Epithelial Antigen Research Articles

Prevalence of Helicobacter pylori cagA and iceA Genes and Their Association with Gastrointestinal Diseases.

H. pylori infection causes peptic ulcer, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. It has several virulence factors such as cytotoxin-associated gene A(cagA) and the induced by contact with epithelium antigen (iceA). We aimed to explore the relationship between cagA and iceA of H. pylori and gastrointestinal diseases. One hundred and eighteen patients who attended Gastrointestinal Endoscopy Unit at Zagazig University Hospitals, Egypt, were included in this study. Two gastric biopsies were collected and evaluated by rapid urease test (RUT) and PCR. cagA and iceA genes were amplified by PCR. We found that 54 patients (45.76%) were positive by both RUT and PCR. cagA and iceA genes were present in 57.4% and 46.29% of the studied patients, respectively. cagA was the most prevalent gene in gastritis (33.3%) and peptic ulcer (68.7%). iceA1/iceA2 positive genes were the most prevalent in gastric cancer (75%). iceA1 gene was present in 38.7% of cagA positive cases, but iceA2 gene was present in 45.2% of cagA positive cases. iceA1/iceA2 positive genes were present in 29% of cagA positive cases. In conclusion, cagA and iceA genes could be used as markers for severe gastrointestinal diseases. iceA gene was strongly related to cagA gene.

Analysis of Circulating Tumor Cells in Ovarian Cancer and Their Clinical Value as a Biomarker

Background/Aims: Monitoring the appearance and progression of tumors are important for improving the survival rate of patients with ovarian cancer. This study aims to examine circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC) patients to evaluate their clinical significance in comparison to the existing biomarker CA125. Methods: Immuomagnetic bead screening, targeting epithelial antigens on ovarian cancer cells, combined with multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR) was used to detect CTCs in 211 samples of peripheral blood (5 ml) from 109 EOC patients. CTCs and CA125 were measured in serial from 153 blood and 153 serum samples from 51 patients and correlations with treatment were analyzed. Immunohistochemistry was used to detect the expression of tumor-associated proteins in tumor tissues and compared with gene expression in CTCs from patients. Results: CTCs were detected in 90% (98/109) of newly diagnosed patients. In newly diagnosed patients, the number of CTCs was correlated with stage (p=0.034). Patients with stage IA-IB disease had a CTC positive rate of 93% (13/14), much higher than the CA125 positive rate of only 64% (9/14) for the same patients. The numbers of CTCs changed with treatment, and the expression of EpCAM (p=0.003) and HER2 (p=0.035) in CTCs was correlated with resistance to chemotherapy. Expression of EpCAM in CTCs before treatment was also correlated with overall survival (OS) (p=0.041). Conclusion: Detection of CTCs allows early diagnose and expression of EpCAM in CTC positive patients predicts prognosis and should be helpful for monitoring treatment.

Nanoscale flow cytometry of patient plasma for the detection of prostate cancer-associated extracellular vesicles

Introduction Prostate cancer is the predominant cancer in men, affecting one in seven men during their lifetime. Current tests for prostate cancer include the digital rectal exam and the prostate-specific antigen (PSA) test. Extracellular vesicles (EVs) are submicron particles that participate in intercellular cross-talk by releasing cell mediators such as microRNA, carbohydrates and proteins. While they are known to express the broad tetraspanin family of proteins, i.e. CD9/CD63, prostate cancer-derived EVs have also been found to express PSA and six transmembrane epithelial antigen of the prostate (STEAP1). Traditionally, scientists have purified these EVs through ultracentrifugation. Here we propose a tandem purification of patient plasma, followed by nanoscale flow cytometry (A50+) as a novel method to detect tumour-derived EVs. Materials and Methods Plasma was obtained from healthy volunteers, patients with benign prostatic hyperplasia (BPH), and patients with metastatic prostate cancer. CD9, CD63, PSA and STEAP1 were used as primary antibodies for the purification of EVs from neat plasma. To perform the purification in tandem, Protein G immunoprecipitation using CD9 and PSA was carried out first, followed by immunoaffinity purification with biotinylated CD63 and STEAP1. First and second elutions were collected for the enumeration of dual positive events by A50+. Initial histogram overlays and bivariate plots of neat and purified plasma were computed, then exported as comma-separated values for mathematical modelling by MATLAB. Results Strong dual positive EV populations from patient plasma were optimised, demonstrating that the method enriches tumour-derived EVs from neat samples of patient plasma. This was observed for HVs, patients with benign prostatic hyperplasia, and prostate cancer patients with Gleason 4+4. Enrichment in these purified samples was measured by A50+ and demonstrated by overlaying purified and non-purified histoplots by MATLAB. Additional results show that PSA and STEAP1 can be adapted to detect single or dual positive populations of tumour-associated EVs in prostate cancer patients. Discussions and Conclusions This study suggests that tandem purification of tumour-associated EVs and A50+ analysis from plasma of prostate cancer patients can lead to earlier diagnosis and risk stratification, compared to traditional screening tests and aspiration cytology. Future studies will be directed toward optimizing this detection method for markers of other cancer types to achieve better outcomes in cancer detection and prognosis.

Clinical and functional significance of STEAP4-splice variant in CD14+ monocytes in patients with rheumatoid arthritis.

Tumour necrosis factor alpha (TNF)-α-induced adipose-related protein (TIARP) is a negative regulator of inflammation in arthritis model mice. In humans, six-transmembrane epithelial antigen of prostate 4 (STEAP4) (human counterpart of TIARP) is also expressed in CD14+ monocytes from patients with rheumatoid arthritis (RA). Recently, highly levels of exon 3-spliced variant STEAP4 (v-STEAP4) expression have been observed in porcine lung. The aim of this study is to elucidate the expression and functional role of v-STEAP4, comparing it with that of STEAP4, in the pathogenesis of arthritis. We identified v-STEAP4 in CD14+ cells. The expression of STEAP4 and v-STEAP4 was higher in patients with RA than in healthy participants. We also found that STEAP4 and v-STEAP4 were correlated positively with C-reactive protein and that their expression was decreased after treatment with an interleukin (IL)-6 antagonist in patients with RA. To investigate further the role of STEAP4 and v-STEAP4, we produced STEAP4 and v-STEAP4 over-expressing human monocytic cell lines (THP-1) for functional analysis. In the v-STEAP4 over-expressing cells, the production of IL-6 was suppressed significantly, but TNF-α was increased significantly through lipopolysaccharide (LPS) stimulation. Immunoblot analysis revealed that phosphorylated (p-)nuclear factor kappa B (NF-κB) was increased after LPS stimulation and degradation of nuclear factor kappa B inhibitor alpha (IκBα) was sustained, whereas p-signal transducer and activator of transcription 3 (STAT-3) was decreased with v-STEAP4. We identified specific up-regulation of v-STEAP4 in RA monocytes. V-STEAP4 might play a crucial role in the production of TNF-α and IL-6 through NF-κB and STAT-3 pathways, resulting in the generation of RA.

Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.

The immune mechanism of Mycoplasma hyopneumoniae 168 vaccine strain through dendritic cells

BackgroundMycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure.ResultsThe antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone.ConclusionThese findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.

CCR6 promotes steady-state mononuclear phagocyte associationwith the intestinal epithelium, imprinting and immune surveillance.

The intestinal lamina propria (LP) contains antigen-presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and invivo models to test and confirm the importance of LP-MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP-epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP-MNPs from CCR6-/- mice did not display defects in acquiring antigen and stimulating T-cell responses in exvivo assays or in responses to antigen administered systemically. However, LP-MNPs from CCR6-deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP-MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.

Gypenosides induce cell death and alter gene expression in human oral cancer HSC-3 cells.

Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.

Abstract 3782: Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen

Abstract Background Genetic analysis of circulating tumor cells (CTCs) is useful as a liquid biopsy. However, there are 3 challenging issues in processing of CTC samples for clinical use of the analysis: [1] numerous residual blood cells in processed samples, [2] loss of CTCs that do not express epithelial markers, and [3] very laborious process. Here, we developed a novel system capable of overcoming all of these problems. Methods Our CTC analysis system is composed of 3 steps: [1] filtering of whole blood followed by immunostaining and magnetic labeling of cells trapped on the filter, [2] depletion of white blood cells (WBCs) in the cells recovered from the filter by magnetic separation, and [3] trapping the resultant cells at an observation chamber in a microfluidic enrichment device using dielectrophoresis, followed by recovering them as a concentrated sample after fluorescence microscope observation. SNV and CNV analysis was conducted using the collected concentrated sample, and the system performance was substantiated. Genetic mutations of the cells in the collected sample were detected by the Quenching Probe method. On the other hand for CNV analysis, cells in the collected concentrated sample were immobilized on slide glass and analysis of gene amplification conducted by FISH. Results With SNV analysis, our system successfully detected EGFR, KRAS or PIK3CA mutations of cancer cell lines spiked in 8 mL of whole blood (Table). The detection sensitivity of our method was 1 cell/mL, and both the cancer cells and their genetic mutations were detected within 9 hours of starting the processing of whole blood. Additionally, with CNV analysis, HER2 gene amplification was confirmed with cell lines spiked in 8 mL of blood. This detection result was obtained within 48 hours. Conclusion Our system would be useful for the analysis of gene mutations in a wide range of CTC types independent of certain epithelial antigens, such as CK and EpCAM, and can be used for various genetic analyses. Table Results of spiking test for SNV analysisSpiked cell linesExpected count[cells/8ml]No. of detected spiked cells [cells]No. of detected remained WBCs [cells]Detected genotypesNCI-H197512979EGFR L858RNCI-H16508225EGFR exon19 deletionSW6208648KRAS codon12, 13SNU-1124154KRAS codon12, 13MCF-777341PIK3CA G1633A Citation Format: Hidenori Takagi, Masahiro Kozuka, Hiroshi Ito, Soo Hyeon Kim, Mitsuharu Hirai, Teruo Fujii. Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen [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 3782. doi:10.1158/1538-7445.AM2017-3782

Abstract 5425: The role of six transmembrane epithelial antigen of the prostate 2 in hepatocellular carcinoma

Abstract Introduction: The incidence of hepatocellular carcinoma (HCC) in Hispanics is three times higher than non-Hispanic whites, and even higher in South Texas (STX) Hispanics. This is attributed to a higher prevalence of hepatitis C, diabetes, obesity and perhaps genetic and epigenetic alterations. Knowledge regarding genetic alterations in Hispanics is sparse as demonstrated by the lack of Hispanics with HCC in The Cancer Genome Atlas (TCGA). Therefore, our group sequenced paired adjacent liver and HCC tumor samples from STX Hispanics, which highlighted a gene over-expressed in tumors of Hispanics, called the Six Transmembrane Epithelial Antigen of the Prostate 2 (STEAP2). STEAP2 is a metalloreductase of iron and copper and is implicated in increased iron and reactive oxygen species in the liver which can lead to the progression of inflammation and cirrhosis. STEAP2 may play an important oncogenic role in HCC, especially in the setting of obesity. We propose to test the hypothesis that overexpression of STEAP2 will lead to malignant property in HCC cells resulting in enhanced proliferation, survival, invasiveness, and eventually development of HCC, especially in obese hosts. Methods: Hispanic paired tissue continues to be collected from our institution for RNA sequencing and establishment of Hispanic HCC cell lines. STEAP2 RNA and protein expression levels in Hispanic paired samples versus Caucasian paired samples were evaluated by RT-PCR, Western blot, and immunohistochemistry. Knockdown and overexpression of STEAP2 were established in HCC cell lines and in primary Hispanic HCC cell lines to examine the effects on iron levels, oxidative stress, proliferation, invasiveness, apoptosis and cell cycle in vitro. Results: Hispanic HCC RNA sequencing data compared to TCGA HCC RNA sequencing data (no Hispanics) demonstrated the overexpression of STEAP2 in HCC tumors in Hispanic patients, which were validated by RT-PCR data and Western blot data. Lipid peroxidation product, 4-hydroxynonenal, and copper levels were higher in HCC tumor versus adjacent tissue. Iron levels were higher in adjacent tissue versus tumor tissue in Hispanics. Knockdown of STEAP2 in SNU398 cells decreased proliferation and migration, while in HUH7 cells STEAP2 knockdown only decreased migration. Conclusions: STEAP2 is specifically overexpressed in HCC tumors in Hispanics in comparison to HCC tumors in non-Hispanic whites and appears to play a malignant-promoting role in HCC cells. Further studies to establish the role of STEAP2 as a tumor promoter in HCC and the mechanisms by which it promotes carcinogenesis are underway. The proposed studies will likely yield mechanistic insights into the molecular mechanisms that drive HCC development and progression in South Texas Hispanics and potential therapeutic targets involving STEAP2-mediated metal ion metabolism and oxidative stress. Citation Format: Carla R. Zeballos, Hakim Bouamar, Xiang Gu, Yidong Chen, Francisco G. Cigarroa, LuZhe Sun. The role of six transmembrane epithelial antigen of the prostate 2 in hepatocellular carcinoma [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 5425. doi:10.1158/1538-7445.AM2017-5425

Abstract 3792: Improved isolation and detection of circulating tumor cells of pancreatic cancer patients with characterization by mutational analysis

Abstract Introduction: Isolation of circulating tumor cells based on cell surface markers is often hindered by epithelial to mesenchymal transition with loss of epithelial antigens. Low numbers of CTCs and fewer cases of CTC positive pancreatic cancer compared to other cancers (e.g. breast cancer) lead to the assumption that pancreatic tumors do not release CTCs as frequently or that the pancreatic CTCs lack EpCAM expression. Isolation of CTCs based on their size is independent of EMT-like phenotypical changes. We therefore compared a filtration-based isolation method with an EpCAM-based isolation method. We optimized CTC detection by using a highly sensitive anti-cytokeratin antibody panel for the detection of cancer cells with moderate cytokeratin expression. In order to use isolated CTCs as liquid biopsy for tumor characterization and treatment selection, downstream analysis is necessary. We here show the feasibility of mutational analysis of isolated CTCs by castPCR. Methods: EpCAM high, medium and low cells were enriched and isolated by an EpCAM-based immunomagnetic procedure (IsoFlux) and a filtration device (Siemens) to determine recovery rates of both methods. Isolated cells were detected by an improved immunofluorescent staining with an anti-Cytokeratins, anti-EpCAM and anti-PBMC panel and characterized by competitive allele-specific TaqMan PCR (castPCR) for KRAS mutations. Results: Cytokeratin expression is crucial for detection of CTCs in a high background of blood cells. Improvement of the staining protocol helps to increase the sensitivity of detection. The filtration based approach is superior to the surface antigen-based isolation. With the size-dependent method we obtained a recovery rate of 52 % even for EpCAM-low cells compared to only 1 % based on the immune-affinity purification. Cells isolated by filtration can be characterized for expression of therapeutic targets by immunostaining. The utility of the size dependent platform for subsequent functional characterization of the CTCs was also demonstrated by detection of k-ras mutations in single isolated CTCs by castPCR. Conclusion: For CTCs undergoing EMT, filtration yields higher recovery compared to the standard surface antigen (EpCAM)-based methods. Isolation by filtration also allows for mutational analysis which can be used to confirm the identity of the isolated cell as CTC. In addition, mutational analysis of CTCs can be used to guide the treatment of patients. Use of liquid biopsies for treatment selection will facilitate truly personalized medicine. Citation Format: Oliver von Ahsen, Nora Brychta, Thomas Krahn. Improved isolation and detection of circulating tumor cells of pancreatic cancer patients with characterization by mutational analysis [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 3792. doi:10.1158/1538-7445.AM2017-3792

STEAP4: its emerging role in metabolism and homeostasis of cellular iron and copper.

Preserving energy homeostasis in the presence of stressors such as proinflammatory cytokines and nutrient overload is crucial to maintaining normal cellular function. Six transmembrane epithelial antigen of the prostate 4 (STEAP4), a metalloreductase involved in iron and copper homeostasis, is thought to play a potentially important role in the cellular response to inflammatory stress. Genome-wide association studies have linked various mutations in STEAP4 with the development of metabolic disorders such as obesity, metabolic syndrome and type 2 diabetes. Several studies have shown that expression of Steap4 is modulated by inflammatory cytokines, hormones and other indicators of cellular stress and that STEAP4 may protect cells from damage, helping to maintain normal metabolic function. STEAP4 appears to be particularly relevant in metabolically oriented cells, such as adipocytes, hepatocytes and pancreatic islet cells. These cells struggle to maintain their function in iron or copper overloaded states, presumably due to increased oxidative stress, suggesting STEAP4's role in metal homeostasis is critical to the maintenance of cellular homeostasis in general, and in preventing the onset of metabolic disease. In this review, we explore genetic associations of STEAP4 with metabolic disorders, and we examine STEAP4 tissue expression, subcellular localization, regulation, structure and function as it relates to metabolic diseases. We then examine how STEAP4's role as a regulator of cellular iron and copper may relate to type 2 diabetes.

Inter-tumor validation, through advanced MRI and circulating biomarkers, of plasma Tie2 as the vascular response biomarker for bevacizumab.

11521 Background: VEGF inhibitor (VEGFi) use is compromised by lack of predictive/ response biomarkers. Previously, we identified plasma Tie2 (pTie2) as a vascular response biomarker (VRB) for bevacizumab (bev) in ovarian cancer (OC). Here, we applied dynamic contrast-enhanced MRI (DCE-MRI) and circulating biomarkers in colorectal cancer (CRC), to validate pTie2 as the first tumor VRB. Methods: Seventy patients were recruited, with untreated, mCRC and ≥1 lesion of 3-10cm diameter for DCE-MRI. Patients received bev 10mg/kg for 2 weeks to elicit a biomarker response and then FOLFOX6/bev until progressive disease (PD) Thirteen circulating and 6 imaging biomarkers were measured before and during treatment and at PD. Unsupervised correlation analysis identified bev-induced biomarker correlations. Biomarkers were evaluated by clustered parameter-time course studies to determine their epithelial or vascular origin. Clinical significance was determined by relating the biomarker data to tumor 3D volumetric change assessed by MRI and PFS. The emergent vascular biomarker signal was modelled with epithelial biomarkers to assess the independent contribution of the vascular compartment to PD. Results: Bev induced significant correlations between pTie2, Ang2 and Ktrans. Cluster analysis of Tie2 concentration-time course curves showed that pTie2 reflected tumor Ktransbut not CK18, an epithelial antigen, i.e. changes in pTie2 reflected tumor vascular biology Patients who had the greatest area under the pTie2-time curve had tumors with high Ktransand/or low pVEGFR2, pre-treatment. They also had the greatest reduction in tumor volume and longest PFS. Fusion of pTie2 and CK18 data significantly improved modelling of PD. Conclusions: Bev impacts tumor vasculature causing proportional changes in pTie2. Information from pTie2 adds clinical value to that derived from the epithelial compartment. Thus (i) pTie2 is the first vascular response biomarker for bev and probably all VEGFi and (ii) demonstration of separate vascular and epithelial compartments in ovarian and CRC validates the vascular compartment as a target. This work identifies the first assay that could optimise use of VEGFi. Clinical trial information: 2009-011377-33.

Do pancreas cancer stem cells play crucial role in survival outcome?

e15721 Background: Recent studies indicate that pancreatic cancer stem cells (CSC) may predict disease behavior and survival outcomes of pancreatic ductal adenocarcinomas (PDACs) patients (pts). Several CSC markers have been reported in PDAC (Fitzgerald, TL, 2014). We herein evaluated the impact of CSC markers including CD44 and Epithelial Specific Antigen (ESA) on survival outcome of PDAC pts who had liver or lung metastasis after initial surgical resection (IR). Methods: Clinicopathologic features and survival of 59 PDACs were analyzed. Pts with IRB approval, and whom had available primary tumor tissue, were included. All neoplasms were immuno labeled with CD44 and ESA. Staining intensity was scored as weak (1), moderate (2), strong (3), while the staining pattern was scored as: few (1), patchy (2), and diffuse (3). The expression for CD44 and ESA was accepted positive if total score ≥4. Time from relapse to death (TRD) was estimated using the Kaplan-Meier method censoring patients that were alive at the last follow up. Survival curves were compared using the log-rank test Results: Of 59 pts, 42 (71 %) had liver, 10 (17%) had lung and 7 (12%) had both liver and lung metastasis. M/F = 34/25; mean age = 64.2 (range, 34-90). Patients were subcategorized as follows; thirteen cases were CD44 (+)/ESA (-) (group1) and 13 were CD44 (-)/ESA (+) (group 2). Eight (61.5%) of group 1 tumors and 2 (15.3%) of group 2 were poorly differentiated. At last follow-up, except one with 63 months survival, all pts died of disease with 23.3 months (range, 3-67) median OS. No significant difference in TRD was observed between group 1 (6.9 months) and 2 (13.8 months) (p = 0.62). However, we observed group 1 tumors had worse OS (12 months) compared to group 2 (36 months). Conclusions:A worse outcome trend was observed for pts with CD44 (+)/ESA (-), albeit not statistically significant and likely limited by small numbers. Further studies are warranted to evaluate the robustness of this observation.

STEAP4 expression in human islets is associated with differences in body mass index, sex, HbA1c, and inflammation.

STEAP4 (six-transmembrane epithelial antigen of the prostate 4) is a metalloreductase that has been shown previously to protect cells from inflammatory damage. Genetic variants in STEAP4 have been associated with numerous metabolic disorders related to obesity, including putative defects in the acute insulin response to glucose in type 2 diabetes. We examined whether obesity and/or type 2 diabetes altered STEAP4 expression in human pancreatic islets. Human islets were isolated from deceased donors at two medical centers and processed for quantitative polymerase chain reaction. Organ donors were selected by status as non-diabetic or having type 2 diabetes. Site 1 (Edmonton): N = 13 type 2 diabetes donors (7M, 6F), N = 20 non-diabetic donors (7M, 13F). Site 2 (Virginia): N = 6 type 2 diabetes donors (6F), N = 6 non-diabetic donors (3M, 3F). STEAP4 showed reduced islet expression with increasing body mass index among all donors (P < 0.10) and non-diabetic donors (P < 0.05) from Site 1; STEAP4 showed reduced islet expression among type 2 diabetes donors with increasing hemoglobin A1c. Islet STEAP4 expression was also marginally higher in female donors (P < 0.10). Among type 2 diabetes donors from Site 2, islet insulin expression was reduced, STEAP4 expression was increased, and white blood cell counts were increased compared to non-diabetic donors. Islets from non-diabetic donors that were exposed overnight to 5 ng/ml IL-1β displayed increased STEAP4 expression, consistent with STEAP4 upregulation by inflammatory signaling. These findings suggest that increased STEAP4 mRNA expression is associated with inflammatory stimuli, whereas lower STEAP4 expression is associated with obesity in human islets. Given its putative protective role, downregulation of STEAP4 by chronic obesity suggests a mechanism for reduced islet protection against cellular damage.

STEAP-1: A Potential Biomarker, Promising Target?

STEAP1 is one of six-transmembrane epithelial antigen of prostate (STEAP) highly expressed in human prostate cancer and is up-regulated in multiple cancers, including bladder, colon, breast, Ewing and lung cancers. Immunohistochemical analysis demonstrates significant STEAP1 expression at the intercellular communication between adjacent cells suggesting that this antigen must be a channel, or a transport protein indicating its potential role in tumor cell intercellular communication increasing the potential of STEAP1 as a diagnostic, prognostic, prophylactic and/or therapeutic target for new immunotherapeutic strategies. In prostate cancer, overexpression relates adenocarcinoma and prostatic intraepithelial neoplasia scores suggest that STEAP1 could be involved in tumor initiation and progression and may be of clinical usefulness in early disease diagnosis. Also, STEAP1 can serve as a biomarker of worse prognosis because of its association with higher Gleason score, seminal vesicle invasion, biochemical recurrence, and worse outcome. Therapeutically, STEAP1 is one of a few prostate cancer antigens that meet the appropriate criteria and represent an attractive target for antibody cancer therapy. It can be a target of anti-tumor CD8, or serve as a tool for vaccination as shown by xenograft models and phases I/II studies. STEAP1 could also serve as a new marker of tumor angiogenesis in lung cancer, indicate for occult residual tumor cells in patients with Ewing Sarcoma and also be used as a cell surface antigen for the development of breast cancer immunotherapy. The standardization of its evaluation as well as the validation through randomized trials would be necessary.

Expression of Epithelial Mesenchymal Transition and Cancer Stem Cell Markers in Circulating Tumor Cells.

The characterization of circulating tumor cells (CTC) has the potential not only to provide important insights into molecular alterations of advanced tumor disease but also to facilitate risk prediction. Epithelial mesenchymal transition (EMT) has been discovered as important process for the development of metastases and the dissemination of tumor cells into the blood stream. In different tumor types, CTC with a mesenchymal phenotype have been reported that have presumably underwent EMT. Moreover, CTC with stem-cell like characteristics have been postulated as important drivers of tumor progression. Different platforms have been introduced to allow CTC enrichment independent of expression of epithelial antigens, as these may be downregulated in EMT- or stem-cell-like CTC. Both for CTCs with EMT- or stem-cell features different markers have been proposed. However, there is still a lack of evidence on the association of these markers with functional features and characteristics for stem cells and cells undergoing EMT.

ED14.05 Immunotherapy of Small Cell Lung Cancer

ED14.05 Immunotherapy of Small Cell Lung Cancer

Impact of Prebiotics, Probiotics and Gut Derived Metabolites on Host Immunity

Increasing evidence indicates that gut microorganisms impact multiple aspects of the innate and adaptive mucosal immune system. Current research focuses on the potential of prebiotics (non-digestible fibres that nourish beneficial bacteria) and probiotics (beneficial live bacteria) to promote health, prevent disease, and for use as a treatment strategy for a variety of immune-mediated conditions. The immune modulatory effects of probiotics and prebiotics are strain- or structure-specific and vary with disease state, age, and sex. Prebiotics and live beneficial bacteria, including their metabolic products or soluble mediators, have the ability to affect the composition of the intestinal microbiota. As well, they influence the integrity and functions of intestinal epithelial cells and antigen presenting cells, including dendritic cells and macrophages, by both direct and indirect mechanisms of action.Statement of novelty: This review serves to highlight select advances related to the impact of prebiotics, p...

Improving the Accuracy of Mesothelioma Diagnosis in China

IntroductionIn the Western world, malignant mesothelioma (MM) is most prevalent in the pleura of older males who have been professionally exposed to asbestos. Information about MM from rapidly industrializing countries such as China is minimal. There is concern that a proportion of MM diagnoses in China may be incorrect because most Chinese physicians do not have experience diagnosing this rare cancer. We recently reported an unusually high incidence of peritoneal MM among eastern Chinese female patients. Here, we review the accuracy of MM diagnoses in China and provide suggestions to improve the accuracy of diagnosis. MethodsWe reviewed 92 pathological diagnosis of MM in 2002–2015 from two reference centers in the province of Zhejiang in eastern China. We performed a large set of immunohistochemistry analyses to increase the reliability of the diagnosis. ResultsWe confirmed the MM diagnosis in 12 of 34 of the pleural tumors (35.3%), in 38 of 56 of the peritoneal tumors (67.9%), and in two of two of the MMs of the tunica vaginalis (100%). MMs were characterized by tumor cells showing nuclear Wilms tumor 1 and calretinin staining and by strong membranous staining for cytokeratin CAM5.2. The results of staining for the epithelial markers carcinoembryonic antigen, thyroid transcription factor-1, MOC31, BerEP4, p63, p40, paired box 8, ER and PR were negative. BRCA1 associated protein 1 nuclear staining was lost in percentages similar to what has been reported for samples from Western countries. ConclusionsOur findings suggest that MM—especially in its pleural localization—is often misdiagnosed in eastern China. Identifying pitfalls and possible solutions in the pathological diagnosis of MM will affect both the standard of care and research in China.