Search

Eosinophilic colitis is a rare condition that usually presents with non specific abdominal symptoms. Very uncommonly it presents with an acute surgical emergency such as peritonitis or haemorrhage. We present a rare presentation of eosinophilic colitis with toxic hemorrhagic colitis and ischaemic bowel requiring laparotomy and bowel resection.

Resistance to therapy is one of the major causes of cancer-associated death. While a number of mechanisms for the development of resistance to epidermal growth factor receptor, EGFR-targeting drugs in lung cancer have been described, most genomic and transcriptomic studies have focused on analyzing bulk samples that can only provide an average measurement over the entire mixture of cell populations of a tumor. With this approach, it is difficult to resolve cell-to-cell variability of drug resistance within a heterogeneous tumor. In order to accurately describe and eventually delineate the underlying causes of cancer progression, it requires the analysis of single cells on both, the transcriptomic and the genomic level. Only the co-detection of mutations and expression features in individual cells allows to define the connection between the two. We therefore aimed to establish a new methodology for concurrent evaluation of transcriptomic and genomic features within the same single cell on the Fluidigm C1 system. We applied this protocol to an EGFR-mutant lung cancer cell line, PC9, that has developed resistance to tyrosine kinase inhibitor, TKI treatment after prolonged exposure to sublethal doses of Gefitinib. We amplified cDNA and selected genomic EGFR regions of the TKI responsive and resistant PC9 clones from a total of ≈300 single cells. We used our in-house single cell’s analysis pipeline for parallel processing of transcriptome and variants detection. In addition, a novel algorithm, NODES was used to normalize the single cell RNAseq data and detect differentially expressed genes. The differential transcriptome and emergence of the EGFR T790M resistance mutation in relation to the TKI response were evaluated. We observed up-regulation of receptor tyrosine kinase AXL in the resistant cell lines. This observation is in agreement with previous findings that AXL is the key player that promotes TKI resistance in lung cancer. In addition, we found up-regulation of other genes that have not been previously described. Cumulatively, this method allows us to dissect the underpinnings of the TKI resistance mechanism and enables us to identify biomarker for specific cellular features that are connected with resistance and thereby with lung cancer patient’s response to TKI treatment. Citation Format: Say Li Kong, Huipeng Li, Dave Ruff, Joyce An Yi Tai, Elise T. Courtois, Huay Mei Poh, Dawn Pingxi Lau, Audrey Ann Liew, Gek San Tan, Tony Kiat Hon Lim, Daniel Shao Weng Tan, Shyam Prabhakar, Axel M. Hillmer. Transcriptome differences in tyrosine kinase inhibitor-resistant clones of EGFR-mutant lung cancer using a new microfluidic assay for concurrent single-cell RNA and targeted DNA sequencing [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 3153. doi:10.1158/1538-7445.AM2017-3153

Objectives This purpose of this study was to examine clinical-pathologic factors – particularly smoking and brain metastases – in EGFR mutation positive (M+) lung adenocarcinoma (ADC) to determine their impact on survival in patients treated with first line EGFR TKI. Methods A retrospective review of EGFR mutation reflex testing experience for all ADC diagnosed at a tertiary Asian cancer centre from January 2009 to April 2013. Amongst this cohort, patients with advanced EGFR M+ ADC treated with first line EGFR TKI were identified to determine factors that influence progression free and overall survival. Results 444/742 (59.8%) ADC reflex tested for EGFR mutations were EGFR M+. Amongst never-smokers (n=468), EGFR M+ were found in 74.5% of females and 76.3% of males, and amongst ever smokers (n=283), in 53.3% of females and 35.6% of males. Exon 20 mutations were found more commonly amongst heavy smokers (> 50 pack years and > 20 pack years, Pearson’s chi square p=0.044, and p=0.038 respectively). 211 patients treated with palliative first line TKI had a median PFS and OS of 9.2 and 19.6 months respectively. 26% of patients had brain metastasis at diagnosis. This was significantly detrimental to overall survival (HR 1.85, CI 1.09-3.16, p=0.024) on multivariate analysis. There was no evidence that smoking status had a significant impact on survival. Conclusions The high prevalence of EGFR M+ in our patient population warrants reflex testing regardless of gender and smoking status. Smoking status and dosage did not impact progression free or overall survival in patients treated with first line EGFR TKI. The presence of brain metastasis at diagnosis negatively impacts overall survival.

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained.

Background: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis., Singapore-MIT Alliance for Research and Technology

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in " normal" tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.

We report a case of prostatic ductal adenocarcinoma which expressed thyroid transcription factor-1 (TTF-1) on immunohistochemical examination, and highlight the fact that this aberrant staining is a potential pitfall in the evaluation of this lesion. Prostatic core biopsy specimens from a 65-year-old man revealed an adenocarcinoma with large-calibre glands lined by stratified hyperchromatic nuclei. There was no accompanying conventional acinar adenocarcinoma and TTF-1 was distinctly positive in the nuclei of the malignant cells. This led to a search for possible lung and other primaries. When a thorough systemic investigation disclosed no evidence of other primary tumours, a radical prostatectomy was performed which revealed predominant ductal adenocarcinoma with a minor component of acinar adenoarcinoma. TTF-1 measurement was repeated which showed staining confined to the ductal adenocarcinoma. Prostate specific antigen (PSA), PSAP and racemase were also found. Our findings underscore the increasing spectrum of lesions that may express TTF-1 on immunohistochemical examination. TTF-1 is part of a family of homeodomain transcription factors with restricted expression in the thyroid and lung.1–3 Its immunohistochemical detection has been used in diagnostic surgical pathology to distinguish thyroid and lung tumours from those of other organs.4,5 Specifically, antibodies to TTF-1 have been reported to be useful in distinguishing pulmonary adenocarcinoma from other primary carcinomas.6 TTF-1 is expressed by differentiated thyroid neoplasms.7 It can assist in the differential diagnostic investigation of mesothelioma from pulmonary adenocarcinoma, small cell lung carcinoma from Merkel cell carcinoma,2,3 and neuroendocrine tumours of the lung from well-differentiated neuro-endocrine tumours at other sites.1,8 TTF-1 mRNA has been discovered in orbital tissues,9 C cells and parathyroid cells.10 More recently, nuclear TTF-1 protein expression was reported in primary and metastatic colonic adenocarcinoma,11,12 primary ovarian epithelial neoplasms,13 thyroid-like nasopharyngeal papillary adenocarcinoma,7 gastric …

Colorectal cancer is currently the third in cancer incidence worldwide and the fourth most common cause of cancer deaths. Mortality in colorectal cancer is often ascribed to liver metastasis. In an effort to elucidate the proteins involved in colorectal cancer liver metastasis, we compared the proteome profiles of the human colon adenocarcinoma cell line HCT-116 with its metastatic derivative E1, using the iTRAQ labelling technology, coupled to 2D-LC and MALDI-TOF/TOF MS. A total of 547 proteins were identified, of which 31 of them were differentially expressed in the E1 cell line. Among these proteins, the differential expressions of translationally controlled tumour protein 1, A-kinase anchor protein 12 and Drebrin (DBN1) were validated using Western blot. In particular, DBN1, a protein not previously known to be involved in colorectal cancer metastasis, was found to be overexpressed in E1 as compared to HCT-116 cells. The overexpression of DBN1 was further validated using immunohistochemistry on colorectal cancer tissue sections with matched lymph node and liver metastasis tissues. DBN1 is currently believed to be involved in actin cytoskeleton reorganisation and suppresses actin filament cross-linking and bundling. Since actin reorganisation is an important process for tumour cell migration and invasion, DBN1 may have an important role during colorectal cancer metastasis.

Non-small-cell lung cancer (NSCLC) is a heterogeneous disease, with a burden of genomic alterations exceeding most other tumors. The goal of our study was to evaluate the frequencies of co-occurring mutations and copy-number aberrations (CNAs) within the same tumor and to evaluate their potential clinical impact. Mass-spectrometry based mutation profiling using a customized lung cancer panel evaluating 214 mutations across 26 key NSCLC genes was performed on 230 nonsquamous NSCLC and integrated with genome-wide CNAs and clinical variables. Among the 138 cases having at least one mutation, one-third (41, 29.7%) showed two or more mutations, either in the same gene (double mutation) or in different genes (co-mutations). In epidermal growth factor receptor (EGFR) mutant cancers, there was a double mutation in 18% and co-mutations in the following genes: TP53 (10%), PIK3CA (8%), STK11 (6%) and MET (4%). Significant relationships were detected between EGFR mutation and 1p, 7p copy gains (harboring the EGFR gene) as well as 13q copy loss. KRAS mutation was significantly related with 1q gain and 3q loss. For Stage I, tumors harboring at least one mutation or PIK3CA mutation were significantly correlated with poor prognosis (p-value = 0.02). When combining CNAs and mutational status, patients having both KRAS mutation and the highest related CNA (3q22.3 copy loss) showed a significant poorer prognosis (p-value = 0.03). Our study highlights the clinical relevance of studying tumor complexity by integrative genomic analysis and the need for developing assays that broadly screen for both "actionable" mutations and copy-number alterations to improve precision of stratified treatment approaches.

Positron emission tomography (PET) using ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) is an imaging modality which reflects cellular glucose metabolism. Most malignant cells accumulate and trap ¹⁸F-FDG, allowing the visualisation of increased uptake. It is hence widely used to differentiate malignant from benign lesions. "False positive" findings of hepatic lesions have been described in certain instances such as hepatic abscesses, but are rare in cases involving hepatocellular adenomas. To our knowledge, there have been only 7 reports in the English literature documenting PET-avid hepatocellular adenomas; 6 of the 7 reports were published in the last 3 years with the first report by Patel et al. We report the case of a 44-year-old Chinese female patient with a history of cervical adenocarcinoma, referred for a hepatic lesion noted on a surveillance computed tomography (CT) scan. A subsequent CT-PET performed showed a hypermetabolic lesion (standardized uptake value 7.9) in segment IVb of the liver. After discussion at a multi-disciplinary hepato-pancreato-biliary conference, the consensus was that of a metastatic lesion from her previous cervical adenocarcinoma, and a resection of the hepatic lesion was performed. Histology revealed features consistent with a hepatocyte nuclear factor-1 α inactivated steatotic hepatocellular adenoma.

Gene regulation of many key cell-cycle players in S-, G2 phase, and mitosis results from transcriptional repression in their respective promoter regions during the G0 and G1 phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle–dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma. Mol Cancer Ther; 12(8); 1651–64. ©2013 AACR.

Mucinous epithelial ovarian cancer has a poor prognosis in the advanced stages and responds poorly to conventional chemotherapy. We aim to elucidate the clinicopathological factors and incidence of HER2 expression of this cancer in a large Asian retrospective cohort from Singapore. Of a total of 133 cases, the median age at diagnosis was 48.3 years (range, 15.8–89.0 years), comparatively younger than western cohorts. Most were Chinese (71%), followed by Malays (16%), others (9.0%), and Indians (5%). 24% were noted to have a significant family history of malignancy of which breast and gastrointestinal cancers the most prominent. Majority of the patients (80%) had stage I disease at diagnosis. Information on HER2 status was available in 113 cases (85%). Of these, 31 cases (27.4%) were HER2+, higher than 18.8% reported in western population. HER2 positivity appeared to be lower among Chinese and higher among Malays patients (p = 0.052). With the current standard of care, there was no discernible impact of HER2 status on overall survival. (HR = 1.79; 95% CI, 0.66–4.85; p = 0.249). On the other hand, positive family history of cancer, presence of lymphovascular invasion, and ovarian surface involvements were significantly associated with inferior overall survival on univariate and continued to be statistically significant after adjustment for stage. While these clinical factors identify high risk patients, it is promising that the finding of a high incidence of HER2 in our Asian population may allow development of a HER2 targeted therapy to improve the management of mucinous ovarian cancers. Published version

While EGFR tyrosine kinase inhibitors (TKIs) can elicit significant tumor shrinkage in the clinic, drug resistance inevitably ensues, likely due to a persistent subpopulation. T790M mutation is a common resistance mechanism found in 60% of tumors progressing on TKI. The aim of this study is to uncover novel mechanisms of gefitnib-resistant (GR) NSCLC by selecting TKI naïve PC9 single cell clones (SCC) differing in stemness and vulnerabilities to gefitinib (G), and inducing resistance through sublethal drug exposures. A high throughput drug screen was performed in 4 SCC to discover alternate therapies to target T790M negative GR clones. 231 SCC of PC9 were isolated and confirmed to be T790M negative (0.3% sensitivity) using Agena SABER assay. All clones were characterized by their vulnerability to G (viable after 48 hour exposure to 0.1uM) and ALDH1 activity using the ALDEFLUOR assay and flow cytometry. Four “diverse” clones (stratified according to ALDH1 and sensitivity to TKI) were selected to generate GR cell lines. Agglomerative hierarchical clustering of differentially expressed genes revealed that the clone exhibiting extreme low survival and ALDH1 signature clusters significantly apart from the rest. Subsequent iterations of this experiment (in total 5 per clone) revealed unique (and mostly stochastic) patterns of acquiring T790M across all clones. GR clones were then subjected to mutational analysis using the Ion AmpliSeq™ Colon and Lung Cancer Panel v1, and showed that 11/20 (55%) PC9 SCC GR lines have acquired the T790M mutation. T790M positive (AF range: 2.4 - 41.0%) GR lines were sensitive to Afatinib and WZ4002, a 2nd and 3rd generation EGFR TKI, with EGFR1086 phosphorylation being completely absent in all non-responding lines. T790M negative GR lines further acquired NRAS Q61K, G12D mutation and KRAS Q22K mutation. A high throughput drug screen with kinase inhibitor library from Selleck Chemicals (n = 273 kinases, n = 349 anticancer library) confirmed the selectivity of MEK inhibitors, as well as identified a subset of T790M-ve lines that are sensitive to Aurora kinase inhibitors. Our study highlights the potential for pEGFR to complement T790M status as a selection biomarker, particularly where low tumor cell concentration may yield false negative results. Taken together, we have uncovered potential therapeutically tractable subsets of T790M negative EGFR TKI resistant cell lines. pEGFR may identify a subset of T790M negative tumors that remain driven by and may be an alternate selection biomarker to 2nd/3rd gen TKI. Combinational approaches with MEK and aurora kinase inhibitors could be further explored in T790M negative/ pEGFR negative tumors. Citation Format: Dawn Pingxi Lau, Shivaji Rikka, Hui Sun Leong, Gek San Tan, Eleni G Christodoulou, Sai Sakktee Krisna, Shen Yon Toh, Xue Lin Kwang, Fui Teen Chong, Audrey Ann Liew, Tony Kiat Hon Lim, DasGupta Ramanuj, N Gopalakrishna Iyer, Daniel Shao-Weng Tan. Clonal heterogeneity of EGFR TKI-resistant NSCLC revealed through single-cell dilutional cloning and high-throughput functional screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2109.

e23086Background: Fluorescence in-situ hybridization (FISH) is the reference assay for ROS1 gene rearrangement. Molecular detection of ROS1 rearrangement in lung cancer has therapeutic relevance in...

In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC-MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of1.5 and0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein.

Double cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment.Post-thaw cryopreserved CBUs from cord blood banks, hereinafter termed "banked" CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days.When DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma(null) (NOD/SCID-IL2γ(-/-), NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0-66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0-92.9%) for mice receiving an expanded unit (P0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture.MSC-supported ex vivo expansion of "banked" CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.

Objective Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors and reveal novel therapeutic targets. The authors aimed to produce a comprehensive inventory of gene expression programmes expressed in primary GCs, and to identify those expression programmes significantly associated with patient survival. Design Using a network-modelling approach, the authors performed a large-scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. The authors analysed a training set of 428 GCs and 163 nonmalignant gastric samples, and a validation set of 288 GCs and 61 non-malignant gastric samples. Results The authors identified 178 gene expression programmes (‘modules’) expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs and clinicopathological parameters. Expression of a transforming growth factor β (TGF-β) signalling associated ‘super-module’ of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. The proportion of intra-tumoural stroma, quantified by morphometry in tissue sections from gastrectomy specimens, was also significantly associated with stromal super-module expression and GC patient survival. Conclusion Stromal gene expression predicts GC patient survival in multiple independent cohorts, and may be closely related to the intra-tumoural stroma proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting the GC stroma may merit evaluation.

The oncological results of close distal resection margins (DM) have been mixed due to variations in perioperative treatment protocols and surgical expertise. With the increased application of sphincter-saving surgery in the management of rectal cancer, “close shave” DM is an increasingly encountered phenomenon. Our center aims to examine the oncological outcomes of “close shave” DM in the absence of neoadjuvant therapy in the surgical treatment of rectal cancer. A prospective database of 320 patients who underwent curative surgical resection for primary rectal cancer between 1999 and 2007 was reviewed. One hundred forty-eight patients had “close shave” DM (DM

Gastric cancer remains highly fatal due to a dearth of diagnostic biomarkers for early stage disease and molecular targets for therapy. Plasma membrane proteins, including cluster of differentiation (CD) proteins and receptor tyrosine kinases (RTKs), are a rich reservoir of biomarkers. Recognizing that interrogating plasma membrane proteins individually overlooks extensive interactions among them, we have systematically investigated the membrane proteomes and transcriptomes of six gastric cancer cell lines. Our data revealed aberrantly high expression of proteins whose functions accurately reflect the clinical phenotype of gastric cancer, and prioritized critical RTKs and CD proteins in gastric cancer. Expression of selected surface proteins was confirmed by flow cytometry and immunostaining of clinical gastric cancer tissues. Close to 90% of the gastric cancer tissues in a cohort showed up-regulation of at least one of four proteins, that is, MET, EPHA2, FGFR2, and CD104/ITGB4. All intestinal type gastric cancer tumors in this cohort overexpressed at least one of a panel of three proteins, MET, FGFR2, and EPHA2. This study reports the first quantitative global landscape of the surface proteome of gastric cancer cells and provides a shortlist of gastric cancer biomarkers.

Background Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC. Methods HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided. Results TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002). Conclusions TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

Compartmental tracer kinetic models currently used for analysis of dynamic contrast-enhanced MRI data yield poor fittings or parameter values that are unphysiological in necrotic regions of the tumor, as these models only describe microcirculation in perfused tissue. In this study, we explore the use of Fick's law of diffusion as an alternative method for analysis of dynamic contrast-enhanced MRI data in the necrotic regions. Xenografts of various human cancer cell lines were implanted in 14 mice that were subjected to dynamic contrast-enhanced MRI performed using a spoiled gradient recalled sequence. Tracer concentration was estimated using the variable flip angle technique. Poorly perfused and necrotic tumor regions exhibiting delayed and slow enhancement were identified using a k-means clustering algorithm. Tracer behavior in necrotic regions was shown to be consistent with Fick's diffusion equation and the in vivo gadolinium diffusivity was estimated to be 2.08 (±0.88) × 10−4 mm2/s. This study proposes the use of gadolinium diffusivity as an alternative parameter for quantifying tracer transport within necrotic tumor regions. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e.

Hepatitis E, usually an acute hepatitis in the immunocompetent, has a chronic form described in immunocompromised hosts. We report the clinical course and outcome of an adult liver transplant recipient whose posttransplant period was complicated by chronic hepatitis E, Epstein-Barr virus infection, and cellular rejection of the graft.

High macrophage infiltration into tumours often correlates with poor prognoses; in colorectal, stomach and skin cancers, however, the opposite is observed but the mechanisms behind this phenomenon remain unclear. Here, we sought to understand how tumour-associated macrophages (TAMs) in colorectal cancer execute tumour-suppressive roles. We found that TAMs in a colorectal cancer model were pro-inflammatory and inhibited the proliferation of tumour cells. TAMs also produced chemokines that attract T cells, stimulated proliferation of allogeneic T cells and activated type-1 T cells associated with anti-tumour immune responses. Using colorectal tumour tissues, we verified that TAMs in vivo were indeed pro-inflammatory. Furthermore, the number of tumour-infiltrating T cells correlated with the number of TAMs, suggesting that TAMs could attract T cells; and indeed, type-1 T cells were present in the tumour tissues. Patient clinical data suggested that TAMs exerted tumour-suppressive effects with the help of T cells. Hence, the tumour-suppressive mechanisms of TAMs in colorectal cancer involve the inhibition of tumour cell proliferation alongside the production of pro-inflammatory cytokines, chemokines and promoting type-1 T-cell responses. These new findings would contribute to the development of future cancer immunotherapies based on enhancing the tumour-suppressive properties of TAMs to boost anti-tumour immune responses.

Kuttner Tumour is a chronic inflammatory disease diagnosed histologically. It is under-diagnosed due to lack of awareness of the disease. We present a case of a 40-year old female with recurrent upper lid swelling. Biopsy revealed the diagnosis of Kuttner Tumours of the lacrimal glands. Although surgical excision is a treatment option, it can be treated with a course of steroids. Our patient opted for conservative management. We describe the presentation and course of the case, with an emphasis on the histological features of the tumour.

Aim To determine the expression pattern and prognostic value of heparan sulfate in gastric cancer. Method The 10E4 antiheparan sulfate monoclonal antibody was used to examine the expression pattern of heparan sulfate in tissue microarrays consisting of 162 cases of gastric carcinoma by immunohistochemistry. The immunoreactivities of both epithelial and stromal components of the specimens were examined and analysed statistically for significant associations with clinicopathological parameters, including histological grade of the tumour, extent of cancer infiltration and presence of lymph-node metastases, lymphovascular invasion, perineural invasion, perforation of gastric wall and stromal reaction. The potential use of heparan sulfate as a predictive factor for patient survival was also evaluated. Results Reduced expression of heparan sulfate in the epithelial component was associated with higher histological grades of gastric cancer as well as the presence of more extensive tumour infiltration. Furthermore, this decrease in heparan sulfate expression was found to be predictive of reduced patient survival after tumour recurrence. Conclusion The data suggest that heparan sulfate may play an important role in regulating the biology of gastric cancer, and that it may be a useful prognostic marker of this tumour.

Background & Aims Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs. Methods We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed. Results Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Lauren's histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil–based therapy. Conclusions Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.

Background: The incidence of mismatch repair (MMR) deficiency in young colorectal cancers (CRC) remains unknown in Asians. This preliminary study assessed the clinicopathological features and efficacy of screening for MMR protein deficiency in young Asian CRC patients. Methods: From January 2006 to October 2009, patients under the age of 50 with immunohistochemical (IHC) staining for MMR proteins in resected CRC specimens were retrieved from a prospective computerised database. Results: Eighty unrelated patients comprising predominantly 80% Chinese (n = 64), with median age of diagnosis at 41 years (range 22–50 years) had IHC performed. Twenty-three per cent (n=18) of the patients had abnormal IHC staining. Loss of staining for MLH1, MSH2 and MSH6 proteins were observed in 18%, 2% and 6% of tumours respectively. Of the 15 patients who had abnormal staining of MLH1, three had concomitant equivocal staining for MSH6. One tumour specimen had abnormal staining in all 3 proteins. Multivariate analysis revealed that family history was the only significant predictive factor for defective MMR detection (OR 8.06, 95% CI 1.69–38.35, p=0.002). However if Amsterdam criteria alone were to be used, 72% (n=12) of the cohort would have not been detected for MMR gene defects. Conclusion: The overall burden of germline MMR deficiency in the Singapore population may be as high as 23%. Amsterdam criteria alone are insufficient to detect hereditary non-polyposis colorectal cancer (HNPCC) related patients. The use of IHC staining of at least 3 MMR proteins is a useful screening strategy for HNPCC diagnosis and routine screening of mismatch repair deficiency may be recommended for all young Asian CRC patients.

A 69-year-old man presented with intestinal obstruction and peritonism. Chest and abdominal X-rays showed massive pneumoperitoneum with large bowel obstruction (Figs. 1, 2). Exploratory laparotomy revealed closed loop obstruction secondary to sigmoid volvulus with extensive pneumatosis cystoids intestinalis of non-dilated small bowel (Figs. 3, 4), without evidence of perforation. We postulate that rupture of a submucosal cyst in the small bowel resulted in pneumoperitoneum. Total colectomy was performed, with ileocecal anastomosis and defunctioning ileostomy. Histology confirmed pneumatosis cystoides intestinalis of small bowel without full-thickness perforation (Figs. 5, 6).

Hereditary mixed polyposis syndrome (HMPS) is characterized by polyps of mixed adenomatous/hyperplastic/atypical juvenile histology that are autosomal dominantly inherited and that eventually lead to colorectal cancer (CRC). Although CRC with adenomatous polyps is initiated by inactivating adenomatous polyposis coli (APC), the initiating event of CRC with mixed polyps remains unclear. We aimed to identify the underlying germline defect in HMPS.We screened for bone morphogenesis protein receptor 1A (BMPR1A) mutation by exonic sequencing, reverse-transcriptase polymerase chain reaction (PCR) followed by cDNA sequencing, and multiplex ligation-dependent probe amplification (MLPA) analysis in eight Singapore Chinese HMPS families.Germline BMPR1A defects were found in four (50%) families. In two families, it is shown to co-segregate with the disease phenotype in all affected members over three generations, indicating that it is the disease-causing mutation. CRC incidence is 75%. The most defining characteristic is the presence of mixed hyperplastic-adenomatous polyps. Juvenile polyps are rarely reported, and if present, are usually of mixed components. Detailed histology of the polyps from one patient over 11 years distinguishes HMPS from juvenile polyposis syndrome (JPS). We report further the first cases of Wilms' tumor and papillary thyroid carcinoma associated with BMPR1A germline defect.Germline BMPR1A defect is the disease-causing mutation in 50% of the HMPS families. If patients present with mixed morphology polyps in the large bowel that are autosomal dominantly inherited and corresponding absence of upper gastrointestinal abnormalities, the gene to begin mutation screening should be BMPR1A rather than APC.

Background/Aims The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. Methods Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. Results Different mechanisms accounted for Ras pathway activation in HCC. H- ras was up-regulated during different steps of hepatocarcinogenesis. B- raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo , the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. Conclusions Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B- raf ). Sorafenib blocks signaling and synergizes with rapamycin in vivo , preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

A 71-year-old man presented to our hospital with 3-week history of fever in the background of loss of both weight and appetite over the past 3 months. He was found to have a large 10-cm mass in the right lobe of the liver on a triple-phase computed tomographic scan. The tumor showed a distinct fatty component, with areas of arterial enhancement and venous washout suggestive of hepatocellular carcinoma (HCC), another component showing progressive and late enhancement suggestive of cholangiocarcinoma (CC), and a third component showing persistent hypoenhancement relative to the liver parenchyma. He underwent surgical resection. This was histopathologically a biphasic tumor composed of areas showing hepatocytic differentiation, in contiguity with areas showing infiltrative glands set within fibrous stroma in keeping with combined hepatocellular and cholangiocarcinoma (cHCC-CC). A third component of pleomorphic spindle and epithelioid appearance in keeping with sarcomatous transformation was also found intimately related to the CC component. The patient developed extensive thoracic and abdominal metastases 2 months after surgery and died shortly after.

The authors describe multiple telangiectatic or inflammatory adenomas in a 53-year-old woman with steatohepatitis who presented with acute right upper quadrant abdominal pain. Magnetic resonance imaging revealed 6 lesions consistent with multiple hepatic adenomas, 2 of which showed hemorrhage. She underwent right lobectomy and nonanatomical segment 2 liver resections and seven nodules ranging in size from 1.0 to 5.0 cm were identified. All nodules contained portal-like structures and ductular reaction, features seen in focal nodular hyperplasia, as well as significant inflammation, telangiectatic sinusoids and immunoreactivity for serum amyloid A, placing them according to a recently described classification systems as telangiectatic or inflammatory adenomas. The diffuse positivity of the serum amyloid A staining results in this case suggests an important diagnostic role of this stain in smaller tissue samples, such as in core biopsy specimens.

A diagnosis of pulmonary adenoid cystic carcinoma on exfoliative cytology specimen is very uncommon. The diagnostic cytologic material typically is obtained following a tissue biopsy. No previous report of the diagnosis has been made on bronchial brushing cytologic material when the procedure preceded a tissue biopsy.A 44-year-old man who used to smoke cigarettes and was otherwise well complained of persistent cough for the past 6 months. A chest radiograph revealed a mass lesion in the left hilum. Computed tomography of the chest disclosed an irregular and spiculated soft tissue mass in the left apical anterior segment. Bronchial brushing via bronchoscope was performed, revealing carcinoma cells consistent with an adenoid cystic carcinoma on cytology. A bronchial biopsy and subsequent left upper lobectomy were performed, confirming the diagnosis of adenoid cystic carcinoma of the lung associated with tumor extension to the epithelial surface.A diagnosis of bronchial adenoid cystic carcinoma is possible on bronchial brushing. However, as a method in exfoliative cytology, the usefulness of bronchial brushing in diagnosing this tumor is limited by the neoplasm's proximity to the mucosal surface and whether the mucosa has been breached.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridization (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data was also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirms that RTK/RAS components are mutually exclusively amplified in GC, and demonstrates the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient, before a definitive diagnosis can be attained. Citation Format: Kakoli Das, Bavani Gunasegaran, Iain Beehuat Tan, Niantao Deng, Kiat Hon Lim, Patrick Tan. Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH. [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 4911. doi:10.1158/1538-7445.AM2015-4911

Biliary stricture is a rare sequela of biliary tuberculosis (TB) infection. Early recognition with prompt treatment can result in complete resolution. A 56-year-old man was diagnosed with biliary TB after analysis of bile from ERCP showed positive results for TB. Bile duct brushings were inconclusive. Despite completing 7 months of anti-TB treatment, the patient’s liver function test results were still abnormal. MRCP showed dilated intrahepatic ducts with a stricture at the hilar bifurcation

e22134 Background: Research biopsies play a critical role in facilitating biomarker driven clinical trials. The increasing requirement to perform comprehensive molecular analyses necessitates procu...