyun Myong Kim, Sun Young Rha, Hyun Cheol Chung. Novel gastric cancer cell lines established from diffuse type gastric cancer patients for potential subgroup-specific therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 139.

The Efficacy of Taxanes- and Oxaliplatin-Based Chemotherapy in the Treatment of Gastric Cancer After D2 Gastrectomy for Different Lauren Types.

To investigate the efficacy of Taxanes- and Oxaliplatin-based chemotherapies (TC and OC) in the treatment of gastric cancer patients after D2 gastrectomy with different Lauren types. In this study, 299 patients of gastric adenocarcinoma with D2 lymph node dissection were reviewed between 2007 and 2014. Chemotherapies were classified as Oxaliplatin-based and Taxanes-based regimen. Treatment outcomes were analyzed according to different Lauren types, such as the intestinal type, diffuse type, and mixed type groups, respectively. The disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan–Meier method. The log-rank test was used for univariate analysis, and Cox regression was used for multivariate analysis. In diffuse type gastric cancer, the Oxaliplatin-based arm had a longer median DFS and OS compared with Taxanes-based arm (DFS: 47.0 vs 28.6 months, P = 0.04; OS: 51.9 vs 34.5 months, P = 0.048). The chemotherapy regimen was an independent prognostic factor for DFS and OS of diffuse type gastric cancer patients by multivariate analysis (P = 0.01). In the intestinal type, although the DFS and OS of intestinal type patients in TC group were higher than those in OC group (DFS: 53.4 vs 42.4 months; OS: 69.7 vs 57.8 months), there was no statistical significance observed (both P > 0.05). For the mixed type, the 2 different chemotherapy regimens achieved similar median DFS and OS. In a conclusion, the patients of diffuse type were more sensitive to OC, and the intestinal type patients may be benefit from TC. Therefore, it will be of benefit for gastric patients by introducing Lauren classification clinically and to help the choice of chemotherapy regimen for gastric patients after D2 gastrectomy.

Combined Secretomics and Transcriptomics Revealed Cancer-Derived GDF15 is Involved in Diffuse-Type Gastric Cancer Progression and Fibroblast Activation.

Gastric cancer is classified into two subtypes, diffuse and intestinal. The diffuse-type gastric cancer (DGC) has poorer prognosis, and the molecular pathology is not yet fully understood. The purpose of this study was to identify functional secreted molecules involved in DGC progression. We integrated the secretomics of six gastric cancer cell lines and gene expression analysis of gastric cancer tissues with publicly available microarray data. Hierarchical clustering revealed characteristic gene expression differences between diffuse- and intestinal-types. GDF15 was selected as a functional secreted molecule owing to high expression only in fetal tissues. Protein expression of GDF15 was higher in DGC cell lines and tissues. Serum levels of GDF15 were significant higher in DGC patients as compared with healthy individuals and chronic gastritis patients, and positively correlated with wall invasion and lymph node metastasis. In addition, the stimulation of GDF15 on NIH3T3 fibroblast enhanced proliferation and up-regulated expression of extracellular matrix genes, which were similar to TGF-β stimulation. These results indicate that GDF15 contributes to fibroblast activation. In conclusion, this study revealed that GDF15 may be a novel functional secreted molecule for DGC progression, possibly having important roles for cancer progression via the affecting fibroblast function, as well as TGF-β.