Sporadic Gastric Cancer Research Articles

Expression of DNA Damage Response Markers in Early-Onset or Familial Gastric Cancers

Background:Early-onset or familial gastric cancer (GC) is known to have clinicopathologic profiles different from those of sporadic GC. We aimed to compare DNA damage response marker expression between early-onset or familial GC and sporadic GC.Methods:GC samples were obtained from patients who underwent gastrectomy for GC at Seoul National University Hospital. Immunohistochemical analyses of various DNA damage response markers, including BRCA1, BRCA2, MRE11, RAD51C, and γH2AX, were performed using 54 early-onset GC, 59 familial GC, and 337 sporadic GC tissue microarray samples. Correlations between marker expression and clinicopathologic features were evaluated by univariate and multivariate analyses, and overall survival was analyzed.Results:The rate of γH2AX positivity was significantly higher (p < 0.001) in early-onset or familial GC than in sporadic GC. In contrast, the rates of MRE11 negativity and RAD51C negativity were significantly higher in sporadic GC than in early-onset or familial GC. BRCA1 negativity was associated with decreased overall survival in sporadic GC (p = 0.002), and MRE11 negativity was associated with decreased overall survival in sporadic GC (p = 0.012).Conclusion:Our results show significant differences in DNA damage response marker expression between early-onset or familial GC and sporadic GC.

Clinical significance of BRCA1 and BRCA2 mRNA and protein expression in patients with sporadic gastric cancer.

The purpose of the present study was to investigate the clinical significance of BRCA1/BRCA2 DNA repair associated (BRCA1/BRCA2) gene expression in patients with sporadic gastric cancer (GC) who had received postoperative adjuvant chemotherapy. Breast cancer type 1 and 2 susceptibility protein (BRCA1 and BRCA2) expression and BRCA1/BRCA2 mRNA expression were evaluated using immunohistochemistry (IHC) and in-situ hybridization (ISH) on tissue GC microarray tissues, in addition to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results were analyzed for clinicopathological associations. A total of 367 cases of sporadic GC (stages II and III) were subjected to BRCA1 and BRCA2 expression analysis, and for BRCA1 and BRCA2 IHC, 360 cases were informative. A total of 61 cases (16.9%) displayed a loss of BRCA1 and 63 (17.5%) displayed a loss of BRCA2. BRCA1 and BRCA2 ISH results were obtained in 364 cases, of which 98 (26.9%) presented with low expression of BRCA1 mRNA and 148 (40.7%) with low expression of BRCA2 mRNA. In 72 of the 367 cases, BRCA1 and BRCA2 mRNA expression levels were assessed using RT-qPCR, of which 50 (69.4%) and 56 (77.8%) displayed low expression of BRCA1 and BRCA2, respectively. Positive IHC expression of BRCA2 was associated with advanced tumor stage; however, BRCA1 expression was not associated with any clinicopathological parameters. Associations between the RT-qPCR and ISH methods were not significant for either BRCA1 or BRCA2. The results of Kaplan-Meier survival analysis with stage subgrouping revealed no significant differences with regard to survival rate. Of the multivariate analyses, neither BRCA1 nor BRCA2 IHC results were independent prognostic factors. In summary, the present study indicated that BRCA1 and BRCA2, as assessed by IHC, may be used as clinicopathological biomarkers to evaluate the prognosis of sporadic GC.

CDH1 somatic alterations in Mexican patients with diffuse and mixed sporadic gastric cancer

BackgroundDiffuse gastric cancer (DGC) is associated with the reduction or absence of the expression of the cell adhesion protein E-cadherin (encoded by the CDH1 gene). Molecular characteristics are less well described for mixed gastric cancer (MGC). The main somatic alterations that have been described in the CDH1 gene are mutations, loss of heterozygosity (LOH) and promoter methylation. The aim was to analyze CDH1 somatic alterations in Mexican patients with diffuse and mixed gastric cancer.MethodsWe searched for mutations in the CDH1 gene in tumor DNA from DGC (n = 13) and MGC (n = 7) patients by next generation sequencing (NGS). Validation of findings was performed using Sanger sequencing. LOH was analyzed using dinucleotide repeat markers surrounding the CDH1 gene, and methylation was investigated by DNA bisulfite conversion and sequencing. E-cadherin protein deficiency was analyzed by immunohistochemistry.ResultsSeventeen point variants were identified by NGS, 13 of them were validated by Sanger sequencing. Only 1/13 had not been previously reported (c.-137C > A), and 12/13 were already reported as polymorphisms. Two DGC cases presented LOH at the locus 16q22.1 (13.3%). CDH1 promoter methylation was positive in (7/11) 63.6% and (4/6) 66.6% of the cases with DGC and MGC, respectively. E-cadherin protein deficiency was observed in 58.3% of DGC cases while 100% in MGC cases.ConclusionsWhile no pathogenic somatic mutations were found that could explain the diffuse histology of gastric cancer in DGC and MGC, methylation was the most common somatic inactivation event of the CDH1 gene, and LOH was rare. The previously unreported c.-137C > A variant modify the CDH1 gene expression since it alters the binding sites for transcription factors.

CDH1 Gene and Hereditary Diffuse Gastric Cancer Syndrome: Molecular and Histological Alterations and Implications for Diagnosis And Treatment.

Gastric cancer, a group of common malignancies, results in the most cancer mortality worldwide after only lung and colorectal cancer. Although familial gastric cancers have long been recognized, it was not until recently that they were discovered to be associated with mutations of specific genes. Mutations of CDH1, the gene encoding E-cadherin, are the most common germline mutations detected in gastric cancer and underlie hereditary diffuse gastric cancer (HDGC) syndrome. All reported HDGCs are the pure diffuse type by Lauren classification and are associated with dismal prognosis once the tumor invades the submucosa. Because CDH1 germline mutations are inherited in an autosomal-dominant fashion and have high penetrance, the International Gastric Cancer Linkage Consortium (IGCLC) developed criteria to facilitate the screening of CDH1 mutation carriers; these criteria have been proven to have excellent sensitivity and specificity. Recent histologic studies suggest that HDGC progresses through several stages. Even when the tumor becomes “invasive” in lamina propria, it may stay indolent for a long time. However, the molecular mechanisms that induce the transitions between stages and determine the length of the indolent phase remain to be determined. Although the standard management for CDH1 mutation carriers is prophylactic total gastrectomy, many questions must be answered before the surgery can be done. These include the optimal surveillance strategy, the best strategy to choose surgical candidates, and the ideal time to perform surgery. In addition to increasing the risk of gastric cancer, CDH1 germline mutations also increase the risk of invasive lobular carcinoma of the breast, and possibly colorectal adenocarcinoma, and are associated with blepharocheilodontic syndrome (a congenital development disorder). However, the optimal management of these conditions is less established owing to insufficient data regarding the risk of cancer development. This review focuses on molecular and histological findings in HDGC, as opposed to sporadic diffuse gastric cancer, and their implications for the management of CDH1 mutation carriers and the diagnosis and treatment of HDGC. Other conditions associated with CDH1 germline mutations and future research directions are also discussed.

The Prognostic Value of lncRNAs Expression and Clinicopathological Feature in Gastric Cancer: A Field Synopsis of Observational Studies

Background: Data on prognostic value of long non-coding RNAs (lncRNAs) to sporadic gastric cancer have been published at a growing pace. However, there has been no comprehensive quantitative overview has been available. We aim to investigate the association between the expression of long non-coding RNAs (lncRNAs), clinicopathological factors and prognosis of gastric cancer. Methods: We explored correlation of lncRNA with clinicopathological features and prognosis. The pooled hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS), disease-free survival (DFS)/progression-free survival (PFS)/Disease Specific Survival (DSS)/recurrence-free survival (RFS) were calculated to estimate the association strength. Results: Literature search identified 80 eligible studies comprising 2205 cases which investigated senven gastric cancer associated clinicopathological factors. Overall, a significant association was observed between high lncRNAs level and poor OS in patients with gastric cancer (HR = 1.46, 95% CI: 1.31-1.63, P<0.001). We conduct stratified analysis based on statistical approach showing that high expression of lncRNAs in both log rank (HR=1.64, 95%CI: 1.32-2.03, P<0.001) and multivariate analysis (HR=1.37, 95%CI: 1.21-1.55, P<0.001) were significant associated with poor OS, and the pooled HR of association between elevated lncRNAs expression level and DFS/PFS/DSS/RFS in gastric cancer patients was 1.60 (95% CI: 1.29-1.98, P<0.001). For the OS, the results showed that the lncRNA expression level was signifcantly associated the clinicopathological parameters including TNM stage (P<0.001), tumor size (P<0.001), pathological differention (P=0.02), lymph nodes metastasis (P<0.001), distance metastasis (P<0.001) and invasion depth (P<0.001), and there was significant association in TNM stage (P<0.001), lymph nodes metastasis (P<0.001), distance metastasis (P<0.001), and invasion depth (P=0.02) for DFS. Conclusion: High expression of lncRNA could predict poor prognosis in gastric cancer, and abnormal expression of lncRNAs supported the clinical value regarding the clinicopathological factors. Funding: This study was funded by National Natural Science Foundation of China (81701536, 81373097). Declaration of Interest: The authors declare no conflict of interest.

Familial aggregation of gastric cancer with microsatellite instability*

Background: Microsatellite instability (MSI) is currently a new molecular subtype of gastric cancer (GC). About 90% of GC cases appear sporadically. MSI seems to be responsible for both sporadic and familial GC. The aim of this study was to analyze the frequency of MSI in GC with familial history of GC.Methods: The MSI analysis was conducted using five quasi-monomorphic mononucleotide repeats: BAT-26, BAT-25, NR-24, NR-21 and NR-27. From our database, we analyzed 457 patients in terms of cancer history across family members, particularly focusing on GC.Results: MSI status in patients without familial history of GC was present in 22.1% of the cases, whereas in the patients with familial history of GC it was present in 28% of the cases (p = 0.220). For 1st or 2nd degree family members with GC, MSI was observed in 27.6% and in 30.8%, respectively (p = 0.812). MSI was observed in hereditary gastric cancer (HGC) in 33.3% and in familial gastric cancer (FGC) in 30%. No difference in survival rates was observed between the analyzed groups.Conclusions: In our publication, we could not find any link between familial background and the MSI status in GC patients. More detailed molecular and genetic analysis of subgroups of these patients is required.

Outcomes after completion total gastrectomy for gastric remnant cancer: experience from a Canadian tertiary centre

There is controversy about the safety and outcomes of completion total gastrectomy (CTG) for gastric adenocarcinoma. We compared a cohort of patients who underwent CTG for gastric remnant cancer (GRC) after partial gastrectomy for benign disease with patients who underwent primary total gastrectomy (PTG) for sporadic gastric cancer. We retrospectively reviewed a single-institution, prospectively maintained clinical database of patients who had undergone gastrectomy from 2005 to 2016 for demographic, surgical, clinical and tumour pathology data, as well as postoperative, pathologic and oncologic outcomes including complications, length of stay, disease-free survival and overall survival. We used the χ2 and Wilcoxon rank-sum tests to compare groups and performed the Mantel-Cox log-rank test for Kaplan-Meier survival estimates. We compared the CTG group to all patients in the PTG group and to a 5:1 propensity-matched PTG cohort. We analyzed data for 64 patients (9 CTG, 55 PTG). The groups were equivalent at baseline and had similar operative, perioperative treatment and pathologic characteristics. After propensity matching, the reoperation rate for complications was higher after CTG than PTG (22% v. 0%, p = 0.03), but there was no significant difference in the overall complication rate or length of stay. At 5 years, there was no difference in disease-free survival (28% v. 58%, p = 0.4) or overall survival (33% v. 44%, p = 0.7). Our findings suggest that CTG for gastric adenocarcinoma can be undertaken safely a priori with no additional risk of recurrence or death compared to PTG for sporadic gastric cancer.

Abstract 1570: Loss of mismatch repair protein MSH6 sensitizes diffuse-type gastric cancer cells to DNA-PKcs inhibitor KU60648

Abstract Diffuse-type gastric cancer (DGC) is a histologic subtype characterized by poor cellular cohesiveness, increased metastasis and poor prognosis. As preclinical models of DGC that reliably predict clinical activity of novel compounds are lacking, we generated patient-derived xenograft (PDX) models using malignant ascites from DGC patients that closely recapitulated the primary cancer. To uncover novel therapeutic targets, we applied the concept of synthetic lethality and asked if DGCs require DNA repair proteins for survival. Major DNA repair kinases ATM, ATR and DNA-PKcs in established DGC cell lines and ex vivo cell lines from PDXs (GAGA3, GAGA6 and GAS24) were inhibited using small-molecule inhibitors KU55933, VE-821 and KU60648, respectively. Interestingly, most diffuse-type gastric cells exhibited elevated lethality when DNA-PKcs, a central kinase that regulates the non-homologous end joining pathway (NHEJ), was inhibited by KU60648 as compared to the nontransformed gastric epithelial line, HFE-145. We confirmed that genetic depletion of DNA-PKcs by siRNA resulted in cell death in two PDX lines GAS24 and GAGA6 cells but not in HFE-145 cells, suggesting the dependence of these cells on DNA-PKcs for survival. Two mechanisms appear to sensitize DGC cells to DNA-PKcs inhibition; firstly, impaired homologous recombination repair in GAS24 and GAGA6 as shown by compromised ability to resolve radiation-induced γH2AX foci, and secondly, transcriptional downregulation of the mismatch repair protein, MSH6, in GAGA3 and GAGA6. Knockdown of MSH6 in GAS24 cells enhanced their sensitivity to KU60648, suggesting a functional link between MSH6 and DNA-PK function. Lastly, to develop MSH6 as a biomarker for predicting DNA-PKcs sensitivity, we analyzed MSH6 levels by immunohistochemical staining in formalin-fixed, paraffin-embedded (FFPE) tissue sample sections derived from PDXs. In these preliminary studies, MSH6 expression was lower with high heterogeneity in GAGA3 and GAGA6 compared to GAS24. In this study, we show for the first time the downregulation of DNA mismatch protein MSH6 in sporadic diffuse gastric cancer, and its potential as a therapeutic predictor for DNA-PKcs inhibition treatment. Citation Format: Vaidehi Krishnan, Wen Min Lau, Eileen Teng, Jin Wei Tan, Haresh Sankar, Debbie Xiu En Lim, Crystal Kok Yan Pang, Ming Teh, Jimmy Bok Yan So, Patrick Tan, Yoshiaki Ito, Shing Leng Chan. Loss of mismatch repair protein MSH6 sensitizes diffuse-type gastric cancer cells to DNA-PKcs inhibitor KU60648 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1570.

Association between Krüppel like factor 6 intervening sequence 1-27 G > A and cancer susceptibility: A meta-analysis.

It has been reported that Krüppel like factor 6 intervening sequence (KLF6 IVS) 1-27 G > A might be associated with cancer susceptibility. Here, we conducted a meta-analysis to summarize and clarify this association. A systematic search of studies on the association between KLF6 IVS 1-27 G > A, and cancer susceptibility was conducted in databases. Odds ratios and 95% confidence intervals were used to pool the effect size. Seven articles were included in our meta-analysis. Overall and in prostate cancer, population-based subgroup overall and Caucasian subgroup overall, no evidence was found for the association between KLF6 IVS 1-27 G > A polymorphism and cancer susceptibility in any genetic model and the results showed stability in sensitivity analyses. KLF6 IVS 1-27 G > A may not be associated with cancer susceptibility, especially the susceptibility of unselected prostate cancer. However, there was insufficient data to fully confirm the association between KLF6 IVS 1-27 G > A and familial prostate cancer, sporadic prostate cancer, gastric cancer, and cancers from different ethnicity, and the results should be interpreted with caution.

Novel Variants and Copy Number Variation in CDH1 Gene in Iranian Patients with Sporadic Diffuse Gastric Cancer.

The aim of this study was to survey the nucleotide changes and copy number variations (CNV) in the CDH1 gene in Iranian patients with sporadic diffuse gastric cancer (SDGC). In this study, 28 patients were examined who upon gastrectomy had been diagnosed with SDGC according to the familial history and histopathological criteria which was confirmed by the pathologist. DNA extraction was performed from formalin-fixed paraffin-embedded tissues using a phenol-chloroform method following xylene deparaffinization. Determination of DNA sequence by Sanger was performed using PCR amplification of 16 exons and boundaries of intron/exon of CDH1 gene. Multiplex ligation-dependent probe amplification (MLPA) was performed on patients with pathogenic disorders in the sequence. In total, patients included 20 males and 8 females. Of all patients, 12 patients were under 45years old (early onset gastric cancer, EODC) and 16 patients were older. The tumor was diagnosed in the early TNM stage (I, II) in six patients and in late stages (III, IV) in 19 cases. Altogether, 16 variants (three exonic with one new variant and 13 intronic with nine new variants) were found in DNA sequencing of the CDH1 gene in five samples. Also, using MLPA, a new duplication in exon 9 and one deletion in exon 2 were detected in two other patients. Altogether, CDH1 variants were identified in seven out of 28 patients (25%). Our study revealed several novel somatic variants in the CDH1 gene in Iranian patients with sporadic diffuse GC. Our data supports the hypothesis that mutations in CDH1 gene, and particularly the mutations we describe, should be considered, even in sporadic cases of gastric cancer. The presence of these mutations in patients raises important issues regarding genetic counseling and diagnostic test in DGC patients.

Merging perspectives: genotype-directed molecular therapy for hereditary diffuse gastric cancer (HDGC) and E-cadherin\u2013EGFR crosstalk

Hereditary diffuse gastric cancer is a cancer predisposition syndrome associated with germline mutations of the E-cadherin gene (CDH1; NM_004360). Male CDH1 germline mutation carriers have by the age of 80 years an estimated 70% cumulative incidence of gastric cancer, females of 56% for gastric and of 42% for lobular breast cancer. Metastatic HDGC has a poor prognosis which is worse than for sporadic gastric cancer. To date, there have been no treatment options described tailored to this molecular subtype of gastric cancer. Here we review recent differential drug screening and gene expression results in c.1380del CDH1-mutant HDGC cells which identified drug classes targeting PI3K (phosphoinositide 3-kinase), MEK (mitogen-activated protein kinase), FAK (focal adhesion kinase), PKC (protein kinase C), and TOPO2 (topoisomerase II) as selectively more effective in cells with defective CDH1 function. ERK1-ERK2 (extracellular signal regulated kinase) signaling measured as top enriched network in c.1380delA CDH1-mutant cells. We compared these findings to synthetic lethality and pharmacological screening results in isogenic CDH1−/− MCF10A mammary epithelial cells with and without CDH1 expression and current knowledge of E-cadherin/catenin–EGFR cross-talk, and suggest different rationales how loss of E-cadherin function activates PI3K, mTOR, EGFR, or FAK signaling. These leads represent molecularly selected treatment options tailored to the treatment of CDH1-deficient familial gastric cancer.

Abstract 3806: Integrative omics analysis in gastric cancer reveals SLC7A5 functional related to DNA mismatch repair system as a potential therapeutic biomarker in GC

Abstract Background: Metastasis is a major cause leading to high mortality rate and poor prognosis in Gastric Cancer (GC) patients. Our previous studies showed that SLC7A5 is an amino acid transporter gene meditated by a pivotal post-transcriptional tumor suppressor mir-126 and associated with tumor metastasis in GC. Purpose: (a) to further determine the SLC7A5 functional mechanisms in GC by involving more bio-omics data such as DNA methylation, copy number variation (CNV) and mRNA expression; b) to study the association of SLC7A5 bio-omics data with the clinicopathologic features, prognosis and subtypes in GC patients. Methods: We intensively performed the genome-wide association study for bio-omics data of GC patients available from The Cancer Genome Atlas (TCGA, n=421) and Gene Expression Omnibus published studies (GEO, GSE13911, n=69, GSE15459, n=209). We did Kaplan-Meier survival analysis and univariate Cox regression model analyses in SLC7A5 mRNA expression from curated database (http://www.kmplot.com) with 882 GC patients for overall survival and 646 GC patients for progression free survival analysis. Results: CNV of SLC7A5 is positively correlated with mRNA expression (p=3.18e-07), which suggests that SLC7A5 is potentially a target driver gene for GC. Further integrative analysis identified two important methylation probes in promotor region (TSS200 position) negatively correlated with mRNA expression of SLC7A5 (p=1.03e-3). In addition, the aberrant high methylation and expression of SLC7A5, which were significantly higher than the corresponding nonmalignant tissues, were identified in a subset of gastric cancer patients. Interestingly, SLC7A5 showed statistically significant correlation with the mismatch repair-deficient phenotype in sporadic microsatellite instability gastric cancer (p=2.12e-5). Data mining from two independent datasets of GEO validated SLC7A5 is positively correlated with DNA mismatch repair genes MLH1, BRCA1 and BRCA2. And further Gene Ontology (GO) pathway analysis suggested that SLC7A5 and DNA mismatch repair genes (MMR) together shared the same metabolic processes. Importantly, the overall survival from 882 GC patients and progression free survival analysis from 646 GC patients showed SLC7A5 high expression is associated with poor prognosis tumorigenesis, which demonstrated that SLC7A5 is a potential independent biomarker for GC. Conclusion: We found SLC7A5 has positive interaction with DNA mismatch genes through database mining, which result in microsatellite instability phenotype in GC patients. We also found SLC7A5 over expression is associated with poor prognosis and potentially an pivotal therapeutic target in GC. Note: This abstract was not presented at the meeting. Citation Format: Yunyun Zhou, Xuehua Chen, Zhenggang Zhu, Junqing Wang. Integrative omics analysis in gastric cancer reveals SLC7A5 functional related to DNA mismatch repair system as a potential therapeutic biomarker in GC [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 3806. doi:10.1158/1538-7445.AM2017-3806

Abstract 4270: Family history in first degree relatives and risk of gastric cancer in the alpha-tocopherol, beta-carotene cancer prevention study

Abstract Background: Studies of the familial associations of gastric cancer risk have largely been limited to case-control approaches which are susceptible to recall bias and cannot distinguish temporality. Furthermore, associations with family history could be explained by shared exposure to Helicobacter pylori infection, the most important environmental risk factor for gastric cancer. Notably, the risks of early onset disease and diffuse-type tumors are thought to have a greater genetic component. We therefore examined in a prospective study the associations of gastric cancer risk with history of gastric cancer in first degree relatives, controlling for H. pylori infection. Methods: We selected 405 incident gastric cancer cases and 4,129 controls with known H. pylori serology status from the Finnish Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study among male smokers aged 50-69 at baseline. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using logistic regression with adjustment for H. pylori and other potential confounders. All statistical tests were two-sided. Results: Family history of gastric cancer was associated with increased risk (OR=1.84, 95% CI=1.32-2.58). The association appeared stronger with gastric cancer in siblings (OR=2.87, 95% CI=1.64-5.03) than with gastric cancer in parents (OR=1.53, 95% CI=1.05-2.24). Distinguishing cases by anatomic subsite, the family history association was significant for noncardia gastric cancer (n=305; OR=2.15, 95% CI=1.50-3.07), but not for cardia cancer (n=100; OR=1.25, 95% CI=0.63-2.49). With regard to histological subtype, family history was significantly associated with both diffuse-type (n=90; OR=2.12, 95% CI=1.10-4.12) and intestinal-type (n=153; OR=1.70, 95% CI=1.02-2.83) gastric cancer. In age-specific analyses, the association with family history was strongest in the youngest age-group at baseline 50-54 years (OR=3.42, 95% CI=1.85-6.30) and absent in the oldest age group 65-69 years (OR=0.85, 95% CI=0.28-2.58). Conclusion: The results suggest that certain subtypes of sporadic gastric cancer are strongly associated with family history of gastric cancer. These associations do not appear to be explained by the associations with H. pylori infection. Citation Format: Minkyo Song, Maria Constanza Camargo, Stephanie J. Weinstein, Demetrius Albanes, Charles S. Rabkin. Family history in first degree relatives and risk of gastric cancer in the alpha-tocopherol, beta-carotene cancer prevention study [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 4270. doi:10.1158/1538-7445.AM2017-4270

Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset Cancers

BACKGROUND & AIMS:Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients.METHODS:We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays.RESULTS:We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5–7.7). RHOA activity was reduced by an R5W substitution—the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells.CONCLUSIONS:In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer

BackgroundPatients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease.MethodsIn order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells.ResultsUnsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1–ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function.ConclusionIntegrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

Effect of histone modifications on hMLH1 alternative splicing in gastric cancer.

hMLH1 is one of the mismatch genes closely related to the occurrence of gastric cancer. Epigenetic regulation may play more important roles than gene mutations in DNA damage repair genes to drive carcinogenesis. In this article, we discuss the role of epigenetic changes, especially histone modifications in the regulation of hMLH1 alternative splicing. Our results showed that hMLH1 delEx10, delEx11, delEx10-11, delEx16 and delEx17 transcripts were ubiquitous in sporadic Chinese gastric cancer patients and gastric cancer cell lines. Lower level of H4K16ac and H3ac was detected in hMLH1 exon 10-11 region in gastric cancer cell lines when compared with human gastric mucosal epithelial cell line GES-1. A significant decrease of hMLH1 delEx11 and delEx10-11 was observed in gastric cancer cell lines after trichostatin A treatment. H3K36me3 and H3K4me2 levels were lower in hMLH1 exon 10-11 and exon 16-17 regions in gastric cancer lines when compared with GES-1. Aberrant transcripts such as hMLH1 delEx11 and delEx10-11 were significantly higher in gastric cancer cell lines after small interfering RNA-mediated knockdown of SETD2 (the specific methyltransferase of H3K36). The hMLH1 delEx10 and delEx10-11 transcripts were increased after interference of SRSF2. Taken together, our study demonstrates that lower level of histone acetylation and specific histone methylation such as H3K36me3 correlate with aberrant transcripts in hMLH1 exon 10-11 region. SRSF2 may be involved in these specific exons skipping as well.

T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth

ABSTRACTT-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709–718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRIImut peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRIImut-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.

Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients With Gastric Cancer

Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer.

Expression of Mismatch Repair Proteins in Early and Advanced Gastric Cancer in Poland.

BackgroundMutations in DNA of mismatch repair (MMR) genes result in failure to repair errors that occur during DNA replication in microsatellites, resulting in accumulation of frameshift mutations in these genes and leading to DNA mismatch replication errors and microsatellite instability. Gastric cancers (GCs) with high MSI (MSI-H) are a well-defined subset of carcinomas showing distinctive clinicopathological features. In this study we investigated the rate of MSI and the correlation between MSI status and clinicopathological features of GC.Material/MethodsThe study included 107 patients with GCs: 61 with advanced gastric cancers (AGC) and 46 with early gastric cancer (EGC). MSI deficiency in GCs was assessed by the immunohistochemical analysis of expression of MMR proteins – MLH1, MSH2, MSH6, and PMS2 – using formalin-fixed and paraffin-embedded tissue.ResultsA total of 6 (5.6%) MSI-H were observed. The loss of MMR proteins expression was associated with the intestinal type of GC in Lauren classification, and tubular and papillary architecture in WHO classification. There was no statistically significant association between negative MMR expression and other selected clinical parameters: age, sex, tumor location, depth of invasion (EGC and AGC), lymph nodes status, presence of the ulceration, and lymphocytic infiltrate.ConclusionsIn the present era of personalized medicine, the histological type of GC and MMR proteins status in cancer cells are very important for the proper surveillance of patients with familial GC and sporadic GCs, as well as for selecting the proper follow-up and treatment. Larger collaborative studies are needed to verify the features of MSI-H GCs in Poland.

Abstract 3814: Synthetic lethal approaches targeting E-cadherin-deficient cancers

Abstract E-cadherin is a cellular adhesion protein that is frequently mutated in lobular breast cancer and diffuse gastric cancer. The E-cadherin protein which is encoded by the CDH1 gene has key roles in establishing and maintaining cell polarity and differentiation, the organization of cell migration and architecture and the mediation of signaling through various proliferation and survival pathways. E-cadherin also has a tumor suppressor role and its loss in cancer cells would preclude drug targeting by conventional therapy. To circumvent this, we have taken a synthetic lethal approach to exploit any vulnerability created by the loss of E-cadherin. In the therapeutic setting, synthetic lethality refers to a combination of a mutated gene and a drug targeted at a second gene or protein causing cell death (specifically in cancer cells). To identify the vulnerabilities created by E-cadherin loss, we performed a genome-wide siRNA knockdown screen in isogenic MCF10A cell lines with and without E-cadherin expression. From the functional screen, we identified broad classes of G-protein-coupled receptor (GPCR) signaling proteins and families of cytoskeletal proteins which were highly enriched among the synthetic lethal candidates. Indeed, we identified drug classes with linkages to several of the GPCR and cytoskeletal targets that showed evidence of E-cadherin synthetic lethality when we performed a 4,057 known drug screen. These included certain PI3K inhibitors (PI-103), anti-glucocorticoid (mifepristone), tyrosine kinase inhibitor (saracatinib) and multiple histone deacetylase inhibitors (vorinostat and entinostat). Interestingly, the combination of saracatinib and mifepristone gave a synergistic effect (combination index &amp;lt; 1.0) in targeting E-cadherin-deficient MCF10A cells. These results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both of sporadic and familial lobular breast cancer and diffuse gastric cancer. Citation Format: Augustine Chen, Bryony J. Telford, Andrew Single, Henry Beetham, Kaylene J. Simpson, Parry Guilford. Synthetic lethal approaches targeting E-cadherin-deficient cancers. [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 3814.