Mutations In Gastric Cancer Research Articles

Integrated DNA and RNA sequencing to reveal early drivers of metastasis in gastric cancer.

e16096 Background: Gastric cancer (GC) is the second cause of cancer-related death and metastasis is an important cause of death. Considering difficulties in searching for metastatic driver mutations, we tried a novel strategy to identify early drivers in primary gastric tumors. In this study, we conducted an integrative genomic analysis on GC patients and identified early drivers that lead to metastases. Methods: Tumors and matched normal tissues from 400 Chinese GC patients were characterized by whole-exome sequencing (WES), transcriptome sequencing and targeted exome sequencing (TES), especially the comparative analysis results between higher metastatic potential (HMP) group with T1 stage and lymph-node metastases, and lower metastatic potential (LMP) group without lymph-nodes or distant metastases. Functional experiments on candidate driver mutations were applied in GC cell lines to explore their roles in metastasis and immune escape. Results: HMP group presented higher mutation load and heterogeneity, enrichment in immunosuppressive signaling, lower tumor purity, more stromal and immune cell infiltration than LMP group. We identified 262 differentially expressed mRNA, lncRNA and miRNA between HMP and LMP group (with more than twice proportion of 2-fold increased or decreased tumor than normal tissue in HMP than LMP group, and with significantly 2-fold higher or lower in HMP tumors than LMP tumors). 168 candidate prometastatic mutations were found by WES and 8 were selected for following TES to detect their association with distant metastasis. We found that four candidate mutations were related to distant metastasis and mutated TP53 and MADCAM1 were significantly associated with poor metastasis-free survival. It’s worth noting that though with low mutational rate, MADCAM1 mutations were only observed in patients with metastasis within three years after surgery or at diagnosis. We demonstrated that MADCAM1-mutated proteins act as GOF mutants and could not only directly promote cancer cells migration, but also could trigger tumor metastasis by establishing complicated immunosuppressive microenvironment, including promoting PD-L1-mediated immune escape and reprogramming tumor-associated macrophages (TAMs). Conclusions: Our results showed that the GCs with different metastatic potential are distinguishable at the genetic level. A number of potential metastatic driver mutations was revealed in this study including known cancer-associated genes TP53. We also discovered some novel driver mutations, such as MADCAM1. The results indicate driver mutations in early-onset metastatic GC could promote metastasis not only by directly empower cancer cells to disseminate but also by establishing an immunosuppressive microenvironment. This study increased the understanding of molecular landscape of GC patients and provided possibility for future target therapy.

Geographical Distribution of E-cadherin Germline Mutations in the Context of Diffuse Gastric Cancer: A Systematic Review.

Simple SummaryE-cadherin (CDH1 gene) germline mutations are associated with the development of the autosomal cancer syndrome known as hereditary diffuse gastric cancer. About 30% of families fulfilling the clinical criteria established by the International Gastric Cancer Linkage Consortium have constitutional alterations of the CDH1 gene. Different patterns of CDH1 germline mutations have described as truncating, deletion, insertion, splice site, non sense, silence, and at last, missense alterations. The frequency of the different E-cadherin germline mutations in countries with different incidence rates for gastric carcinoma has reported extremely variable. In this study we aimed to assess the worldwide frequency of CDH1 germline mutations in gastric cancers coming from different geographical areas, using a systematic approach.Hereditary diffuse gastric cancer (HDGC) is a complex and multifactorial inherited cancer predisposition syndrome caused by CDH1 germline mutations. Nevertheless, current CDH1 genetic screening recommendations disregard an unbalanced worldwide distribution of CDH1 variants, impacting testing efficacy and patient management. In this systematic review, we collected and analyzed all studies describing CDH1 variants in gastric cancer patients originating from both high- and low-prevalence countries. Selected studies were categorized as family study, series study, and unknown study, according to the implementation of HDGC clinical criteria for genetic testing. Our results indicate that CDH1 mutations are more frequently identified in gastric cancer low-incidence countries, and in the family study group that encompasses cases fulfilling criteria. Considering the type of CDH1 alterations, we verified that the relative frequency of mutation types varies within study groups and geographical areas. In the series study, the missense variant frequency is higher in high-incidence areas of gastric cancer, when compared with non-missense mutations. However, application of variant scoring for putative relevance led to a strong reduction of CDH1 variants conferring increased risk of gastric cancer. Herein, we demonstrate that criteria for CDH1 genetic screening are critical for identification of individuals carrying mutations with clinical significance. Further, we propose that future guidelines for testing should consider GC incidence across geographical regions for improved surveillance programs and early diagnosis of disease.

Identification of the RP11-21C4.1/SVEP1 gene pair associated with FAT2 mutations as a potential biomarker in gastric cancer

ABSTRACT Gastric cancer (GC) is one of the most common malignancies worldwide. Despite rapid advances in systemic therapy, GC remains the third leading cause of cancer-related deaths. We aimed to identify a novel prognostic signature associated with FAT2 mutations in GC. We analyzed the expression levels of FAT2-mutant and FAT2-wildtype GC samples obtained from The Cancer Genome Atlas (TCGA). The Kaplan–Meier survival curve showed that patients with FAT2 mutations showed better prognosis than those without the mutation. Sixteen long non-coding RNAs (lncRNAs) and 62 messenger RNAs (mRNAs) associated with FAT2 mutations were correlated with the prognosis of GC. We then constructed a 4-mRNA signature and a 5-lncRNA signature for GC. Finally, we identified the most relevant RP11-21 C4.1/SVEP1 gene pair as a prognostic signature of GC that exhibited superior predictive performance in comparison with the 4-mRNA or 5-lncRNA signature by weighted gene correlation network analysis (WGCNA) and Cox proportional hazards regression analysis. In this study, we constructed a prognostic signature of GC by integrative genomics analysis, which also provided insights into the molecular mechanisms linked to FAT2 mutations in GC.

Studying potential PKCδ loss of function mutation and its downstream effects in gastric cancer progression

PKC isozymes are involved in the modulation of cellular pathways related with tumor progression, acting as a suppressor or promoter. In cancer cells, PKCs are mutated, and most common type is loss of function. This paper focuses on the effect of PKCδ mutation in gastric cancer. LOF mutation occurs throughout catalytic and kinase domains of PKCδ, disrupting activation and function of kinase. In catalytic domain, there are various potential mutation targets, such as binding groove and zinc finger. Mutation residues detected in the kinase domain, such as DFG and APE motifs, can alter catalytic function, causing interruption of activation. Also, a critical region, called hinge region, modulates caspase-3 dependent cleavage, and such tyrosine mutation in this region reduces cleavage activity, inhibiting fully activation of kinase. Importantly, LOF mutation affects cellular activity of downstream protein, p53, through inhibiting transcription, localization, and phosphorylation. For instance, C1 domain mutant suppresses binding capacity with p53, reducing transcription of p53. Disruption of cellular component, tight junction, assembling related to PKC mutation. As identified, PKCδ correlates with ZO-1, and LOF mutation prevent translocation of ZO-1 to TJ area, leading to errors in TJ assembling, promoting tumor invasion.

Wild-Type TP53 Predicts Poor Prognosis in Patients with Gastric Cancer.

TP53 gene is often mutated in gastric cancer (GC), nonetheless its relationship with clinicopathological characteristics and prognosis is still unclear. Here, we sought to ascertain the difference in clinical phenotypes between TP53 wild-type and mutant tumors in confirmed gastric cancer patients. To this end, we analyzed TP53 mutation status of 415 TCGA GC patients in relation to their clinical and pathological features as well as prognosis. Longrank Test showed that the survival rate of gastric cancer patients with TP53 WT was significantly lower than that of TP53 mut. Compared with TP53 mut gastric cancer patients with low mRNA expression, TP53 WT patients with low mRNA expression have lower overall survival rate. The death risk of TP53 WT gastric cancer patients is 1.395 times that of TP53 mut gastric cancer patients. The death risk of TP53 mut gastric cancer patients is not related to age, and advanced age is not a risk factor. However, the death risk of TP53 WT patients with gastric cancer increases with age, and the death risk of patients over 70 years old is 1.899 times that of patients under 60 years old. These results suggest that the prognosis of elderly gastric cancer patients with TP53 WT is worse.Conclusion:our results indicate that the status of TP53 mutation in GC is significantly correlated with clinical or molecular categories and that the prognosis of GC patients with WT TP53 is worse than that of patients with mutant TP53. Therefore, our data emphasize the importance of distinguishing TP53 WT to predict poor overall survival and relapse-free survival in patients with GC.

Signal transduction pathway mutations in gastrointestinal (GI) cancers: a systematic review and meta-analysis

The present study was conducted to evaluate the prevalence of the signaling pathways mutation rate in the Gastrointestinal (GI) tract cancers in a systematic review and meta-analysis study. The study was performed based on the PRISMA criteria. Random models by confidence interval (CI: 95%) were used to calculate the pooled estimate of prevalence via Metaprop command. The pooled prevalence indices of signal transduction pathway mutations in gastric cancer, liver cancer, colorectal cancer, and pancreatic cancer were 5% (95% CI: 3–8%), 12% (95% CI: 8–18%), 17% (95% CI: 14–20%), and 20% (95% CI: 5–41%), respectively. Also, the mutation rates for Wnt pathway and MAPK pathway were calculated to be 23% (95% CI, 14–33%) and 20% (95% CI, 17–24%), respectively. Moreover, the most popular genes were APC (in Wnt pathway), KRAS (in MAPK pathway) and PIK3CA (in PI3K pathway) in the colorectal cancer, pancreatic cancer, and gastric cancer while they were beta-catenin and CTNNB1 in liver cancer. The most altered pathway was Wnt pathway followed by the MAPK pathway. In addition, pancreatic cancer was found to be higher under the pressure of mutation compared with others based on pooled prevalence analysis. Finally, APC mutations in colorectal cancer, KRAS in gastric cancer, and pancreatic cancer were mostly associated gene alterations.

P53 immunostaining cannot be used to predict TP53 mutations in gastric cancer: results from a large Central European cohort

Four molecular subgroups of gastric cancer (GC) have been proposed, ie, Epstein-Barr virus (EBV)-positive, microsatellite instable, chromosomal instable (CIN), and genomically stable GC. Based on the complex relationship between chromosomal instability and TP53 mutational status, we hypothesized that the typical clinicopathological characteristics caused by chromosomal instability are correlated with the p53 expression that is detected by immunohistochemistry. Four hundred sixty-seven whole-tissue sections of patients with therapy-naive GC were stained with anti-p53 antibody. The histoscore and staining pattern were analyzed for each slide. Different algorithms of immunohistochemistry evaluation were formed and correlated with clinicopathological characteristics. The algorithms were validated by assessing the mutational status of TP53 in 111 cases. Four hundred forty-two GCs were p53 positive, and 25 were negative, including 414GCs with a homogeneous pattern and 53 GCs with a heterogeneous staining pattern. There was no correlation with overall or tumor-specific survival. In comparison with clinicopathological characteristics, the algorithm highversus lowshowed correlations with microsatellite instability, hepatocyte growth factor receptor (MET), and TP53 mutational status. The algorithm Q1/Q4versus Q2/Q3appeared to be correlated with the phenotype as per the Laurén classification, microsatellite instability, EBV status, and p53 expression pattern. The algorithm <90%=0 and<50%=3+versus ≥90%=0 or ≥50%=3+showed correlations with the EBV status, microsatellite instability, grading, and p53 expression pattern. The algorithm homogeneousversus heterogeneousdid not correlate with any clinicopathological characteristic. Our results showed that the immunohistochemistry of p53, TP53 mutational status, and CIN subtype were connected. However, different algorithms for p53 immunohistochemical evaluation cannot be used to predict TP53 mutations in CIN GCs in individual cases.

Abstract 1079: Distinct DNA methylation targets by aging and chronic inflammation

Abstract Aberrant DNA methylation is involved in many types of human cancers, and is induced by aging. In addition, it is known to be induced by chronic inflammation, and this inflammation-induced DNA methylation is considered as acceleration of age-related methylation. However, few studies addressed target genomic regions of age-related and inflammation-induced DNA methylation in a genome-wide manner. We previously demonstrated that Helicobacter pylori(HP) infection induces aberrant DNA methylation potently in human and gerbil gastric mucosa [Niwa, Cancer Res, 70:1430, 2010; Ushijima, Clin Cancer Res, 18:923, 2012]. Here, taking advantage of the potent methylation induction, we aimed to clarify the targets of methylation induction by aging and inflammation. We collected gastric mucosae from i) HP-never infected young individuals (under 40 years; n=4; G1), ii) HP-currently infected young individuals (n=4; G2), iii) HP-never-infected old individuals (above 60 years; n=4; G3), and vi) HP-currently-infected old individuals (n=4; G4). DNA methylation of 482,421 CpG probes was analyzed by Infinium Human Methylation 450K, and the probes were grouped into 270,249 genomic blocks. It was revealed that high levels of methylation were induced in 44,461 (16.5%) genomic blocks by current inflammation (G2/G1), even after correction of the influence of leucocyte infiltration. 61.8 % of the hypermethylation was acceleration of age-related methylation (G3/G1) while 21.6 % was specific to inflammation. Regions with H3K27me3 were frequently hypermethylated both by aging and inflammation. Basal methylation levels were essential for age-related hypermethylation while even regions with little basal methylation were hypermethylated by inflammation. When limited to promoter CpG islands, being a microRNA gene and high basal methylation levels strongly enhanced hypermethylation while H3K27me3 strongly enhanced inflammation-induced hypermethylation. Inflammation was capable of overriding active transcription. In young gastric mucosae (G2/G1), genes with high expression and frequent mutations in gastric cancers were more frequently methylated than in old ones (G4/G3).Methylation by inflammation was not simple acceleration of age-related methylation. Targets of aberrant DNA methylation were different between young and old gastric mucosae, and driver genes were preferentially methylated in young gastric mucosa [Yamashita, Clin Epigenet, in press]. Citation Format: Satoshi Yamashita, Sohachi Nanjo, Emil Rehnberg, Naoko Iida, Hideyuki Takeshima, Takayuki Ando, Takao Maekita, Toshiro Sugiyama, Toshikazu Ushijima. Distinct DNA methylation targets by aging and chronic inflammation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1079.

PTEN Protein Loss and Loss-of-Function Mutations in Gastric Cancers: The Relationship with Microsatellite Instability, EBV, HER2, and PD-L1 Expression.

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.

CDH1 mutations in gastric cancers are not associated with family history

CDH1 mutation is the most frequent genetic alteration in hereditary diffuse gastric cancer (GC) and early onset diffuse GC patients. However, the incidence of CDH1 mutations in sporadic GC with or without family history has not been studied. This retrospective study includes a total of 993 Korean patients with primary advanced GC who underwent surgery and received palliative chemotherapy. Targeted deep sequencing was performed in all cases and family history of GC was searched with survival analysis. We found CDH1 alterations in 146 of 993 patients (14.7 %) and 8 were germline (0.8 %). Out of 146 patients with CDH1 mutations, 25 (17.1 %) had a family history of GC in one of their first relatives, and 12 patients (8.2 %) were diagnosed with familial GC (FGC). All cases with FGC were diffuse type by Lauren classification, and only one harbored a previously reported germline mutation of CDH1 (c.2638 G > A) and the remaining 11 harbored known somatic CDH1 mutations. Among all patients with CDH1 mutation, there was no significant survival difference between patients with family history or FGC. In the 847 patients without CDH1 mutation, 189 (22.3 %) had a family history of GC and 92 patients (10.9 %) were FGC. CDH1 mutations were more frequent in patients with early onset (<45 years) GC (45.5 %) compared with patients with late onset GC (10.9 %) (p = 0.001), but were not significantly associated with the family history of GC (p > 0.05). CDH1 mutations are mostly somatic and typically are not associated with family history.

The clinical signiicance of ARID1A mutations in gastric cancer patients

Background: ARID1A is a key component of the SWI/SNF chromatin remodeling complex, which has been identified in various cancers. Loss of ARID1A expression is correlated with poor prognosis in gastric cancer (GC); however, the clinical relevance of ARID1A mutations in GC has not yet been reported. Materials and Methods: A total of 518 GC patients receiving gastrectomy were enrolled. The analysis of 13 mutations of the ARID1A gene using mass spectrometric single-nucleotide polymorphism genotyping technology was conducted. The clinicopathological features of GC with and without ARID1A mutations were compared. Results: Among the 518 GC patients, 59 (11.4%) had ARID1A mutations. For diffuse-type GC, patients with ARID1A-mutated tumors were older and had fewer poorly differentiated tumors, fewer incidence of Epstein–Barr virus infection, a higher likelihood of ARID1A expression loss, more microsatellite instability-high tumors, a lower prevalence of peritoneal recurrence, and better survival rates than those with ARID1A nonmutant tumors. For intestinal-type GC, patients with ARID1A-mutant tumors had more PI3K/AKT pathway genetic mutations than patients with ARID1A nonmutant tumors. Multivariate analysis showed that ARID1A mutations are an independent prognostic factor in diffuse-type GC. Conclusion: ARID1A mutations are associated with a better prognosis in diffuse-type GC.

Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer

The oncogenic receptor tyrosine kinase family ErbB consists of four members (ErbB1, ErbB2, ErbB3 and ErbB4); they are involved in the tumorgenesis of diverse cancers. A variety of missence mutations have been clinically observed in ErbB kinases, which would shift drug sensitivity to these kinases and cause drug resistance in targeted cancer therapy. In this study, systematic inhibitor response to ErbB missense mutations in gastric cancer (GC) is investigated by combining computational analysis and experimental assay. The response profile is created for 6 ATP-competitive, reversible inhibitors against 9, 17, 5 and 17 GC-associated missense mutations of ErbB1, ErbB2, ErbB3 and ErbB4 kinase domains, respectively. From the profile a number of potential resistant and sensitive responses are identified theoretically. It is suggested that most ErbB mutations have only a modest effect on inhibitor binding, but few that are located around the kinase active site can influence the binding significantly. Structural examination reveals that steric hindrance and allosteric effect are primarily responsible for inhibitor resistance and sensitivity, respectively. Two ErbB2 mutations, namely V777L and T862A, are predicted to cause effective resistance on inhibitors TAK285 and Lapatinib, respectively. Kinase assays consistently observe that the mutations can reduce inhibitor activity by 4.9-fold and 2.4-fold, with IC50 changing from 29 to 16 nM (wild type) to 83 and 39 nM (mutant) for TAK285 and Lapatinib, respectively.

Distinct DNA methylation targets by aging and chronic inflammation: a pilot study using gastric mucosa infected with Helicobacter pylori

BackgroundAberrant DNA methylation is induced by aging and chronic inflammation in normal tissues. The induction by inflammation is widely recognized as acceleration of age-related methylation. However, few studies addressed target genomic regions and the responsible factors in a genome-wide manner. Here, we analyzed methylation targets by aging and inflammation, taking advantage of the potent methylation induction in human gastric mucosa by Helicobacter pylori infection-triggered inflammation.ResultsDNA methylation microarray analysis of 482,421 CpG probes, grouped into 270,249 genomic blocks, revealed that high levels of methylation were induced in 44,461 (16.5%) genomic blocks by inflammation, even after correction of the influence of leukocyte infiltration. A total of 61.8% of the hypermethylation was acceleration of age-related methylation while 21.6% was specific to inflammation. Regions with H3K27me3 were frequently hypermethylated both by aging and inflammation. Basal methylation levels were essential for age-related hypermethylation while even regions with little basal methylation were hypermethylated by inflammation. When limited to promoter CpG islands, being a microRNA gene and high basal methylation levels strongly enhanced hypermethylation while H3K27me3 strongly enhanced inflammation-induced hypermethylation. Inflammation was capable of overriding active transcription. In young gastric mucosae, genes with high expression and frequent mutations in gastric cancers were more frequently methylated than in old ones.ConclusionsMethylation by inflammation was not simple acceleration of age-related methylation. Targets of aberrant DNA methylation were different between young and old gastric mucosae, and driver genes were preferentially methylated in young gastric mucosa.

Abstract C106: New therapeutic strategy by integrative analysis of germline, somatic genetic alterations and tumor mutational burden (TMB) in deficient mismatch repair (dMMR) gastric cancer

Abstract Background: The prevalence of microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) and Lynch syndrome with germline MMR mutations in gastric cancer (GC), are 5% and 1.6%, respectively. According to TCGA data, BRCA mutations, one of DNA Damage Response (DDR) pathway genes, occur in about 10% of GC (BRCA1 2.7%, BRCA2 7%), but the incidence is increased in MSI-H GC (BRCA1 16%, BRCA2 34%). Our study focused on integrative analysis of germline and somatic genetic alterations according to dMMR status for new therapeutic strategy. Methods: Retrospective comparison analysis of advanced gastric cancer patients between 20 dMMR and 48 proficient MMR (pMMR) was performed using targeted next-generation sequencing (NGS) for somatic genetic alterations and Global Screening Array (Illumina©) to detect germline alterations. PD-L1 expression using immunohistochemistry 22C3 pharmDx was evaluated. TMB was calculated based on somatic nonsynonymous missense mutations and DDR genes were compared. DNA pyrosequencing was used to determine promoter methylation status of MLH1 gene. Results: dMMR was associated with higher TMB (median 64 mut/Mb, IQR 54 -73 vs. 42 mut/Mb, IQR 34 - 50, P&amp;lt;0.001), but not with PD-L1 overexpression (p=0.7). The promoter methylation of MLH1 gene was observed in 50% of total dMMR (n=10/20), and had higher TMB (73 mut/Mb, IQR 63 - 88) compared to MLH1 unmethylated dMMR group (57 mut/Mb, IQR 52 – 62) (P&amp;lt;0.001). Germline or somatic gene alterations associated with MMR expression were not different between dMMR and pMMR (p=0.7, p=0.3, respectively). Somatic DDR alterations were elevated in dMMR compared to pMMR (median 45 mut vs. 36 mut, P=0.004), and in high TMB group with &amp;gt; 50 mut/Mb (n=29, p&amp;lt;0.001). In dMMR group, highly mutated ATM (E166D), CDK12 (E711Q, E716Q, R712T, R713T), PARLB2 (Q559R), STAG2 (V343L) and SMARCA4 (A314P) were observed, implying dMMR GC patients’ sensitivity to PARP inhibitor. Conclusions: There were no differences in germline variations according to MMR status, suggesting no beneficial information from germline testing in advanced GC without family history. However, we observed elevated DDR gene alterations in dMMR GC with higher TMB. These findings may be applicable in strategies to combine immune checkpoint inhibitors with PARP inhibitors in patient with dMMR and/or higher TMB GC. Citation Format: Garden Lee, Jihyun Hwang, Sejung Park, Woo Sun Kwon, Jinseul Park, Jii Bum Lee, Choong-kun Lee, Hyun Cheol Chung, Sun Young Rha. New therapeutic strategy by integrative analysis of germline, somatic genetic alterations and tumor mutational burden (TMB) in deficient mismatch repair (dMMR) gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C106. doi:10.1158/1535-7163.TARG-19-C106

Abstract 5026: The association of genomic features and immunosuppression in gastric cancer

Abstract While the clinical efficacy of anti-PD-1 antibody against gastric cancer has been shown, the clinical response is not satisfied. More detailed analyses of immune status in gastric cancer therefore are required. Here, we show that a sub-population of gastric cancer with a specific driver gene mutation develops immunosuppressive tumor microenvironment (TME), not only inhibiting migrations of CD8+ T cells, but also recruiting regulatory T cells (Tregs). The immunological phenotypes of TME were examined with multi-color flow-cytometry and RNA sequencing of surgically resected samples from 22 gastric cancer patients. Gastric cancers were clustered into two groups according to immunologic gene signatures: high- and low-immunogenic tumors. The low-immunogenic group was further divided into two groups with the ratio of effector Tregs to CD4+ T cells ≥20% or &amp;lt; 20%. Among the low-immunogenic group with high effector Tregs to CD4+ T cells ratio (n=5), two patients harbored a specific driver gene mutation. In order to elucidate the detail mechanism by which the specific driver gene mutation in gastric cancer inhibited migrations of CD8+ T cells and recruited Tregs, we evaluated comprehensive gene expressions using the specific driver gene mutation-overexpressed cell lines. Lower expression of chemokines crucial for CD8+ T-cell recruitment (CXCL10) was detected in the specific driver gene mutation-overexpressed cell lines than in the specific driver gene wild-type-overexpressed cell lines. In addition, Gene Set Enrichment Analysis (GSEA) demonstrated enrichment of gene sets associated with fatty acid synthesis in the specific driver gene mutation-overexpressed cell lines compared with the specific driver gene wild-type-overexpressed cell lines. Indeed, abundant fatty acids were detected in TME of driver gene mutation-overexpressed tumors and were dominantly utilized by Tregs, contributing the compelling survival of Tregs in the TME than effector T cells. These results suggested that gastric cancer cells with the specific driver gene mutation reduce the production of chemokines recruiting CD8+ T cells, produce a favorable TME for Tregs. We then developed mouse models and explored TILs and confirmed that the specific driver gene mutation-overexpressed tumors have lower CD8+ T cells and higher Tregs than wild-type-overexpressed tumors. Efficacy of anti PD-1 antibody was ameliorated in the specific driver gene mutation-overexpressed tumors compared with wild-type-overexpressed tumors. Our findings propose that certain types of cancer cells with the specific driver gene mutation inhibit immigration of CD8+ T cells, recruit Tregs, developing an immune suppressive TME associated with the resistance to anti PD-1 antibody. Further studies are required to develop new therapeutic methods of anti PD-1 antibody in combination with inhibitors which block down-streams of the specific driver gene mutation signaling. Citation Format: Shogo Kumagai, Yosuke Togashi, Kohei Shitara, Takahiro Kinoshita, Katsuya Tsuchihara, Hiroyoshi Nishikawa. The association of genomic features and immunosuppression in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5026.

Abstract 4664: Massively parallel sequencing-based analysis of synchronous gastric cancer and lung cancer

Abstract Synchronous gastric cancer(GC) and lung cancer(LC) have been suggested to represent independent primary tumors rather than metastatic disease. We subjected sporadic synchronous GC/LC from five patients to whole-exome massively parallel sequencing, which revealed a median of and 1174 and 830 nonsynonymous somatic mutations in the synchronous GC and LC repectively. DNA mutations range between 292 to 2421 in lung samples and 287 to 1995 in stomach sample. Common mutations varies between 41 and 145. The substitution of C&amp;gt;A, C&amp;gt;T and T&amp;gt;C accounts for the most common mutations in both lung and stomach cancers. Tumor mutation burden (TMB ) varies from 2.7 to 11.0 Mut/Mb in Lung cancer and from 1.8 to 30.4 Mut/Mb in gastric cancer. We further did pathway analysis based on the list of mutant genes. Top 20 pathways enriched in each patient were analyzed. We found MAPK signaling pathway was most commonly enriched pathways in lung cancer patients, whereas PI3K-Akt signaling and Hedgehog signaling pathway were most commonly enriched pathways in stomach cancer patients. This result suggests the potential benefit of targeted therapeutic treatments involved in these pathways. Citation Format: Nandie Wu, Rutian Li, Yang Shao, Lifeng Wang, Baorui Liu, Jia Wei. Massively parallel sequencing-based analysis of synchronous gastric cancer and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4664.

C-Met-dependent phosphorylation of RhoA plays a key role in gastric cancer tumorigenesis.

RhoA has been identified as having a gain-of-function mutation in approximately 20% of diffuse gastric cancer patients. However, the carcinogenic role of RhoA mutations in gastric cancer (GC) is unclear. In the present study, we report that RhoA directly interacts with c-Met and can be phosphorylated by c-Met at Y42 before subsequent K48-linked polyubiquitination and proteasome-mediated protein degradation. Y42C-mutated RhoA exhibits higher protein levels and promotes the proliferation and motility of GC cells. Interestingly, a c-Met inhibitor significantly repressed the growth of GC cells transfected with WT RhoA but not RhoA mutated at Y42 in vivo and in vitro. Analyses of human GC tissues showed that the combined levels of p-c-Met and p-RhoA are a better predictor for prognosis than either factor alone. Taken together, our findings unravel the mechanism by which the RhoA Y42 mutant is linked to poor prognosis in GC. Moreover, this study helps to identify a strategy for patient stratification and optimization of targeted c-Met therapy. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of KRAS gene mutation associated with Helicobacter pylori infection in patients with gastric cancer.

Objective(s):KRAS proto-oncogene mutation can be considered a diagnostic factor for treating various malignancies. Helicobacter pylori infection, a risk factor for stomach cancer, may cause DNA damage and genetic changes. The aim of the current study was to assess the association of gastric cancer and KRAS mutation, demographic factors, and H. pylori infection.Materials and Methods:DNA was extracted from a total of 140 FFPE gastric cancer tissue samples. detection of KRAS mutation (codons 12 and 13) in tumors was performed by PCR amplification, followed by gel electrophoresis and DNA sequencing. PCR diagnosed any H. pylori infection.Results:KRAS mutation was detected in 6 of the 140 (4.2%) gastric cancer tissue samples. 18 samples (12.8%), all of which were male (P<0.05), tested positive for H. pylori infection. KRAS mutations were present in 22.2% (4/18) of the samples with H. pylori infection (P<0.05). The mean age of patients was 62.25±12.61 years (range: 30–93 years). A male predominance (2.5 to 1) was reported in the gastric cancers, and at diagnosis, women were significantly younger than men (P=0.004). No association was observed between age or gender and KRAS mutation. Neither was one found between age and H. pylori infection. Tumors from H. pylori+ subjects were significantly more likely to have KRAS mutation than tumors from H. pylori- subjects (OR=17.1).Conclusion:The data suggest that H. pylori infection when compared with the absence of H. pylori infection, is associated with a higher prevalence of KRAS mutation in gastric cancer.

Associations of PIK3CA mutations with clinical features and prognosis in gastric cancer.

Aim: The clinical implications of PIK3CA mutations in gastric cancer (GC) are not conclusive. Materials & methods: A systematic searching of the previous publications and related studies in The Cancer Genome Atlas (TCGA) database were performed to investigate the clinical implications of PIK3CA mutations in GC. Results: Twenty-six independent cohort studies including six studies with original data were identified. Meta-analysis suggested PIK3CA mutations were associated with high T stage, poor differentiation and microsatellite instability, but not with prognosis in overall. However, PIK3CA mutation was found to be associated with favorable overall survival in subgroup of patients with low PIK3CA mutation prevalence. Conclusion: PIK3CA mutations might be involved in GC development and might be used as favorable prognostic factor in GC population with low PIK3CA mutations prevalence.

Initial and crucial genetic events in intestinal-type gastric intramucosal neoplasia.

Gastric cancer (GC) is one of the most common and life-threatening malignancies. The course of disease and tumor aggressiveness vary among GCs, although how early fate is determined and by what factors remains elusive. To solve this question, we collected 43 gastric intramucosal neoplasias (GINs), comprising dysplasia/intraepithelial neoplasia (D/IEN; a premalignant lesion) and minute GC (miGC; ≤10 mm) of intestinal histotype and performed targeted deep DNA sequencing of 67 GC-related genes derived from large-scale data. Gastric D/IEN was classified into low or high grade (LG-D/IEN or HG-D/IEN). The most frequent mutations in D/IENs included APC (19/25; 76%), ARID2 (6/25; 24%) and MUC6 (5/25; 20%). All LG-D/IENs had APC mutation (12/12) and APC hotspot mutations affecting R1450 and E1554 were noted in both LG-D/IEN and HG-D/IEN. ARID2 mutation always co-occurred with APC mutation, whose tumor variant allele frequency (TVAF) was higher than that of ARID2 in D/IEN. APC and TP53 mutations were mutually exclusive in D/IEN (p = 0.031 [main cohort], p = 0.025 [expanding cohort]) and TP53-mutated D/IEN was exclusively HG-D/IEN (4/4). TP53 mutations were highly recurrent (11/14; 79%) in MLH1-positive miGCs and were detected even in two microscopic lesions measuring 1 and 3 mm, respectively. Furthermore, TVAF analyses suggested that TP53 mutation is the initial event in the TP53-mutated miGCs. In contrast, TP53 mutation was absent (0/4) in MLH1-negative small intramucosal carcinoma (8-24 mm). Advanced GC data suggested that early mutations (APC and TP53) may affect the potential of cancerous progression from D/IEN. This study revealed somatic mutational landscape and initial mutations of GINs, and we report for the first time that TP53 mutations precede other mutations in intestinal-type GC. Our results also indicate that molecular subtyping based on APC/TP53 mutations would be a high-priority approach for determining and predicting the malignant potential of GIN, including D/IEN. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.