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Background & Aims Almost all gastric cancers are adenocarcinomas, which have considerable heterogeneity among patients. We sought to identify subtypes of gastric adenocarcinomas with particular biological properties and responses to chemotherapy and targeted agents. Methods We compared gene expression patterns among 248 gastric tumors; using a robust method of unsupervised clustering, consensus hierarchical clustering with iterative feature selection, we identified 3 major subtypes. We developed a classifier for these subtypes and validated it in 70 tumors from a different population. We identified distinct genomic and epigenomic properties of the subtypes. We determined drug sensitivities of the subtypes in primary tumors using clinical survival data, and in cell lines through high-throughput drug screening. Results We identified 3 subtypes of gastric adenocarcinoma: proliferative, metabolic, and mesenchymal. Tumors of the proliferative subtype had high levels of genomic instability, TP53 mutations, and DNA hypomethylation. Cancer cells of the metabolic subtype were more sensitive to 5-fluorouracil than the other subtypes. Furthermore, in 2 independent groups of patients, those with tumors of the metabolic subtype appeared to have greater benefits with 5-fluorouracil treatment. Tumors of the mesenchymal subtype contain cells with features of cancer stem cells, and cell lines of this subtype are particularly sensitive to phosphatidylinositol 3-kinase−AKT−mTOR inhibitors in vitro. Conclusions Based on gene expression patterns, we classified gastric cancers into 3 subtypes, and validated these in an independent set of tumors. The subgroups have differences in molecular and genetic features and response to therapy; this information might be used to select specific treatment approaches for patients with gastric cancer.

The incidence of mismatch repair deficiency in colorectal cancer (CRC) in young people remains unknown in Asians. The present study assessed the clinicopathological features and efficacy of immunohistochemistry screening for Lynch syndrome in young Asian CRC patients. This was a retrospective review conducted in Singapore General Hospital between January 2006 and December 2010 of 240 unrelated patients under the age of 50. All patients had immunohistochemical (IHC) staining for mismatch repair proteins in resected CRC specimen data retrieved from a prospective computerized database. A total of 21 % (n = 51) of the patients had abnormal IHC staining. Loss of staining for MLH1, MSH2, MSH6, and PMS2 proteins was observed in 10, 4, 6, and 13 % of tumors, respectively. Of the 22 patients who had abnormal staining of MLH1, 13 had concomitant abnormal staining for PMS2. One tumor specimen had abnormal staining in all four proteins. If the Amsterdam criteria alone were to be used, 86 % (n = 44) of the cohort would have not been detected for mismatch repair gene defects. The overall burden of germline mismatch repair deficiency in the Singapore population may be as high as 21 %. The Amsterdam criteria alone are inadequate to detect Lynch syndrome patients. The use of IHC staining of at least four mismatch repair proteins is a useful screening strategy for Lynch syndrome diagnosis. Routine screening of mismatch repair deficiency may be recommended for all young Asian CRC patients.

Chemoprevention—the use of medication to prevent cancer—can be augmented by the consumption of produce enriched with natural metabolites. However, chemopreventive metabolites are typically inactive and have low bioavailability and poor host absorption. Here, we show that engineered commensal microbes can prevent carcinogenesis and promote the regression of colorectal cancer through a cruciferous vegetable diet. The engineered commensal Escherichia coli bound specifically to the heparan sulphate proteoglycan on colorectal cancer cells and secreted the enzyme myrosinase to transform host-ingested glucosinolates—natural components of cruciferous vegetables—to sulphoraphane, an organic small molecule with known anticancer activity. The engineered microbes coupled with glucosinolates resulted in >95% proliferation inhibition of murine, human and colorectal adenocarcinoma cell lines in vitro. We also show that murine models of colorectal carcinoma fed with the engineered microbes and the cruciferous vegetable diet displayed significant tumour regression and reduced tumour occurrence.

Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) National Medical Research Council (NMRC) National Research Foundation (NRF) This research is supported by the National Research Foundation, Singapore (NRF-NRFF2015-04), the National Medical Research Council, Singapore (LCG17MAY004; NMRC/OFIRG/0064/2017; NMRC/TCR/007- NCC/2013; OFYIRG16nov013), the Agency for Science, Research and Technology, Singapore (1331AEG071; 334I00053; SPF 2012/001), and the Singapore Ministry of Education under its Research Centers of Excellence initiative.

Supplemental Digital Content is available in the text, In this study, a transcriptomic group classification based on a European population is tested on a Singapore cohort. The results highlight the genotype/phenotype correlation in a Southeast Asian population. The G1–G6 transcriptomic classification derived from hepatocellular carcinoma (HCC) resected from European patients, robustly reflected group-specific clinical/pathological features. We investigated the application of this molecular classification in Southeast Asian HCC patients. Gene expression analysis was carried out on HCC surgically resected in Singapore patients who were grouped into G1–G6 transcriptomic categories according to expression of 16 predictor genes (illustrated in Supplementary Table 1 and Supplementary Fig. 1) using quantitative reverse transcription polymerase chain reaction (RT-PCR). Univariate and multivariate polytomous logistic regression was used to investigate association between clinical variables and pooled transcriptomic classes G12, G3, and G456. HCC from Singapore (n = 82) were distributed (%) into G1 (13.4), G2 (24.4), G3 (15.9), G4 (24.4), G5 (14.6), and G6 (7.3) subgroups. Compared to the European data, the Singapore samples were relatively enriched in G1–G3 versus G4–G6 tumors (53.7% vs 46.3%) reflecting the higher proportion of hepatitis B virus (HBV) patients in Singapore versus Europe samples (43% vs 30%). Pooled classes were defined as G12, G3, and G456. G12 was associated with higher alpha-fetoprotein (AFP) concentrations (OR = 1.69, 95% CI: 1.30–2.20; P

Cytology and histology forms the cornerstone for the diagnosis of non-small cell lung cancer (NSCLC) but obtaining sufficient tumour cells or tissue biopsies for these tests remains a challenge. We investigate the lipidome of lung pleural effusion (PE) for unique metabolic signatures to discriminate benign versus malignant PE and EGFR versus non-EGFR malignant subgroups to identify novel diagnostic markers that is independent of tumour cell availability. Using liquid chromatography mass spectrometry, we profiled the lipidomes of the PE of 30 benign and 41 malignant cases with or without EGFR mutation. Unsupervised principal component analysis revealed distinctive differences between the lipidomes of benign and malignant PE as well as between EGFR mutants and non-EGFR mutants. Docosapentaenoic acid and Docosahexaenoic acid gave superior sensitivity and specificity for detecting NSCLC when used singly. Additionally, several 20- and 22- carbon polyunsaturated fatty acids and phospholipid species were significantly elevated in the EGFR mutants compared to non-EGFR mutants. A 7-lipid panel showed great promise in the stratification of EGFR from non-EGFR malignant PE. Our data revealed novel lipid candidate markers in the non-cellular fraction of PE that holds potential to aid the diagnosis of benign, EGFR mutation positive and negative NSCLC.

Trithorax-like group complex containing KDM6A acts antagonistically to Polycomb-repressive complex 2 (PRC2) containing EZH2 in maintaining the dynamics of the repression and activation of gene expression through H3K27 methylation. In urothelial bladder carcinoma, KDM6A (a H3K27 demethylase) is frequently mutated, but its functional consequences and therapeutic targetability remain unknown. About 70% of KDM6A mutations resulted in a total loss of expression and a consequent loss of demethylase function in this cancer type. Further transcriptome analysis found multiple deregulated pathways, especially PRC2/EZH2, in KDM6A-mutated urothelial bladder carcinoma. Chromatin immunoprecipitation sequencing analysis revealed enrichment of H3K27me3 at specific loci in KDM6A-null cells, including PRC2/EZH2 and their downstream targets. Consequently, we targeted EZH2 (an H3K27 methylase) and demonstrated that KDM6A-null urothelial bladder carcinoma cell lines were sensitive to EZH2 inhibition. Loss- and gain-of-function assays confirmed that cells with loss of KDM6A are vulnerable to EZH2. IGFBP3, a direct KDM6A/EZH2/H3K27me3 target, was up-regulated by EZH2 inhibition and contributed to the observed EZH2-dependent growth suppression in KDM6A-null cell lines. EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in both patient-derived and cell line xenograft models. In summary, our study demonstrates that inactivating mutations of KDM6A, which are common in urothelial bladder carcinoma, are potentially targetable by inhibiting EZH2.

Non-alcoholic steatohepatitis (NASH) is one of the most common causes of liver failure worldwide. It is characterized by excess fat accumulation, inflammation, and increased lipotoxicity in hepatocytes. Currently, there are limited treatment options for NASH due to lack of understanding of its molecular etiology. In the present study, we demonstrate that the expression of fat mass and obesity associated gene (FTO) is significantly increased in the livers of NASH patients and in a rodent model of NASH. Furthermore, using human hepatic cells, we show that genetic silencing of FTO protects against palmitate-induced oxidative stress, mitochondrial dysfunction, ER stress, and apoptosis in vitro. Taken together, our results show that FTO may have a deleterious role in hepatic cells during lipotoxic conditions, and strongly suggest that up-regulation of FTO may contribute to the increased liver damage in NASH.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Benefit of adjuvant imatinib therapy following curative resection in patients with intermediate-risk gastrointestinal stromal tumor (GIST) is unclear. GIST-specific exon mutations, in particular exon 11 deletions, have been shown to be prognostic. We hypothesize that specific KIT mutations may improve risk stratification in patients with intermediate-risk GIST, identifying a subgroup of patients who may benefit from adjuvant therapy.In total, 142 GIST patients with complete clinicopathologic and mutational data from two sites were included. Risk classification was based on the modified National Institute of Health (NIH) criteria.In this cohort, 74% (n = 105) of patients harbored a KIT mutation; 61% (n = 86) were found in exon 11 of which nearly 70% were KIT exon 11 deletions (n = 60). A total of 18% (n = 25) of cases were classified as having intermediate-risk disease. Univariate analysis confirmed tumor size, mitotic index, nongastric origin, presence of tumor rupture and modified NIH criteria were adversely prognostic for relapse-free survival (RFS). Among KIT/PDGFRA mutants, KIT exon 11 deletions had a significantly worse prognosis (hazard ratio 2.31; 95% confidence interval, 1.30-4.10; P = 0.003). Multivariate analysis confirmed KIT exon 11 deletion (P = 0.003) and clinical risk classification (P0.001) as independent adverse prognostic factors for RFS. Intermediate-risk patients harboring KIT exon 11 deletions had RFS outcomes similar to high-risk patients.The presence of KIT exon 11 deletion mutation in patients with intermediate-risk GIST is associated with an inferior clinical outcome with RFS similar to high-risk patients.

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Diabetes mellitus is characterized by chronic inflammation and increased risk of infections, particularly of tissues exposed to the external environment. However, the causal molecular mechanisms that affect immune cells and their functions in diabetes are unclear. Here we show, by transcript and protein analyses, signatures of glucose-induced tissue damage, chronic inflammation, oxidative stress, and dysregulated expression of multiple inflammation- and immunity-related molecules in diabetic kidneys compared with non-diabetic controls. Abnormal signaling involving cytokines, G-protein coupled receptors, protein kinase C isoforms, mitogen-activated protein kinases, nuclear factor-κB (NFκB), and Toll-like receptors (TLR) were evident. These were accompanied by overexpression of negative regulators of NFκB, TLR, and other proinflammatory pathways, e.g., A20, SOCS1, IRAK-M, IκBα, Triad3A, Tollip, SIGIRR, and ST2L. Anti-inflammatory and immunomodulatory molecules, e.g., IL-10, IL-4, and TSLP that favor TH2 responses were strongly induced. These molecular indicators of immune dysfunction led us to detect the cryptic presence of bacteria and human cytomegalovirus in more than one third of kidneys of diabetic subjects but none in non-diabetic kidneys. Similar signaling abnormalities could be induced in primary human renal tubular epithelial (but not mesangial) cell cultures exposed to high glucose, proinflammatory cytokines and methylglyoxal, and were reversed by combined pharmacological treatment with an antioxidant and a PKC inhibitor. Our results suggest that diabetes impairs epithelial immunity as a consequence of chronic and inappropriate activation of counter-regulatory immune responses, which are otherwise physiological protective mechanisms against inflammation. The immune abnormalities and cryptic renal infections described here may contribute to progression of diabetic nephropathy. Electronic supplementary material The online version of this article (doi:10.1007/s00109-012-0969-x) contains supplementary material, which is available to authorized users.

Bin Tean Teh and colleagues report exome sequencing of Opisthorchis viverrini–related cholangiocarcinoma, a fatal bile duct cancer associated with liver fluke infection. Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets ( FGFR2 , ERBB2 ) and also novel genes in gastric cancer ( KLF5 , GATA6 ). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2 -amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2 -amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the bona fide secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patients.

Gastric cancer is a leading cause of cancer-related mortality, and chemotherapeutic options are currently limited. PIM1 kinase, an oncogene that promotes tumorigenesis in several cancer types, might represent a novel therapeutic target in gastric cancer.We studied the expression and genomic status of PIM1 in human primary gastric normal and tumor tissue samples by immunohistochemistry and array-based comparative genomic hybridization (aCGH). To ascertain whether PIM1 expression predicted susceptibility to PIM1 kinase-specific inhibition, the cytotoxic effect of a previously reported PIM1-specific small molecular inhibitor (K00135) was investigated in two gastric cancer cell lines with high (IM95) and undetectable (NUGC-4) PIM1 expression levels.PIM1 expression was exclusively nuclear in normal gastric epithelial cells, while aberrant expression/localization (decreased nuclear and/or increased cytoplasmic expression) was observed in 75.6% (68/90) of the human gastric cancer tissue samples, with a significant inverse correlation between nuclear and cytoplasmic expression levels. Clinicopathological analyses revealed that decreased nuclear PIM1 expression correlated with poorer survival and greater depth of tumor invasion, while increased cytoplasmic PIM1 expression correlated inversely with the presence of lymphovascular invasion. High-level PIM1 amplification was identified in 10.5% of gastric cancers by aCGH. K00135 impaired the survival of IM95, while it had no significant effect on NUGC-4 survival.Our findings demonstrate the clinical and therapeutic relevance of PIM1 in gastric cancers, and suggest that PIM1 represents a potential therapeutic target.

Objective: Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated. Methods: Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearesttemplate prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182. Results: The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8 + T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death. Conclusion: A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease., published_or_final_version

Citrullinaemia is a urea cycle defect that results from a deficiency of the enzyme arginosuccinate synthetase. Type 1 disease is diagnosed in childhood, whereas Type 2 disease is adult onset. We report the outcome of a patient with citrullinemia Type 2 who received a liver transplant at our center and the implications of this diagnosis in liver transplantation.

The use of rapamycin and thalidomide in a patient with metastatic gastroesophageal carcinoma, which led to disease stability associated with a significant tumor marker response and improved clinical quality of life, is reported.

Conflicting data on the clinicopathological characteristics as well as prognosis and survival of signet ring cell (SRC) and mucinous adenocarcinomas (MA) of the colorectum persist.Consecutive patients (2,764) with sporadic colorectal cancer from 1999 to 2005 were evaluated. The clinicopathological characteristics of these patients were reviewed. Univariate analysis was performed, and survival curves were constructed using the Kaplan-Meier method. Multivariate analysis assessed independent prognostic factors.The incidence of MA and SRC is 6% and 1.1%, respectively. MA and SRC tend to occur in patients agedor=50 years (SRC 33%, MA 22%, ordinary adenocarcinomas (OA) 14%, p = 0.001) and are more commonly right-sided (MA 30%, SRC 27%, OA 19%, p = 0.001) than OA. SRC tend to be poorly differentiated (SRC 77%, MA 26%, OA 6%, p0.0001) and have a higher risk of recurrence (SRC 40%, MA 26%, OA 6%, p0.0001). SRC and MA are more likely to have locally advanced lesions (T3/T4; SRC 100%, MA 90%, OA 83%, p = 0.002) and lymph node metastases (SRC 89%, MA 61%, OA 52%, p0.0001) and present with an advanced stage at diagnosis (stage III/IV SRC 94%, MA 67%, OA 56%, p0.0001). SRC has poorer 5-year cancer-specific survival (CSS; 11.1%, 95% confidence interval (CI) 0-22.9%) compared with MA (46.8%, 95% CI 38.6-55.0%) and OA (58.7%, 95% CI 56.5-60.9%, p0.001). In a multivariate analysis, SRC is an independent poor prognostic factor (HR 1.9, 95% CI 1.1-3.0) but MA is not.SRC and MA demonstrate clinicopathologic characteristics suggestive of a different biology compared with OA. In our dataset, SRC has a significantly poorer CSS whereas survival rates for MA are similar to OA. These characteristics are similar in both Asian and Western studies reported. Asian reports however suggest a lower incidence of MA.

Background: Somatic mutations are attractive therapeutic targets for “individualized neoantigen vaccines” because of lack of host central tolerance and reduced risk of autoimmunity. Here, we perform large-scale-omic analyses to assess the neoantigen landscape of colorectal cancer (CRC), a cancer largely refractory to immune-checkpoint inhibition. Methods: We performed whole genome sequencing (WGS) (60x tumor, 30x normal) and deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 158 colorectal cancers of which 32 are microsatellite instability high (MSI-H) tumours and 126 are microsatellite stable (MSS). Whole exome sequencing (200x tumor, 100x normal) was also performed on 120 tumours. HLA typing, somatic mutations, gene expression and neoepitope predictions were computationally evaluated. Inferred HLA-A alleles were orthogonally validated with Pacbio long-read sequencing. Tissue microarrays (TMAs) with tumour core, tumour edge and normal adjacent tissue of these 158 CRCs were constructed. Histopathological analyses using multiplex immunohistochemistry (mIHC) to simultaneously evaluate 7 markers, i.e. cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, have been performed. Results: The most common HLAs were, by allele count: A*11:01: 56; A*33:03: 38; B*58:01: 33; B*46:01: 29; B*40:01: 26; C*01:02: 41; C*07:02: 33. Inferred HLA-A alleles from WGS data was largely concordant (>90%) with Pacbio long-read sequencing. There were a median of 2,850 (1229-6909) [MSI] & 213 (27-13,835) [MSS] coding variants, from which 10,487 (4,307-27,365) [MSI] & 726.5 (50-59,096) [MSS] possible neoepitopes were derived, after accounting for epitope processing, the normal proteome and general population variome based on dbSNP, Of these, 5,707 (2,608-15,218) [MSI] & 320 (14-25,243) [MSS] neoepitopes are expressed (based on RNA-Seq). Epitope prediction algorithms revealed a median of 423 (17-1,056) [MSI] & 26 (0-1,102) [MSS] bound & expressed neoepitopes. 5 MSS tumors did not have any predicted bound nor expressed neoepitopes, 112 of 126 (89%) of MSS tumors had at least 5 predicted bound, expressed neoepitopes. Histopathological correlations between extent of immune infiltrates in fixed tissues, tumour PD-L1 expression and neoantigen burden is ongoing. Conclusions: There is substantial variability in the neoantigen landscape amongst MSI & MSS CRCs. MSI contains multiple-fold higher neo-antigens. Amongst MSS tumours, 89% of patients have at least 5 predicted bound and expressed neo-epitopes that could be targeted in neoantigen-based vaccines for personalized immunotherapy. Citation Format: Si-Lin Koo, Joe Poh Sheng Yeong, Andy Nguyen, Clarinda Wei Ling Chua, J Zachary Sanborn, Steve Benz, Wah Siew Tan, Choong Leong Tang, Su Yan, Min Hoe Chew, Brian Goh, Chung Yip Chan, Xiao Qing Koh, Alexander Lezhava, Tony Kiat Hon Lim, Shahrooz Rabizadeh, Anders Skanderup, Iain Beehuat Tan. Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5725.

Background: Colorectal cancer (CRC) is a cancer largely refractory to immune checkpoint inhibition. There is substantial interpatient molecular heterogeneity in colorectal cancer reported from studies on tumor RNA and studies on immunohistochemical analyses of fixed tumor tissue. Several major transcriptomic analyses from microarrays and RNA-seq data have identified major transcriptomic subtypes and major activated pathways and deconvoluted cell-type enrichments. Immunohistochemical (IHC) analyses of formalin-fixed, paraffin-embedded (FFPE) tissue microarrays have identified several different histomorphologic/spatial patterns of immune infiltrates in the tumor microenvironment. Here, we perform large-scale -omic analyses on tumor RNA and multiplex IHC simultaneously on 158 Asian colorectal cancers. Methods: We performed whole-genome sequencing (WGS) (60x tumor, 30x normal) and deep whole-transcriptomic sequencing (RNA-seq) (∼200x106 reads per tumor) on 158 colorectal cancers. To evaluate the spatial patterns of the tumor microenvironment, we constructed a tissue microarray comprising the tumor core, tumor edge and normal adjacent tissue of these 158 CRCs. We performed H&E analyses of the TMA and multiplex immunohistochemistry to simultaneously evaluate 7 markers, i.e., cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, using the an Opal Multiplex fIHC kit. Images were acquired using a Vectra 3 pathology imaging system microscope (PerkinElmer, Waltham, MA, USA). Results: 32 are microsatellite instability high (MSI-H) tumors and 126 are microsatellite stable. (MSS). The major transcriptomic subtypes (Consensus molecular subtypes (CMS 1-4) and CRC assigner (Goblet-like, Enterocyte, Stem-like, Inflammatory, Transit-amplifying subtypes) were identified with good concordance between both classification systems. CMS1 and Inflammatory subtypes were enriched amongst MSI-H tumors. Major oncogenic pathway activations (RAS, Wnt), cell-cycle and inflammatory signatures (interferon-rich) were also identified across the populations. We deconvoluted cell-type enrichment scores from the transcriptomic data to identify different cell-type enrichment patterns across the cohort. On the TMAs, we identified cell type populations and immune infiltrate patterns in the tumor core and invasive edge across the cohort. Correlations across these analyses will be presented at the meeting. Conclusions: There is substantial interindividual variability in the transcriptomic landscape and spatial patterns of immune cell infiltrates in CRCs. Citation Format: Clarinda Wei Chua, Engin Cukuroglu, Elisa Fontana, Si Lin Koo, Joe Poh Yeong, Andy Nguyen, J. Zachary Sanborn, Steve Benz, Emile John Tan, Ronnie Mathew, Ee-Lin Toh, Sarah Boon Ng, Tony Kiat Lim, Anders Jacobsen Skanderup, Shahrooz Rabizadeh, Anguraj Sadanandam, Jonathan Göke, Iain Bee Tan. Tumor whole-transcriptome sequencing and multiplex immunohistochemistry of immune cell populations in 158 Asian colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5693.

BACKGROUND:Assessment of severity of liver fibrosis is essential in the management of non-alcoholic fatty liver disease (NAFLD). Second Harmonic Generation (SHG) microscopy is a novel optical tissue imaging system that provides automated quantification of fibrosis based on unique architectural features of collagen. This study aims to develop and validate a SHG-based index for automated staging of liver fibrosis in patients with NAFLD. METHODS:SHG microscopy was performed on archived liver biopsy specimens from 83 patients with NAFLD. A unique algorithm was developed to identify specific SHG parameters that correlated with fibrosis stage. The accuracy of the algorithm was compared against clinical assessment by experienced liver histopathologists using the Brunt fibrosis staging and further validated using the leave-one-out cross-validation method. RESULTS:Mean age of the study cohort was 51.8 ± 11.7 years, with 41% males. A fibrosis index (SHG B-index) was developed comprising 14 unique SHG-based collagen parameters that correlated with severity of NAFLD fibrosis in a continuous fashion. The SHG B-index had excellent correlation with Brunt fibrosis stage (Spearman's correlation 0.820, p1.76 had an overall diagnostic accuracy of 98.5% for prediction of presence of bridging fibrosis (Brunt stage ≥3) with sensitivity of 87.5%, specificity 98.0%, positive predictive value 96.6% and negative predictive value 92.6%. CONCLUSION:The SHG B-index is a unique SHG-based index that provides accurate automated assessment of fibrosis stage in NAFLD patients.

e15597Background: Responses to anti-PD1 is ~35% in microsatellite unstable (MSI-h) and near 0% in microsatellite stable (MSS) Colorectal cancer (CRC). Understanding the composition of immune cell p...

3612Background: Brain metastases (BM) from colorectal cancer (CRC) are a rare but increasing event as patients survive longer. While human epidermal growth factor receptor 2 (HER2) gene amplificati...

Purpose To assess the efficacy of endovenous laser therapy (EVLT) in the treatment of lower-limb venous ulcers secondary to venous reflux. Materials and Methods Forty-four of 139 patients referred for EVLT from January 2004 to August 2007 had nonhealing venous ulcers. Preprocedural duplex ultrasound (US) was performed to document saphenous venous reflux secondary to saphenofemoral/saphenopopliteal junction incompetence, deep venous insufficiency, and deep vein thrombosis. Follow-up intervals were within 1 week, monthly until ulcer healing, and every 6 months thereafter. Mean follow-up period was 35.8 months (range, 8.1–59.3 months). Results Mean great saphenous vein (GSV) diameter and length treated were 9.9 mm (range, 5.5–16.0 mm) and 36.7 cm (range, 20.0–60.0 cm). Mean laser energy used was 3,292 J (range, 1,392–4,971 J). Mean energy deposited per centimeter of vein was 93.6 J/cm (range, 45.2–182.0 J/cm). Mean laser time was 232 seconds (range, 99–347 sec). Fifteen patients with follow-up duplex US had no GSV flow at 6 months, with nonvisualization indicating complete obliteration. Ulcer healing occurred as early as 1 week after the procedure in some patients. Cumulative healing rates at 1, 3, 6, and 12 months were 82.1%, 92.5%, 92.5%, and 97.4%, respectively. No ulcer had recurred at 1 year, but ulcers recurred in five patients at 14, 14, 23, 35, and 52 months after EVLT, respectively. One patient with a nonhealing ulcer 2 years after treatment developed well differentiated squamous cell carcinoma. Conclusions Ulcer epithelization occurred with continued GSV occlusion and loss of flow. Most ulcers healed within 3 months with no recurrence at 1 year. Nonhealing ulcers should undergo biopsy to exclude malignant transformation.

Aim Solitary caecal ulcer syndrome is rare. We describe our experience of 10 patients with the condition. Method A prospectively collected database of patients undergoing colonoscopy or surgery with histology reporting a solitary caecal ulcer was reviewed from 1999 to 2008. Patients with known carcinoma of the colon, cytomegalovirus infection, amoebiasis, inflammatory bowel disease, immunosuppression and history of nonsteroidal anti-inflammatory drug use were excluded. Results Ten patients were found to have a solitary caecal ulcer. All were of Chinese ethnicity, of median age 61 years. The most common presenting symptoms were haematochezia and right-sided abdominal pain. Histological findings included ulceration sharing some features of solitary rectal ulcer syndrome, but with differences to suggest a different aetiology. Conclusion Solitary caecal ulcer syndrome should be included in the differential diagnosis of lower gastrointestinal haemorrhage, right iliac fossa pain or when computed tomography imaging demonstrates caecal wall thickening. The diagnosis can only be made on histopathological examination.

TPS401 Background: The detection of androgen-receptor splice variant 7 (AR-V7) mRNA transcript in circulating tumor cells (CTCs) of progressive metastatic castration-resistant prostate cancer (mCRPC) patients is associated with resistance to abiraterone and enzalutamide. This finding was validated by other studies that found no association between the presence of AR-V7 transcripts and response to both docetaxel and cabazitaxel. The results suggest that AR-V7 could represent a biomarker to guide treatment selection in mCRPC. However, data in Asian population is limited. This multinational study aims to prospectively investigate the incidence and prevalence of ARV7 in Asian mCRPC patients. This is also the first study to evaluate 3 different detection platforms and help provide guidance on which will be the best platform to carry forward in terms of clinical utility. Methods: 101 Asian subjects with histologically confirmed mCRPC whom are going to be treated with AR-directed therapies (abiraterone, enzalutamide) or taxane chemotherapy (docetaxel) will be enrolled from Singapore, Malaysia, Thailand, Hong Kong and Japan. Subjects will be excluded if they plan to receive additional concurrent anticancer therapies. Peripheral-blood samples will be obtained at baseline and at the time of progression. We will use 3 methods to analyse the samples: (i) CTC enrichment platform followed by automated immunofluorescent staining for DNA, cytokeratins, CD45 (lymphocyte common antigen), and AR-V7; (ii) CTC enrichment platform followed by reverse-transcription polymerase chain reaction analysis; and (iii) the AdnaTest platform for molecular characterization of CTCs. Subjects will be followed up to 2 years. The clinical outcomes of AR-V7-positive and AR-V7-negative patients will be compared. PSA response rates will be compared using the Fisher exact test. Time-to-event outcomes (PFS, OS) will be evaluated using Kaplan-Meier analysis, and survival time differences will be compared using log-rank test. Cox regression analyses will be used to assess the effect of AR-V7 status in predicting clinical outcomes. The study is in progress; 32 of 101 planned patients have been enrolled at the end of September 2017.

TPS542 Background: In recent years, Yttrium-90 (Y90) trans-arterial radioembolization (TARE) has emerged as a therapeutic option for intermediate stage hepatocellular carcinoma (HCC). Cancer immunotherapy targeting tumour immune evasion has shown remarkable successes in various cancers. Nivolumab, an immune checkpoint inhibitor of programmed death 1 (PD1), has demonstrated encouraging activity in advanced stage HCC. Similarly, chronic hepatitis B virus (HBV) infection, a strong risk factor for HCC, is characterized by immune escape mechanisms. We hypothesize that TARE will stimulate tumor and/or HBV specific T cell responses that can be boosted using nivolumab. We thus propose an open label phase 2 trial investigating the combination of TARE with nivolumab in BCLC intermediate stage HCC. Methods: Eligible patients (pts) have ECOG performance status ≤ 2, Child-Pugh A score, intermediate stage HCC planned for TARE according to institutional practice with adequate organ function. Pts will be treated with TARE followed by nivolumab 240mg, 21 days after TARE and every 2 weeks thereafter. Pre- and on-treatment tumor biopsies will be taken. Primary end-point is response rate (RR) of combinational TARE and nivolumab. Key secondary end points are: progression free survival, overall survival, safety and quality of life using FACT-HEP score and EORTC QLQ-C30. Exploratory objectives are to evaluate how tumor PD L-1 expression, HCC mutational burden and blood lymphocyte activation/phenotypic profiles correlate with tumor response. Where possible, serial changes in antigen-specific T cell responses to HBV and/or tumour antigens will also be assessed. This study aims to enroll 40 pts as calculated using the Simon two-stage optimal design with 80% power and one sided significance level of 0.05. This will assess whether the addition of nivolumab improves the RR of TARE at 8 weeks from 21% to 41%. Patient accrual was initiated in December 2016 and as of September 2017, 9 pts have been treated. Clinical trial information: 03033446.

572 Background: Comprehensive molecular profiling of CRC can inform treatment decisions by identifying patient subgroups at varying risks of death. Microsatellite instability (MSI) is prognostic in CRC and is used to select patients for immunotherapy. High tumor mutational burden (TMB) is associated with genomic instability and is prognostic in melanoma. Expression of p16 protein is prognostic in many tumor types. We used proteomic and genomic profiling to measure MSI, TMB and p16 in CRC tumors and to assess associations with patient survival. Methods: In archived clinical samples of CRC, 76 proteins were quantitated with mass spectrometry-based proteomics. MSI was measured by WGS and RNA-seq; unstable loci were quantified in tumor and normal samples. Cutoffs were derived via ROC analysis: high TMB was defined as > 4.5 somatic mutations per megabase; p16 as > 108 amol/ug. Patients were grouped by microsatellite status (MSI vs. microsatellite stable [MSS]), TMB (high vs. low), and p16 protein expression level. Survival curves were compared with the Mantel-Cox log-rank test. Results: Of 145 samples, 39 (27%) had high TMB and 29 (20%) had MSI. Patients with MSI tumors had longer OS than patients with MSS tumors (HR: 0.096; p = 0.003). Similarly, patients with high TMB had longer OS than those with low TMB (HR: 0.076; p < 0.001). High p16 expression was prognostic of poor survival (HR: 2.874; p = 0.019). Among patients with MSS tumors or low TMB, those with low p16 levels had longer OS than patients with high p16 (HR: 0.257; p = 0.002 and HR: 0.249; p = 0.002, for MSS and low TMB, respectively). A combination of MSS, low TMB, and low p16 also differentiated between long and short survivors (HR: 0.249; p = 0.002). These associations remained after adjustment for tumor sidedness. Further analyses of clinical correlates will be presented. Conclusions: A combination of MSS, low TMB and low p16 expression characterized a subset of patients with longer survival. This is important because patients with MSS tumors have limited treatment options but may respond to CDK4/6 inhibitors due to low p16 expression. Molecular profiling of CRC may identify patient subgroups with a relatively poor prognosis who could benefit from personalized therapy.

Background/Aims: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the two most common primary malignant liver tumours in children. Hepatic progenitor cells have been described and can be stained with K19, EpCAM and CD117. We investigated the morphology and staining patterns of primary liver tumours in Asian children. Methods: Four pathologists studied slides from 39 paediatric patients from Vietnam and Korea aged 8 months to 16 years. We performed immunohistochemical stains for K19, EpCAM and CD117, and polymerase chain reaction for tissue hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. Results: There was agreement on the diagnosis of 24 cases of HCC and 10 cases of HB (one recurrent case). The diagnosis was split for six cases (HCC/HB). All 20 cases of HCC tested were HBV DNA+ and HCV RNA−. All nine cases of HB tested were HBV DNA−, while one was HCV RNA+. Of four HCC/HB cases tested, three were HBV DNA+ and all were HCV RNA−. By immunohistochemistry, 8/24 (33%) cases of HCC were K19+ and 18/24 (75%) were EpCAM+, 5/10 (50%) cases of HB were K19+ and 7/10 (70%) were EpCAM+ and 3/6 (50%) cases of HCC/HB were K19+ and 5/6 (83%) were EpCAM+. CD117 was negative in all 38 cases tested. Paediatric HCC has a morphology different from adult HCC, sometimes resembling HB, and a larger proportion of paediatric tumours have progenitor cell features. Conclusions: HB and HCC in children may represent malignant transformation at an early stage in the cellular lineage and often arise from hepatic progenitor cells.

Summary Background Free fat grafts have an unpredictable survival rate, which may be dependent on host bed vascularity. Therefore, the authors hypothesized that the presence of growth factors in platelet-rich plasma (PRP), may enhance free fat graft survival. Methods Free fat grafts and autologous PRP were harvested from a healthy female and processed using the Coleman technique and the Medtronic ® Magellan™ system respectively. The experiment comprised two groups of 12 nude mice each with injection of free fat grafts into the scalp. The experimental group comprised the combination of 0.8ml of free fat graft and 0.2ml of PRP. The control group comprised the combination of 0.8ml of free fat graft and 0.2ml of normal saline. The mice were euthanized after 16 weeks and the fat grafts explanted and measured for weight and volume. Histology was performed with Oil Red O stain. Statistical analysis of the weight and volume in between groups was performed using the independent samples T-test (SPSS v11). The Mann-Whitney test was used to compare the ranking of six histological parameters between the two groups. Results The mean weight and volume for the experimental arm were 0.503g and 0.545ml respectively. The mean weight and volume for the control arm were 0.500g and 0.541ml respectively. The weight, volume and histological parameters between the two groups were not statistically significant. A mouse from each group died of unknown causes. Conclusion PRP did not enhance free fat graft survival in the nude mouse.

Summary Background Transient elastography (TE) is a reliable non-invasive predictor of hepatic fibrosis, but data on TE in Asians are limited. Aim To evaluate prospectively the accuracy of TE for diagnosis of hepatic fibrosis in Asians compared with APRI (aspartate transaminase to platelet ratio index). Methods One hundred and twenty consecutive patients who underwent liver biopsy were enrolled. TE (Fibroscan) was performed by two independent operators. Fibrosis was graded by two independent pathologists using the METAVIR classification. Area under receiver operating curves (AUROC) were used to evaluate the accuracy of TE and APRI in diagnosing significant fibrosis (F ≥ 2) and cirrhosis (F4). Results Predominant aetiologies were hepatitis B (48%), non-alcoholic steatohepatitis (14%) and hepatitis C (8%). TE was unsuccessful in five patients (4.2%) because of small inter-costal space (three patients), obesity and ascites. There was good correlation between TE and fibrosis (r = 0.606). AUROC for diagnosis of significant fibrosis was 0.856 (95% CI 0.779–0.932) for TE and 0.673 (95% CI 0.568–0.777) for APRI. AUROC for diagnosis of cirrhosis was 0.924 (95% CI 0.857–0.990) for TE and 0.626 (95% CI 0.437–0.815) for APRI. Optimal TE value was 9.0 kPa for diagnosis of significant fibrosis and 16.0 kPa for cirrhosis with specificity/sensitivity/PPV/NPV/accuracy of 82.6%/85.2%/80.9%/86.7%/84.1% and 88.9%/82.7%/32.0%/98.8%/83.2%, respectively. Conclusions Transient elastography is a reliable predictor of hepatic fibrosis in Asians. Failure of TE in Asians is commonly because of small inter-costal space. TE is superior to APRI for non-invasive diagnosis of hepatic fibrosis and cirrhosis.

Biphasic ductal and myoepithelial lesions of the breast are uncommon. A majority of these rare lesions behave in a benign fashion. Malignancy is rare, and preferentially involves the epithelial component. Malignancy in both epithelial and myoepithelial components is even rarer. We report such a case, employing the use of a range of cytokeratins and myoepithelial markers to delineate the extent of each component. In addition, we apply the relatively novel technique of multi-immunolabeling combined with Adobe Photoshop imaging to highlight the different components of the neoplasm on the same tissue section. These ancillary tests can provide much needed information about the contribution of epithelial or myoepithelial components to malignant tumors, hence providing the gateway to further study into their histogenesis and natural history.

Background 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. Case 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. Conclusion 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.

Purpose The recent discovery and characterization of an oncogenic ROS1 gene rearrangement has raised significant interest because small molecule inhibitors are effective in these tumors. The aim of this study was to determine frequency and clinicopathological features associated with ROS1 rearrangement in patients with cholangiocarcinoma (CCA). Materials and Methods A total of 261 patients who underwent surgery for CCA between October 1997 and August 2013 were identified from an international, multi-institutional database. ROS1 rearrangement was evaluated by break-apart fluorescence in situ hybridization using tissue microarrays of these patients. Results Of 261 CCA evaluated, three cases (1.1%) showed ROS1 rearrangement by fluorescence in situ hybridization (FISH), all of which were derived from intrahepatic origin. ROS1 protein expression was observed in 38 samples (19.1%). Significantly larger tumor size was observed in ROS1 immunohistochemistry (IHC)–negative patients compared with ROS1 IHC–positive patients. ROS1 FISH–positive patients had a single tumor with a median size of 4 cm and well-to-moderate differentiation. Overall, there was no difference in terms of baseline characteristics, overall survival, and recurrence-free survival between ROS1-positive and -negative patients. Conclusion ROS1 rearrangement was detected in 1.1% of CCA patients. Although rare, conduct of clinical trials using ROS1 inhibitors in these genetically unique patients is warranted.

Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra-epithelial (ie)ILC1-like cells that represent a broader category of NK cells in mucosal and non-mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals and tissues. It also provides a global, comprehensive, and detailed description of ILC heterogeneity in humans across patients and tissues.

Background Despite advances in therapeutics, outcomes for hepatocellular carcinoma (HCC) remain poor and there is an urgent need for efficacious systemic therapy. Unfortunately, drugs that are successful in preclinical studies often fail in the clinical setting, and we hypothesize that this is due to functional differences between primary tumors and commonly used preclinical models. In this study, we attempt to answer this question by comparing tumor morphology and gene expression profiles between primary tumors, xenografts and HCC cell lines. Methods Hep G2 cell lines and tumor cells from patient tumor explants were subcutaneously (ectopically) injected into the flank and orthotopically into liver parenchyma of Mus Musculus SCID mice. The mice were euthanized after two weeks. RNA was extracted from the tumors, and gene expression profiling was performed using the Gene Chip Human Genome U133 Plus 2.0. Principal component analyses (PCA) and construction of dendrograms were conducted using Partek genomics suite. Results PCA showed that the commonly used HepG2 cell line model and its xenograft counterparts were vastly different from all fresh primary tumors. Expression profiles of primary tumors were also significantly divergent from their counterpart patient-derived xenograft (PDX) models, regardless of the site of implantation. Xenografts from the same primary tumors were more likely to cluster together regardless of site of implantation, although heat maps showed distinct differences in gene expression profiles between orthotopic and ectopic models. Conclusions The data presented here challenges the utility of routinely used preclinical models. Models using HepG2 were vastly different from primary tumors and PDXs, suggesting that this is not clinically representative. Surprisingly, site of implantation (orthotopic versus ectopic) resulted in limited impact on gene expression profiles, and in both scenarios xenografts differed significantly from the original primary tumors, challenging the long-held notion that orthotopic PDX model is the gold standard preclinical model for HCC. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1814-8) contains supplementary material, which is available to authorized users.

Background GI stromal tumours (GISTs) are clinically heterogenous exhibiting varying degrees of disease aggressiveness in individual patients. Objectives We sought to identify genetic alterations associated with high-risk GIST, explore their molecular consequences, and test their utility as prognostic markers. Designs Exome sequencing of 18 GISTs was performed (9 patients with high-risk/metastatic and 5 patients with low/intermediate-risk), corresponding to 11 primary and 7 metastatic tumours. Candidate alterations were validated by prevalence screening in an independent patient cohort (n=120). Functional consequences of SETD2 mutations were investigated in primary tissues and cell lines. Transcriptomic profiles for 8 GISTs (4 SETD2 mutated, 4 SETD2 wild type) and DNA methylation profiles for 22 GISTs (10 SETD2 mutated, 12 SETD2 wild type) were analysed. Statistical associations between molecular, clinicopathological factors, and relapse-free survival were determined. Results High-risk GISTs harboured increased numbers of somatic mutations compared with low-risk GISTs (25.2 mutations/high-risk cases vs 6.8 mutations/low-risk cases; two sample t test p=3.1×10 −5 ). Somatic alterations in the SETD2 histone modifier gene occurred in 3 out of 9 high-risk/metastatic cases but no low/intermediate-risk cases. Prevalence screening identified additional SETD2 mutations in 7 out of 80 high-risk/metastatic cases but no low/intermediate-risk cases (n=29). Combined, the frequency of SETD2 mutations was 11.2% (10/89) and 0% (0/34) in high-risk and low-risk GISTs respectively. SETD2 mutant GISTs exhibited decreased H3K36me3 expression while SETD2 silencing promoted DNA damage in GIST-T1 cells. In gastric GISTs, SETD2 mutations were associated with overexpression of HOXC cluster genes and a DNA methylation signature of hypomethylated heterochromatin. Gastric GISTs with SETD2 mutations, or GISTs with hypomethylated heterochromatin, showed significantly shorter relapse-free survival on univariate analysis (log rank p=4.1×10 −5 ). Conclusions Our data suggest that SETD2 is a novel GIST tumour suppressor gene associated with disease progression. Assessing SETD2 genetic status and SETD2 -associated epigenomic phenotypes may guide risk stratification and provide insights into mechanisms of GIST clinical aggressiveness.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

Background Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC. Results High depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis. Conclusions Our analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0589-1) contains supplementary material, which is available to authorized users.

There is an error in the order of authors. The correct order is: Soo Fan Ang1*, Elizabeth Shu-Hui Ng1, Huihua Li2,3, Yu-Han Ong1, Su Pin Choo1, Joanne Ngeow1, Han Chong Toh1, Kiat Hon Lim4, Hao Yun Yap5, Chee Kiat Tan6, London Lucien Peng Jin Ooi7, Peng Chung Cheow7, Alexander Yaw Fui Chung7, Pierce Kah Hoe Chow8,9, Kian Fong Foo1, Min-Han Tan1* 1 Division of Medical Oncology, National Cancer Centre Singapore, Singapore, Republic of Singapore, 2 Health Services Research, Singapore General Hospital, Singapore, Republic of Singapore, 3 Centre for Quantitative Medicine, Duke-National University of Singapore Graduate Medical School, Singapore, Republic of Singapore, 4 Department of Pathology, Singapore General Hospital, Singapore, Republic of Singapore, 5 Department of General Surgery, Singapore General Hospital, Singapore, Republic of Singapore, 6 Department of Gastroenterology and Hepatology, Singapore General Hospital, Singapore, Republic of Singapore, 7 Department of Hepatobiliary and Transplant Surgery, Singapore General Hospital, Singapore, Republic of Singapore, 8 Division of Surgical Oncology, National Cancer Centre Singapore, Singapore, Republic of Singapore, 9 Office of Clinical Sciences, Duke-National University of Singapore Graduate Medical School, Singapore, Republic of Singapore.

Background and Aims Surgery is the primary curative option in patients with hepatocellular carcinoma (HCC). Current prognostic models for HCC are developed on datasets of primarily patients with advanced cancer, and may be less relevant to resectable HCC. We developed a postoperative nomogram, the Singapore Liver Cancer Recurrence (SLICER) Score, to predict outcomes of HCC patients who have undergone surgical resection. Methods Records for 544 consecutive patients undergoing first-line curative surgery for HCC in one institution from 1992–2007 were reviewed, with 405 local patients selected for analysis. Freedom from relapse (FFR) was the primary outcome measure. An outcome-blinded modeling strategy including clustering, data reduction and transformation was used. We compared the performance of SLICER in estimating FFR with other HCC prognostic models using concordance-indices and likelihood analysis. Results A nomogram predicting FFR was developed, incorporating non-neoplastic liver cirrhosis, multifocality, preoperative alpha-fetoprotein level, Child-Pugh score, vascular invasion, tumor size, surgical margin and symptoms at presentation. Our nomogram outperformed other HCC prognostic models in predicting FFR by means of log-likelihood ratio statistics with good calibration demonstrated at 3 and 5 years post-resection and a concordance index of 0.69. Using decision curve analysis, SLICER also demonstrated superior net benefit at higher threshold probabilities. Conclusion The SLICER score enables well-calibrated individualized predictions of relapse following curative HCC resection, and may represent a novel tool for biomarker research and individual counseling.

OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

Sir,Gastric adenocarcinoma (GAC) is perceived as a progression from normal epithelium to dysplasia to frankly invasive carcinoma. The pre‐invasive lesions of intestinal type of GAC have been well r...