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Preclinical models of diffuse-type gastric cancer (DGC) that reliably predict clinical activity of novel compounds are lacking. To overcome the problem of poor tumor cellularity in DGC, we used cells from malignant ascites to establish DGC patient-derived xenograft (PDX) models that recapitulate the primary cancer. Cells in PDX model GAGA6 with FGFR2 amplification were sensitive to AZD4547, a potent FGFR inhibitor that is being clinically evaluated for FGFR-aberrant cancer types. Intermittent in vivo treatment of GAGA6 tumors with AZD4547 gave rise to PDX tumors with acquired resistance to AZD4547, GAGA6-R. Surprisingly, there were no mutations in the FGFR2 gene in GAGA6-R, negating gatekeeper mutations as a mechanism of drug resistance. Phosphorylation of FGFR2 and downstream signaling molecules AKT/PKB and MAPK/ERK remained inhibited by AZD4547. Further analysis of signaling pathways identified AKT-independent phosphorylation and inhibition of GSK3β as a mechanism of drug resistance in GAGA6-R cells. Treatment of GAGA6-R cells with protein kinase C (PKC) inhibitor H7 in combination with AZD4547 led to dephosphorylation and activation of GSK3β with concomitant downregulation of MCL-1 and BCL-XL. Combined treatment with AZD4547 and H7 in vitro synergistically enhanced cell death in GAGA6-R but not GAGA6 cells. Furthermore, midostaurin, a multikinase inhibitor with PKC-inhibiting activity, in part reversed resistance of GAGA6-R tumor to AZD4547 in vivo. Our results suggest that upon challenge with FGFR inhibitors, FGFR2-amplified tumors that are highly dependent on FGFR2 signaling for survival rapidly develop resistance by switching to a PKC-mediated inhibition of GSK3β to gain a survival advantage. Mol Cancer Ther; 17(1); 232–42. ©2017 AACR.

Peritoneal recurrence after gastrectomy for gastric cancer is common and the prognosis is dismal. Recent evidence suggests that extensive peritoneal lavage with large volume of normal saline after surgery before abdominal closure can reduce the risk of peritoneal recurrence and improve overall survival. This study aims to evaluate the benefit of extensive intraoperative peritoneal lavage. This is a prospective, open-label, multicentre randomised controlled trial involving 15 international centres in China, Korea, Japan, Malaysia and Singapore. Patients with cT3/4 stomach cancer undergoing curative resection are randomised to either extensive peritoneal lavage (10 l of saline) or standard lavage (≤2 l of saline). The primary outcome is overall survival and secondary outcomes include disease-free survival and peritoneal recurrence. The minimum sample size is 600 subjects with 300 per arm completing 3 years follow-up. The data will be analysed on an intention-to-treat basis, assuming a two-sided test with a 5% level of significance.

Diffuse gastric cancer (DGC) is a poorly differentiated adenocarcinoma characterized by infiltration of signet ring cells into the stomach wall and a lack of tumor mass formation. DGC has rapid disease progression with a dismally poor prognosis and is resistant to chemo- and radiotherapy. There is no available targeted therapy, and preclinical models that reliably predict clinical activity of novel compounds are also lacking. In addition, molecular analyses of DGC have been hampered by difficulty in obtaining primary tumors of sufficient tumor cellularity without contaminating normal fibroblasts. To overcome the scarcity of cancer cells and the abundance of surrounding stromal tissue, we isolated cancer cells from malignant ascites of DGC patients. Red blood cells were removed by Ficoll separation and CD45+ hematopoietic cells were depleted using antibody-conjugated magnetic beads. Exome sequencing was performed on cancer cells and peripheral blood cells from the same patient to identify somatic alterations in DGC. We also generated patient-derived xenografts (PDX) using isolated cancer cells to establish preclinical cancer models for DGC. Exome sequencing revealed FGFR2 and KRAS amplifications as well as p53 mutations. Cancer cells isolated from malignant ascites formed subcutaneous tumors that recapitulated the histology of primary DGC tumors with characteristic signet ring cells and intracellular mucin. FGFR2 amplification in PDX tumors was confirmed by fluorescence in situ hybridization (FISH), with correspondingly high levels of FGFR2 transcripts and protein expression. We performed drug treatments on PDX tumors using FGFR2 inhibitor AZD4547 and found that FGFR2 amplification status alone was sufficient to predict the sensitivity of PDX tumors to AZD4547. Tumor volume of FGFR2-amplified tumors decreased or remained the same following AZD4547 treatment compared to vehicle-treated tumors which showed increased tumor volume. In contrast, AZD4547 treatment had little or no effect on tumor growth of non-FGFR2 amplified tumors. Furthermore, these PDX tumors regardless of FGFR2 amplification status responded poorly to cisplatin, a standard drug currently used for treatment of DGC. In conclusion, we report a high prevalence of FGFR2 gene amplification in DGC and the establishment of reliable PDX models that can predict tumor response to targeted therapy. Citation Format: Wen Min Lau, Zhijiang Zang, Eileen Teng, Kakoli Das, Wei Peng Yong, Ming Teh, Tania Chia, Jin Wei Tan, Amy Tay, Asim Shabbir, Koji Kono, Jimmy So, Patrick Tan, Shing Leng Chan. Clinical relevance of FGFR2 amplification in diffuse gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2014-1228

Background & Aims: VEGF is a key stimulator of angiogenesis and promotes the growth colon tumors via increased nutrient and oxygen supply. A recent study demonstrated that colorectal cancer (CRC) cells have increased expression of VEGF and its receptors VEGFR1 & VEGF-R2, which indicates a possibility of direct stimulation of CRC cell growth by VEGF. However, the role of VEGF in CRC cell proliferation by an autocrine mechanism has not been fully explored in depth. We hypothesized that VEGF secreted by CRC cells binds to VEGF-R1 & VEGF-R2 on CRC cells and: 1) promotes proliferation of these cells through an autocrine pathway, 2) inhibits CRC apoptosis. Methods: We used: (1) human CRC cell lines HCT116 & HT29 and (2) normal colonic epithelial cells NCM356 & NCM460 cultured in nutrient media. Studies: 1) VEGF, VEGF-R1 & VEGF-R2 mRNA and protein expression of by Real-Time RT-PCR and immunoblotting; 2) VEGF165 secretion into the culture media by ELISA; 3) cell proliferation by BrdU assay; 4) apoptosis by TUNEL staining and assessment of activated caspases 3 and 9; 5) Inhibition of VEGF receptor function using AAL993 inhibitor, which blocks function of VEGF-R1 and VEGF-R2; 6) assessment of phosphorylation of EGF receptor (EGF-R) to test its transactivation by VEGF. Results: 1) Normal colonic cells do not secrete VEGF (< 35 pg/ml culture media) while CRC cells express and secrete high levels of VEGF (840 1500 pg/ml culture media); 2) Normal colonic epithelial cell lines expressed no or minimal VEGF-R1 & VEGF-R2 mRNA and protein while CRC cell lines exhibit strong expression of VEGF-R1 & VEGF-R2 vs. normal colonic epithelial cells by 14and 24-fold, respectively (all p < 0.001); 3) CRC cell lines have significantly increased cell proliferation by 31% (p < 0.01) and decreased apoptosis vs. normal colonic epithelial cells; 3) Inhibition of both VEGF-R1 & VEGF-R2 receptors using AAL-993 significantly decreased CRC cell proliferation by (2.2-fold) ; 4) Treatment of CRC cells with AAL-993 decreased phosphorylation of EGF-R by 1.9-fold (p < 0.01). Conclusions: 1) VEGF and its VEGF-R1 & VEGF-R2 receptors are increased in CRC cell lines; 2) CRC cells secrete VEGF, which stimulates CRC cell proliferation via an autocrine mechanism by its VEGF-R1 & VEGF-R2 receptors and also inhibits apoptosis; 3) VEGF receptor signaling pathway is critical for activating EGF-R in CRC cells; 4) These studies suggest a cross-talk between VEGF receptor and EGF-R signaling in CRC cells, which promotes these cells' proliferation.

Cancer stem cells (CSCs) have been discovered in cancers of many tissues but they have not been explored for gastric cancer. A candidate approach was taken to isolate putative gastric cancer stem cells from primary gastric cancer specimens because surface markers have been used to describe CSCs from different cancers. Cells dissociated from biopsy samples extracted from primary tumour and non-tumour sites of the same patient were analyzed by flow cytometry. Cell surface markers surveyed include CD45 for hematopoietic cells, CD31 for endothelial cells, CD44, CD133, CD34, CD117, CD166, CD90 and EpCAM as potential cancer stem cell markers. EpCAM levels were found to be higher in cells isolated from the tumor than non-tumor sites. Using median fluorescence intensity, it was found that cells at the tumor site express 1.2-1.7 fold higher level of EpCAM. This was observed in histopathologically distinct types of gastric tumors, namely intestinal subtypes that are well, moderately and poorly differentiated. Data from diffused type gastric tumors were not as consistent mainly because of sampling difficulties. 100,000 EpCAM-positive cells form xenograft tumors in NOD/SCID mice whereas 500,000 EpCAM-negative cells from a fraction that was not hematopoietic (CD45-negative), endothelial (CD31-negative) nor fibroblastic (CD140b-negative) were unable to do so. To further enrich for gastric cancer stem cells, the EpCAM-positive cells were sorted based on EpCAM+CD44+ or EpCAM+CD133+ cells. To expand the tumor material, we generated xenograft tumors directly from fresh tumor pieces. Histological analyses verified that the xenograft tumors recapitulate those in the patient samples. Cells dissociated from the xenograft tumor were then subjected to CSC identification assays. To date, three lines of xenograft tumors were generated from tumor tissues of well, moderately and poorly differentiated histological subtype of gastric cancers. As few as 1000 EpCAM+CD44+ cells were able to initiate tumors in NOD/SCID mice whereas 20,000 EpCAM+CD44- cells were required to do so. We are currently doing limiting dilution assays to estimate the number of cancer initiating cells in each fraction we isolate from the xenografts. We were unable to see differential tumorigenic potentials in EpCAM+CD133+ and EpCAM+CD133- fractions. In conclusion, we have identified a subpopulation of gastric cancer cells that potentially mark gastric cancer stem cells as characterized by their cancer-initiating potential. 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 3371.