Resected Gastric Cancer Tissues Research Articles

Fusobacterium Nucleatum-Induced Tumor Mutation Burden Predicts Poor Survival of Gastric Cancer Patients

Co-infection of Helicobacter pylori and Fusobacterium nucleatum is a microbial biomarker for poor prognosis of gastric cancer patients. Fusobacterium nucleatum is associated with microsatellite instability and the accumulation of mutations in colorectal cancer. Here, we investigated the mutation landscape of Fusobacterium nucleatum-positive resected gastric cancer tissues using Illumina TruSight Oncology 500 comprehensive panel. Sequencing data were processed to identify the small nucleotide variants, small insertions and deletions, and unstable microsatellite sites. The bioinformatic algorithm also calculated copy number gains of preselected genes and tumor mutation burden. The recurrent genetic aberrations were identified in this study cohort. For gene amplification events, ERBB2, cell cycle regulators, and specific FGF ligands and receptors were the most frequently amplified genes. Pathogenic activation mutations of ERBB2, ERBB3, and PIK3CA, as well as loss-of-function of TP53, were identified in multiple patients. Furthermore, Fusobacterium nucleatum infection is positively correlated with a higher tumor mutation burden. Survival analysis showed that the combination of Fusobacterium nucleatum infection and high tumor mutation burden formed an extremely effective biomarker to predict poor prognosis. Our results indicated that the ERBB2-PIK3-AKT-mTOR pathway is frequently activated in gastric cancer and that Fusobacterium nucleatum and high mutation burden are strong biomarkers of poor prognosis for gastric cancer patients.

The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8(+) T cells in patients with gastric cancer

Objective: To investigate the levels of Th9 cells and interleukin-9 (IL-9), and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer. Methods: Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included. Twenty individuals who received physical examination in the same period were also enrolled. Peripheral blood was collected, and then plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Tumor-infiltrating lymphocytes (TILs) and autologous gastric cancer cells were isolated from resected gastric cancer tissues. CD4(+) T cells, CD8(+) T cells, and CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells were purified from PBMCs and TILs. Plasma IL-9 level was measured by enzyme linked immunosorbent assay (ELISA). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs and TILSs was assessed by flow cytometry. The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1 (PU.1) were semi-quantified by real-time quantitative polymerase chain reaction (RT-qPCR). PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9. Cellular proliferation was measured by cell counting kit-8. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and STAT6 were investigated by western blot. Cytokine production was measured by ELISA. Purified CD8(+) T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9. CD8(+) T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner. The percentage of target cell death was calculated by measuring the lactate dehydrogenase (LDH) level. These cretion of γ-Interferon (γ-IFN) and tumor necrosis factor-α (TNF-α) was measured by ELISA. CD4(+) CCR4(-)CCR6(-)CXCR3(-)cells, CD8(+) T cells, and autologous gastric cancer cells were directly cocultured, and anti-IL-9 neutralizing antibody was added. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.

Selection of RNA-based evaluation methods for tumor microenvironment by comparing with histochemical and flow cytometric analyses in gastric cancer

Understanding the tumor microenvironment (TME) and anti-tumor immune responses in gastric cancer are required for precision immune-oncology. Taking advantage of next-generation sequencing technology, the feasibility and reliability of transcriptome-based TME analysis were investigated. TME of 30 surgically resected gastric cancer tissues was analyzed by RNA-Seq, immunohistochemistry (IHC) and flow cytometry (FCM). RNA-Seq of bulk gastric cancer tissues was computationally analyzed to evaluate TME. Computationally analyzed immune cell composition was validated by comparison with cell densities established by IHC and FCM from the same tumor tissue. Immune cell infiltration and cellular function were also validated with IHC and FCM. Cell proliferation and cell death in the tumor as assessed by RNA-Seq and IHC were compared. Computational tools and gene set analysis for quantifying CD8+ T cells, regulatory T cells and B cells, T cell infiltration and functional status, and cell proliferation and cell death status yielded an excellent correlation with IHC and FCM data. Using these validated transcriptome-based analyses, the immunological status of gastric cancer could be classified into immune-rich and immune-poor subtypes. Transcriptome-based TME analysis is feasible and is valuable for further understanding the immunological status of gastric cancer.

Abstract A005: Clinicopathological factors associated with establishment of gastric cancer PDXs and cell lines

Abstract Background: Patient-derived xenograft (PDX) models has been widely accepted as a suitable preclinical model. However, the number of PDX models as well as cell-lines from gastric cancer (GC) are limited. We started our DEF study project on May 2013 for establishing new GC PDX models and cell lines for accelerating the development of new therapeutics for GC. Methods: Two hundred and fifty (250) patients, including 233 patients underwent gastrectomy and 17 patients received cell-free and concentrated ascites reinfusion therapy (CART) in our hospitals, were enrolled. We subcutaneously engrafted 232 surgically resected gastric cancer tissues into immune-deficient NOG mice. Primary or PDX tumor tissues or ascites from CART patients were cultivated for establishing GC cell lines. Results and Discussion: From 233 surgically resected tumor tissues, we have successfully established 35 gastric cancer PDX models. PDX-establishment rate was 15.1%, and differentiated type adenocarcinomas (DAs) were more effectively established than poorly differentiated type adenocarcinomas (PDAs). The histology of PDX resembled their primary tumors, and the concordance of histological differentiation grade between primary tumors and PDXs was significant (28/35, p&amp;lt;0.01). Twenty-three (23) GC cell lines have been established from surgically resected primary or subsequently transplanted PDX tissues. Another 2 GC lines were established by cultivating primary GC tissues directly. Among 25 GC cell lines, 24 lines could develop subcutaneous tumors (CDXs) in SCID mice. In contrast to PDX, the concordance of histological differentiation grade between primary tumors and CDXs was not significant. In addition, we have also established 7 GC cell lines from ascites from 17 patients received CART, and six of them could develop CDXs. All of the CDXs from CART cases showed PD histology. As the results, we have established both PDX and Cell lines simultaneously from 21 patients, which allowed us to invest a direct histological and molecular comparison between primary, PDX, and CDX tumors. We would also like to discuss about lymphoproliferative lesions, most of which showed proliferation of B cell with various cytological atypia, encountered during the PDX establishment process. Citation Format: Takeshi Kuwata. Clinicopathological factors associated with establishment of gastric cancer PDXs and cell lines [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 A005. doi:10.1158/1535-7163.TARG-19-A005

PO-282 Cancer-associated fibroblast-derived lumican promotes gastric cancer progression via the integrin beta1-FAK signalling pathway

IntroductionGastric cancer (GC) is one of the most frequent malignant tumours worldwide and is associated with high invasiveness, high metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs), residing around tumour cells in tumour stroma, are important modifiers of tumour initiation and progression. However, the molecular mechanisms by which CAF’s modulate tumour development have yet not to be characterised in GC.Material and methodsCAFs and normal fibroblasts (NFs) were cultured from resected gastric cancer tissues and patient matching non-cancerous tissues. The expression of Lumican in gastric CAFs was detected by quantitative RT-PCR and Westerblot. The effects of CAFs, which was interfered with Lumican, on gastric cancer cell proliferation, migration and invasion in vitro were measured. were measured by CCK8, the coculture migration or matrigel invasion assay, respectively. In vivo tumorigenesis and metastasis were performed by injection CAFs and GC cells subcutaneously or peritoneally.Results and discussionsLumican, an extracellular matrix protein, is highly expressed in human gastric CAFs and its expression is positively associated with depth of invasion, lymph node metastasis, TNM stage and poor survival rate of GC. Knockdown of Lumican in CAFs impaired the GC cell proliferation, migration and invasion in vitro, as well as tumorigenesis and peritoneal metastasis in vivo. Further functional studies revealed that integrin beta1-FAK signalling pathway mediated the promoting effects of stromal Lumican on GC cell metastasis potential.ConclusionCAF-derived Lumican contributes to tumorigenesis and metastasis of GC by activating the integrin b1-FAK signalling pathway.Targeting stromal Lumican may provide a potential treatment strategy for GC.

Metastasis-associated protein 1 (MTA1) in gastric cancer tissues is positively associated with poorer prognosis

BackgroundThe present study examined the clinical significance of metastasis-associated protein 1 (MTA1) in the progression and patient survival of gastric cancer. MethodsParaffin-embedded resected tissues of gastric cancer mucosa (n = 436) and adjacent normal mucosa (n = 92) were assessed immunohistochemically for MTA1 protein, and scored according to the percentage of cells positively stained for MTA1 combined with stain intensity. Associations between MTA1 staining scores and clinicopathological factors, including survival time, were evaluated. ResultsThe staining scores for MTA1 were significantly higher in gastric cancer tissues than in matched normal tissues. MTA1 scores positively correlated with tumor size, depth of invasion, presence of lymph node metastasis, lymphatic involvement, venous invasion, distal metastasis, and advanced clinical staging. Patients with high MTA1 scores in gastric cancer tissues had a significantly lower five-year survival rate compared with patients with low MTA1 scores. The multivariate analysis indicated that MTA1 protein levels in resected gastric cancer tissues, as reflected by immunohistochemical staining, are an independent prognostic index of gastric carcinoma (P < 0.01). ConclusionMTA1 immunopositivity was significantly associated with progression of gastric cancer, and may be helpful in gastric cancer prognosis.

Expressions of Ras Homolog Gene Family, Member A (RhoA) and Cyclooxygenase-2 (COX-2) Proteins in Early Gastric Cancer and Their Role in the Development of Gastric Cancer.

BackgroundThis research focused on detecting the expressions of RhoA and cyclooxygenase-2 (COX-2) proteins in early gastric cancer tissues and to explore their role in the development of gastric cancer.Material/MethodsSurgically resected gastric cancer tissues and the paired normal paracancerous tissues were collected from 26 patients with early gastric cancer from January 2015 to November 2015. The expressions of RhoA and COX-2 proteins were detected by using RT-PCR and immunohistochemistry techniques, respectively. Cell proliferation and migration experiments were conducted on the RhoA-silenced A6-B9 cells and COX-2-silenced D7-B8 cells so as to discuss their role in the development of gastric cancer.ResultsRelative mRNA expressions of RhoA and COX-2 in the cancer tissues were 0.823±0.021 and 0.892±0.103, respectively, which showed significant differences compared to the normal cancerous tissues (0.295±0.014 and 0.129±0.037) (p<0.05). Immunohistochemical staining indicated that the expressions of RhoA and COX-2 proteins in tumor tissues were significantly upregulated as compared to normal cancerous tissues (p<0.05). Cell cloning and streaking assays showed that silencing of RhoA and COX-2 gene caused a considerable decline in the proliferation and migration capacities of the gastric cancer cells, respectively (p<0.05).ConclusionsRhoA and COX-2 were upregulated in early gastric cancer tissues, which facilitated the proliferation and migration of gastric cancer cells. Both proteins may be used as potential markers for the diagnosis of early gastric cancer.

CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer.

Cancer-associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor-promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF-mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF-573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

Abstract 423: CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer

Abstract Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2015-423

Expression of lysyl oxidase and vascular endothelial growth factor in gastric cancer and their effects on tumor invasion and metastasis

Objective To study the expression of lysyl oxidase (LOX) and vascular endothelial growth factor (VEGF) in gastric cancer and paracancerous tissues,and their relationshipa with genesis of gastric cancer.Methods Sixty-five samples of freshly surgically resected gastric cancer tissues and the paracancerous tissues 5 cm away from the gastric cancer tissues each were collected in Wuhan Puren Hospital from Sep.2009 to Jan.2013.By using the method of Western blotting,the protein expression of LOX and VEGF in gastric cancer and paracancerous tissues was detected.The Spearman rank correlation analysis was done to analyze the correlation between the LOX and VEGF expression in gastric cancer.Results LOX relative expression in 65 cases of gastric cancer tissues and their paracancerous tissues was 0.4178 ± 0.1135 and 0.2174 ± 0.1524 respectively (P ＜ 0.05).VEGF in 65 cases of gastric cancer tissues and their paracancerous tissues was 0.4793 ± 0.1019 and 0.2116 ± 0.1146 respectively (P ＜ 0.05).Conclusion The expression levels of LOX and VEGF are obviously higher in the gastric cancer tissues than in the paracancerous tissues.LOX and VEGF may play an important role in the pathogenesis of gastric cancer. Key words: Gastric cancer; Lysyl oxidase; Vascular endothelial growth factor

Direct Molecular Tissue Analysis by MALDI Imaging Mass Spectrometry in the Field of Gastrointestinal Disease

*Institute of Pathology, Research Unit Analytical Pathology, Helmholtz Zentrum Munchen, German Research Center for Environmental Health, Neuherberg, Germany; ‡ § Department of Medicine II, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany; Department of Medicine II, Universitatsklinikum Mannheim, University of Heidelberg, Mannheim, Germany; and Institute of Pathology, Technische Universitat Munchen, Munich, Germany

MALDI Imaging Identifies Prognostic Seven-Protein Signature of Novel Tissue Markers in Intestinal-Type Gastric Cancer

Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.

Abstract 3364: Evaluation of CD133/CD44 expression level in resected gastric cancer tissues after neoadjuvant chemotherapy

Abstract BACKGROUND: Increasing evidence suggests that there is a small subset of cells, called cancer stem cells (CSC), that are responsible for cancer initiation, development, metastasis and treatment resistance. Although CD133 and CD44 are thought to be the putative markers to characterize CSC of various solid tumors such as brain cancer, colorectal cancer, breast cancer and prostate cancer, the link between expression status of CD133/44 and the clnical future of resected gastric cancer tissues after neo-adjuvant chemotherapy has not yet studied. We evaluated CD133/44 expression status in resected gastric cancer issue after neoadjuvant chemotherapy. PATIENTS AND METHODS: 32 cases were selected from advanced gastric cancer specimens of patients who underwent neo-adjuvant chemotherapy (S-1 and cisplatin or irinotecan and cisplatin or S-1 monotherapy) and surgical resection between 2001 and 2007 at the National Cancer Center Hospital. Immunohistochemical staining with anti-CD133 and anti-CD44 antibody were performed with these specimens. Immunopositive cells were counted from 10 fields in the representative section and expressed as a percentage. The specimen with necrosis or disappearance of the tumor cells in more than 1/3 of the whole lesion was defined as positive histological response specimen. Correlation of immunoreactivity with clinicopathological variables was evaluated and survival curves were compared between CD133/CD44 positive and negative cases. RESULTS: Patient characteristics: median age 57 years (range 31-74); male/female 22/10; histological type diffuse-type/intestinal-type 25/7. CD133 and CD44 expression (positive cells: 0% &amp;lt;) were detected in 24 specimens (75%), and 29 specimens (91%), respectively. There was no significant difference of histological response rate between the CD133 positive and negative group (17% vs 50%, P=0.152). Median survival times were 30.4months in CD133+ group and 27.7months in CD133- group (P=0.65). Median relapse free survivals were 22.1months in CD133+ group and 17.6months in CD133- group (P=0.33). Histological type of gastric cancer (intestinal type vs diffuse type) was not correlated with CD133/CD44 expression. CONCLUSION: CD133 and CD44 were frequently expressed in gastric cancer tissues after neoadjuvant chemotherapy. This study had limited numbers of patient sample and we were unable to study the CD133/44 status before treatment. Further study to identify the usefulness of CD133 and CD44 to predict the prognosis in gastric cancer patients is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3364.

S1993 Tumor Suppressor At Motif Binding Factor 1 (ATBF1) Negatively Regulates Cell Growth Through the Nuclear Translocation with Runt Domain Transcription Factor 3 (RUNx3) in Cooperation with TGF-β Signal Transduction

Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor (Nat. Genet., 2005) and frequent loss of heterozygosity at ATBF1 locus in gastric cancer was recently reported (Clin Cancer Res., 2007). We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer (Oncogene, 2001) and ATBF1 enhanced the promoter activity of the p21 (Cancer Res., 2003). We also found that ATBF1 translocates between cytoplasm and nucleus (Int. J. Cancer, 2007), but its precise mechanism has not been fully elucidated. In this study, we investigated the mechanism of nuclear translocation of ATBF1 with runt domain transcription factor 3 (RUNX3) in cooperation with TGF-β signal transduction. Materials and Methods: 1. To analyze the expression of ATBF1 in gastric cancer cells, we performed immunohistochemistry of 123 resected gastric cancer tissues. 2. ATBF1 and RUNX3 were over expressed by transfecting myc-tagged ATBF1 expression vector and flag-tagged RUNX3 expression vector. Immunoprecipitation (IP) between ATBF1 and RUNX3 was performed. 3. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3, we examined the subcellular localization of ATBF1 and RUNX3 in SNU-16 gastric cancer cells treated with from 0.2 to 2.0 ng/ml of recombinant TGF-β1 by confocal mirosope. 4. We confirmed ATBF1 and RUNX3 protein expression in cytoplasm and nucleus extracted fraction by western blot. 5. Cell cycle analyses by using FACScan were performed. 6. Dual-luciferase assays were performed by transfecting ATBF1 and RUNX3 with p21 reporter vector. Results: 1. The nuclear localization of ATBF1 was observed in 33 (26.8%) gastric cancers and the cytoplasm localization of ATBF1 was observed in 61 (49.6%) gastric cancers. ATBF1 expression was not observed in 29 (23.6%) gastric cancers. 2. IP revealed that the physical interaction of ATBF1-RUNX3. 3. In SNU-16 gastric cancer cells, ATBF1 and RUNX3 existed in the nucleus before recombinant TGF-β1 stimulation, but after 24 hours of TGF-β1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus by confocal microscopic observation. 4. Western blot also revealed ATBF1 and RUNX3 protein in the nucleus was increased after TGF-β1 stimulation. 5.ATBF1 induced the accumulation in the G0/G1 phase. 6. ATBF1 and RUNX3 synergistically up-regulated p21 promoter activity. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus with RUNX3 by TGF-β signal transduction and works as tumor suppressor and transcription regulator in the nucleus.

Cancer-associated splicing variants of the CDCA1 and MSMB genes expressed in cancer cell lines and surgically resected gastric cancer tissues

Alternative splicing is a molecular mechanism by which different combinations of exons can be alternatively spliced to produce different mRNA isoforms. Recently, several databases have been published to predict the alternative splicing of mRNA; cancer-specific alternative splicing has also been predicted with these databases. Those variants may be potentially useful targets for cancer therapy, however, the accuracy and veracity of these databases have yet to be confirmed. In this study, we analyzed 17 genes by reverse transcriptase-polymerase chain reaction (RT-PCR) that were predicted to have cancer-specific alternative splicing by using the splicing database, the Alternative Splicing Annotation Project (ASAP) by Lee et al, between 38 cancer cell lines from various organs and 9 corresponding normal tissues. By designing 2 types of primer sets for RT-PCR including (1) primers flanking the alternatively spliced exons and (2) primers spanning the exon/exon junctions, cancer-associated splicing variants were investigated. The alternatively splicing events were detected in 15 of 17 genes (88%); 35 of 43 variants (81%) were detected successfully with RT-PCR. Among these variants, M-RIP, HYAL2, CDCA1, and MSMB genes showed differential expressions between cancer cell lines and corresponding normal tissues. Furthermore, RT-PCR with surgically resected gastric cancer tissues (diffuse type, 6; intestinal type, 4) confirmed that 2 variants of CDCA1 were upregulated in cancer tissues, whereas both variants of MSMB were expressed predominantly in normal tissues. Alternative splicing variants, especially in CDCA1, were detected in this study and may be potentially useful as diagnostic markers and/or novel targets for anticancer therapy.

Augmented gp130‐mediated cytokine signalling accompanies human gastric cancer progression

H. pylori infection accounts for most cases of gastric cancer, but the initiating events remain unclear. The principal H. pylori pathogenicity-associated CagA protein disrupts intracellular SHP-2 signalling pathways including those used by the IL-6 family cytokines, IL-6 and IL-11. Imbalanced IL-6 family cytokine signalling in the gp130(757FF) mouse model of gastric cancer arising from hyperactivation of oncogenic STAT3 after altered SHP-2 : ERK1/2 signalling produces dysplastic antral tumours preceded by gastritis and metaplasia. In a cohort of patient gastric biopsies with known H. pylori and CagA status, we investigated whether (i) STAT3 and ERK1/2 activation is altered in H. pylori-dependent gastritis; (ii) these profiles are more pronounced in CagA+ H. pylori infection; and (iii) the expression of pro-inflammatory cytokines that activate STAT3 and ERK 1/2 pathways is associated with progression to gastric cancer. IL-6, IL-11, and activated STAT3 and ERK1/2 were quantified in antral biopsies from gastritic stomach, metaplastic tissue, and resected gastric cancer tissues. We observed significantly increased STAT3 and ERK1/2 activation (p = 0.001) in H. pylori-dependent gastritis, which was further enhanced in the presence of CagA+ H. pylori strains. Of known gastric ligands that drive STAT3 activation, IL-6 expression was increased after H. pylori infection and both IL-6 and IL-11 were strongly up-regulated in the gastric cancer biopsies. This suggests a mechanism by which IL-11 drives STAT3 activation and proliferation during gastric cancer progression. We addressed this using an in vitro approach, demonstrating that recombinant human IL-11 activates STAT3 and concomitantly increases proliferation of MKN28 gastric epithelial cells. In summary, we show increased STAT3 and ERK1/2 activation in H. pylori-dependent gastritis that is likely driven in an IL-6-dependent fashion. IL-11 expression is associated with adenocarcinoma development, but not gastritic lesions, and we identify a novel mechanism for IL-11 as a potent inducer of proliferation in the human gastric cancer setting.