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Gene variations in GAGA samples Single

Immunoblot and Annexin V assays of GAGA6 and GAGA6-R PDX tumors after drug treatment with AZD4547, sunitinib or 1-AKP.

AZD4547 treatment and downstream analysis in GAGA3 and GAGA6 PDX models.

Expression of PKC isoforms after inhibition by PKC inhibitor H7.

Clinicopathological features of GAGA patients

Amplified chromosomal regions in GAGA samples

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.

Comparison of histology and copy number variations of GAGA1, GAGA3 and GAGA6 primary tumors and PDX models

The optimal therapeutic strategy in nucleoside analogue (NA) experienced chronic hepatitis B (CHB) using peginterferon is still unclear; hence we explored a switch to or add-on peginterferon strategy versus continued NA.We conducted a randomized controlled trial of CHB patients on NA12 months with HBV DNA(-) randomized to switch or add-on peginterferon-alpha2b (1.5 μg/kg/weekly) for 48 weeks versus continuing NA (controls) (allocation 2:2:1; Clinicaltrial.gov: NCT01928511) in tertiary Singapore hospitals. The primary composite endpoint at week 72 was hepatitis B e antigen (HBeAg) loss or quantitative HBsAg (qHBsAg)1 log IU/mL reduction, and secondary endpoints were HBsAg loss, HBsAg seroconversion, qHBsAg200 IU/mL, qHBsAg100 IU/mL, HBV DNA(-), viral relapse, and safety. Analysis was by intention-to-treat (ITT).A total of 253 patients (controls 51, switch 103, add-on 99) were randomized. The primary ITT endpoint was achieved in 3.9% of controls, 33.3% of switch, and 26.7% of add-on (P.0001, switch/add-on versus controls). HBsAg loss occurred in 0% of controls, 7.8% of switch, and 10.1% of add-on (ITT, P.001, switch/add-on versus controls). HBeAg(+) patients on peginterferon had higher HBeAg loss than controls but poor HBsAg responses, whereas HBeAg(-) patients on peginterferon achieved better HBsAg responses than controls. Reduction in qHBsAg in HBeAg(+) was 0.14 log IU/mL versus 0.51 log IU/mL in HBeAg(-) (P.0001) in peginterferon-treated patients. Clinical relapse was higher in switch (13.6% overall, 27% in HBeAg(+)) versus 1% add-on and 0% controls. Adverse events were typically interferon-related symptoms, with one death (myocardial infarction unrelated to therapy).ITT analysis showed that either peginterferon strategies were superior to NA for the primary endpoint and HBsAg loss, but add-on peginterferon is preferred to switch due to improved safety and similar efficacy. ClincialTrials.gov number: NCT01928511.

Our objective was to examine differences in cytokine/chemokine response in chronic hepatitis B(CHB) patients to understand the immune mechanism of HBsAg loss (functional cure) during antiviral therapy. We used an unbiased machine learning strategy to unravel the immune pathways in CHB nucleo(t)side analogue-treated patients who achieved HBsAg loss with peg-interferon-α(peg-IFN-α) add-on or switch treatment in a randomised clinical trial. Cytokines/chemokines from plasma were compared between those with/without HBsAg loss, at baseline, before and after HBsAg loss. Peg-IFN-α treatment resulted in higher levels of IL-27, IL-12p70, IL-18, IL-13, IL-4, IL-22 and GM-CSF prior to HBsAg loss. Probabilistic network analysis of cytokines, chemokines and soluble factors suggested a dynamic dendritic cell driven NK and T cell immune response associated with HBsAg loss. Bayesian network analysis showed a dominant myeloid-driven type 1 inflammatory response with a MIG and I-TAC central module contributing to HBsAg loss in the add-on arm. In the switch arm, HBsAg loss was associated with a T cell activation module exemplified by high levels of CD40L suggesting T cell activation. Our findings show that more than one immune pathway to HBsAg loss was found with peg-IFN-α therapy; by myeloid-driven Type 1 response in one instance, and T cell activation in the other.

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.

Detection of pre-neoplastic gastric mucosal changes and early gastric cancer (EGC) by white-light endoscopy (WLE) is often difficult. In this study we investigated whether combined autofluorescence imaging (AFI) and narrow band imaging (NBI) can improve detection of pre-neoplastic lesions and early gastric cancer in high-risk patients.Chinese patients who were 50-years-old or above with dyspepsia were examined by both high-resolution WLE and combined AFI followed by NBI (AFI-NBI), consecutively in a prospective randomized cross-over setting, by two experienced endoscopists. The primary outcome was diagnostic ability of the two methods for patients with pre-neoplastic lesions such as intestinal metaplasia (IM) and mucosal atrophy.Sixty-five patients were recruited. One patient with large advanced gastric cancer was found and excluded from the analysis. Among the remaining 64 patients, 38 (59%) had IM; of these, 26 (68%) were correctly identified by AFI-NBI (sensitivity 68%, specificity 23%) and only 13 (34%) by WLE (sensitivity 34%, specificity 65%). AFI-NBI detected more patients with IM than did WLE (p=0.011). Thirty-one patients (48%) had mucosal atrophy. Ten patients (32%) were identified by AFI-NBI (sensitivity 32%, specificity 79%) and four patients (13%) by WLE (sensitivity 13%, specificity 88%) (p=0.100). No dysplasia or EGC was found.AFI-NBI identified significantly more patients with IM than did WLE. Our result warrants further studies to define the role of combined AFI-NBI endoscopy for detection of precancerous conditions.

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.

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.

Background & Aims It is not clear how the duration of upper endoscopy affects the detection of cancer or premalignant lesions that increase the risk for gastric cancer. We investigated whether the length of time spent performing esophagogastroduodenoscopy (EGD) affects the detection of important pathologic features of the stomach. Methods We collected data from 837 symptomatic patients, during a 3-month period in 2010, who underwent a first diagnostic EGD at a tertiary university hospital in Singapore. Endoscopists were classified as fast or slow based on the mean amount of time it took them to perform a normal EGD examination. We used logistic regression to compare between groups the numbers of intestinal metaplasias, gastric atrophies, dysplasias, and cancers detected, using histologic analysis of biopsy samples collected during endoscopy as the standard. Results Of 224 normal endoscopies, the mean duration was 6.6 minutes (range, 2–32 min). When we used 7 minutes as the cut-off time, 8 endoscopists were considered to have short mean examination times (mean duration, 5.5 ± 2.1 min; referred to as fast endoscopists ), and 8 endoscopists were considered to have long mean examination times (mean duration, 8.6 ± 4.2 min; referred to as slow endoscopists ). Eleven cancers and 81 lesions considered to pose risks for cancer were detected in 86 patients; 1.3% were determined to be cancer, 1.0% were determined to be dysplasia, and 8.7% were determined to be intestinal metaplasia and/or gastric atrophy. Slow endoscopists were twice as likely to detect high-risk lesions as fast endoscopists (odds ratio, 2.50; 95% confidence interval, 1.52–4.12), regardless of whether they were endoscopy staff or trainees. The slow endoscopists also detected 3-fold more neoplastic lesions (cancer or dysplasia; odds ratio, 3.42; 95% confidence interval, 1.25–10.38). Conclusions Endoscopists with mean EGD examination times longer than 7 minutes identified a greater number of high-risk gastric lesions than faster endoscopists. Examination time may be a useful indicator of quality assessment for upper endoscopy. Studies are required to test these findings in different populations.

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.