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OBJECTIVE: Endoscopic surveillance for Barrett's oesophagus (BO) is limited by sampling error and the subjectivity of diagnosing dysplasia. We aimed to compare a biomarker panel on minimal biopsies directed by autofluorescence imaging (AFI) with the standard surveillance protocol to derive an objective tool for dysplasia assessment. DESIGN: We performed a cross-sectional prospective study in three tertiary referral centres. Patients with BO underwent high-resolution endoscopy followed by AFI-targeted biopsies. 157 patients completed the biopsy protocol. Aneuploidy/tetraploidy; 9p and 17p loss of heterozygosity; RUNX3, HPP1 and p16 methylation; p53 and cyclin A immunohistochemistry were assessed. Bootstrap resampling was used to select the best diagnostic biomarker panel for high-grade dysplasia (HGD) and early cancer (EC). This panel was validated in an independent cohort of 46 patients. RESULTS: Aneuploidy, p53 immunohistochemistry and cyclin A had the strongest association with dysplasia in the per-biopsy analysis and, as a panel, had an area under the receiver operating characteristic curve of 0.97 (95% CI 0.95 to 0.99) for diagnosing HGD/EC. The diagnostic accuracy for HGD/EC of the three-biomarker panel from AFI+ areas was superior to AFI- areas (p

Aims—Pyothorax associated lymphoma (PAL) occurs in a clinical setting of longstanding pyothorax or chronic inflammation of the pleura. Like primary effusion lymphoma, it has an association with Epstein-Barr virus (EBV), and is confined to the pleural cavity, but has differing morphological and phenotypic features. Human herpesvirus 8 (HHV-8) has been consistently reported in primary effusion lymphoma. This study examines the immunophenotype of two European cases of PAL, investigates the presence of HHV-8 and its expression profile, and assesses whether PAL is similar to other effusion lymphomas. Methods—Material was obtained from two European cases of PAL. Immunocytochemical analysis was performed using antibodies against CD45, CD20, CD79a, CD45RAA, CD3, CD43, CD45RO (UCHL1), CD30, BCL-2, CD68, epithelial membrane antigen (EMA), BCL-6, p53, Ki-67, κ light chain, λ light chain, and the EBV antigens latent membrane protein 1 (LMP-1) and EBV encoded nuclear antigen 2 (EBNA-2). The cases were examined for HHV-8 by means of polymerase chain reaction in situ hybridisation (PCR-ISH), solution phase PCR, in situ hybridisation (ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26) and viral (v) cyclin encoding regions. The expression profile of HHV-8 in PAL and in BC-1 and BC-3 cells was assessed by RNA TaqMan PCR to the HHV-8 genes encoding v-cyclin, v-IL-6, and G protein coupled receptor (GPCR). Results—Both cases expressed CD24, CD20, CD79a, BCL-2, light chain restriction, and high Ki-67 staining. EBV was identified by EBER-ISH in one case. HHV-8 was not identified by solution phase PCR, but was detected by PCR-ISH (sensitivity of 1 viral genome copy/cell) in 35% of the cells and by TaqMan PCR, which showed 50–100 HHV-8 copies/2000 cell genome equivalents (sensitivity of 1 viral genome in 106 contaminating sequences). HHV-8 v-IL-6, v-cyclin, and GPCR encoded transcripts were identified using RNA TaqMan PCR. v-IL-6 was high in PAL and in BC-1 and BC-3 cells. Conclusion—The presence of HHV-8 in one of two patients with PAL raises interesting questions in relation to the pathobiology of the condition. Clearly, the results indicate that HHV-8 is not an obligate pathogen, necessary for the effusion phenotype, but might contribute to it by its secretion of specific cytokines.

Background —Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposi9s sarcoma (KS), malignant lymphoma (Hodgkin9s and nonHodgkin9s), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. Aim —To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposi9s sarcoma development. Methods —A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). Results —Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. Conclusions —The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

AIMS: To determine whether Abl immunoreactivity correlates with grade and cell kinetics (apoptosis and mitosis) in chondrosarcoma. METHODS: Sections from 16 chondrosarcomas were stained immunohistochemically using a polyclonal antibody to the c-Abl/Bcr-Abl oncoprotein. Apoptotic indices and mitotic indices were assessed in all tumours. Sections from 24 paraffin wax blocks of human fetal rib (gestational ages, 15-42 weeks) were also stained to determine whether the Abl protein is synthesised consistently throughout endochondral ossification. RESULTS: Abl staining in immature fetal rib chondrocytes at all stages of development was predominantly nuclear, and 70% of cells showed moderate to strong staining. Abl immunoreactivity was minimal or absent in hypertrophic chondrocytes about to undergo apoptosis at the growth plate. There was strong Abl staining in grade 1 and grade 2 chondrosarcomas but staining was greatly reduced or absent in grade 3 chondrosarcomas. There was a very significant linear correlation between apoptotic index (mean, 0.68%; range, 0-3.2%) and mitotic index (mean, 0.23%; range, 0-0.9%), and both indices were significantly lower in grade 1 than in grade 2 and grade 3 chondrosarcomas. CONCLUSIONS: These data suggest that abl gene expression is associated with differentiation and apoptosis inhibition in fetal and neoplastic chondrocytes. However, these putative effects cannot be ascribed solely to the Abl protein, because several additional factors contribute to the regulation of both differentiation and apoptosis.

Introduction 1–3% of gastric cancers arise from hereditary predisposition syndromes. E-cadherin gene (CDH1) mutations are the best characterised of these and confer a life-time risk of approximately 80% for patients developing diffuse gastric cancer. Currently, the only definitive treatment available is prophylactic total gastrectomy (PTG). We present our experience of open PTG, focusing on the early outcomes. Method Twenty four consecutive patients with CDH 1 mutation underwent PTG from 2005 to 2014 (median age 33, range 22–51). All patients had at least one gastroscopy with biopsies taken according to the Cambridge Protocol and were assessed by a multi-disciplinary team within a high-volume Cancer Centre. All patients had an open posterior vagal sparing total gastrectomy with stapled oesophagojejunal anastomosis and Roux-en-Y reconstruction by a single surgeon. Twenty three patients had a D0.5 -1 lymphadenectomy; one patient with suspected invasive tumour had a D2 resection. Nasogastric tubes and abdominal drains were not used. Patients were mobilised on the first postoperative day with epidural analgesia. Oral fluids were restarted on postop day 3 and diet on day 5. Results There were two postoperative complications (8%) but no deaths; One patient had a jejuno-jejuno anastomotic leak 10 days after surgery requiring revisional surgery but made a full recovery. Another patient had bleeding which was managed conservatively with transfusion. Two patients developed anastomotic strictures and were successfully treated with endoscopic balloon dilatation. Median follow-up to date is 5 years (range: 3 months to 9 years). Histology showed scattered microscopic foci of intramucosal signet ring carcinoma (pT1a) in 23/24 patients (96%); the median number of foci was 7 (range: 1–182). No invasive tumours were found, all nodes were negative for tumour and all longitudinal margins were clear. The median lymph node yield was 5.5 (range: 1–26) and median hospital stay was 8 days (range: 6–10). Conclusion Open PTG can be performed with acceptable mortality and morbidity. Our challenge for the future is to offer minimally invasive PTG without increasing the risks. Disclosure of interest None Declared.

Introduction Barrett9s oesophagus (BO) increases the risk of developing oesophageal adenocarcinoma (OAC) by around 40-fold. OAC incidence rates have increased sixfold since the 1970s but the bleak prognosis for this disease is improved by early detection. The intermediate dysplastic stage between BO and OAC is the most reliable marker of progression. However the histological presence of dysplasia is subjective due to known sampling bias along with a high inter and intraobserver variability. Aberrant DNA methylation is shown to be characteristic of certain tumours and is known to occur early during transformation. We therefore hypothesise that DNA methylation changes take place early during carcinogenesis in BO patients which could be utilised as progression biomarkers. The study aims were therefore to identify genes showing a variation in methylation through progression and to then validate them on an independent dataset. Methods DNA methylation levels were assessed across 27 578 CpG loci covering 14 000 genes in 24 of each BO and OAC test samples using arrays. Both supervised clustering (GSEA software) and unsupervised clustering (R software) were used to identify genes with a statistically significant difference in methylation in BO versus OAC. Genes present in both the lists were selected for validation. Internal validation and external validation on an independent set of 48 BO, 31 BO with dysplasia and 68 OAC samples was carried out using pyrosequencing assays. Results Supervised clustering came up with 2822 hypermethylated and 645 hypomethylated genes (p≤0.001) and unsupervised with 1563 hypermethylated and 610 hypomethylated genes (p≤0.01). Genes successfully validated include RGN (senescence marker protein-30), SLC22A18 (tumour suppressing subtransferable candidate 5), PIGR (polymeric immunoglobulin receptor), GJA12 (gap junction protein, γ2) and TCEAL7 (transcription elongation factor A – like 7). After internal validation (spearman9s correlation p RGN (ANOVA p SLC22A18 (ANOVA p PIGR (ANOVA p GJA12 (ANOVA p TCEAL7 (ANOVA p=0.008, JT p=0.001). Conclusion High throughput DNA methylation screening using arrays and robust external validation has allowed us to identify novel genes having biomarker potential previously unknown to play a role in Barrett9s carcinogenesis. Further validation in an EDRN phase III study is underway.

Introduction Patients fulfilling the criteria for Hereditary Diffuse Gastric Cancer (HDGC) may undergo endoscopic surveillance to help detect microscopic foci of signet ring cells and guide decisions about prophylactic gastrectomy. Current guidelines suggest a minimum of 30 random gastric biopsies sampled annually in patients fulfilling HDGC criteria. However, the wide distribution and small size of malignant foci makes them difficult to identify. The authors report the first prospective study to examine whether tri-modal imaging can aid detection of microscopic foci. Methods Patients fulfilling the criteria for HDGC attended for trimodal imaging endoscopy. A strict protocol was used with N-acetyl cysteine as a mucolytic, the stomach was inspected with white light followed by autofluorescence (AFI) to highlight areas of concern which were then visualised using narrow band imaging (NBI). A single endoscopist performed all procedures and a second endoscopist confirmed all findings at the time. In addition to any targeted biopsies, 24 random biopsies (prepylorus × 4, antrum × 4, t-zone × 6, body × 4, fundus × 4 and cardia × 2) were taken in each case and all specimens were examined by an expert histopathologist. Results Between October 2007 to March 2009, a total of 19 patients (7 with confirmed genetic CDH1 mutation) underwent 27 endoscopic surveillance procedures. The most common abnormality seen was pale areas in the antrum (8/27). These could be appreciated with white light endoscopy and were further highlighted with AFI with 2 showing abnormal pit patterns on NBI. Additional targeted biopsies were taken in 16 procedures. A total of 9 cases were positive for signet cells (4 fundus, 3 transitional zone, 1 body and 1 unspecified) 7 of the 9 positive cases were picked up solely on random biopsies. The two positive targeted biopsies were from a pale area at the transitional zone in the same patient who did not wish to undergo gastrectomy. Conclusion Careful inspection with standard white light and multiple random biopsies will detect microscopic foci in these high risk patients. Trimodal imaging helps delineate pale areas of concern but did not pick up additional foci.

Introduction Esophageal adenocarcinoma (EAC) has one of the fastest rising incidences of any cancer in the western world. With a 5-year survival below 10% it is one of the most common causes of cancer death in US and UK. Currently little is understood about the genetic alterations that drive the development of OAC. Better understanding of these alterations may allow the development of novel therepeutic approaches Methods We have performed whole genome sequencing on 22 cases. Targeted amplicon resequencing of 27 recurrently mutated genes was performed on a validation cohort of 100 further oesophageal adenocarcinomas. Results In the discover set of 22 OACs we identified recurrent mutations (>3 tumours) in 31 genes including several implicated in tumorigenesis; TP53, CDKN2A, ARID1A. Strikingly in the validation cohort we observed that > 30% of EAC samples harbour mutation of one or both of the SWI/SNF complex members ARID1A and SMARCA4. In addition we identified highly recurrent mutations in several additional genes including TRIM58, SSTR4 and MYO18B. Conclusion Whole genome sequencing provides an unbiased screen of mutational architecture of OAC. This has allowed the identification of several recurrently mutated genes not previously implicated in this disease providing a unique insight to it’s pathogenesis Disclosure of Interest None Declared

Introduction Identifying Barrett’s oesophagus (BE) patients at risk of progressing to oesophageal adenocarcinoma (EA) remains a challenge. Diagnosis of low grade dysplasia is limited by considerable intra and inter-observer variability and inflammation in biopsy samples can lead to a diagnosis of indefinite for dysplasia. Immunostaining using molecular biomarkers would therefore be useful as a diagnostic adjunct in the assessment of dysplasia. The Aim of this study was to identify and validate a molecular biomarker that can objectively determine dysplasia status and thereby determine cancer risk in BE. Methods Biomarkers of interest were identified through mining of a microarray gene expression dataset from 59 oesophageal samples with strict consensus diagnosis by expert pathologists [21 BE with no dysplasia (NDBE), 10 BE with low grade dysplasia (LGD), 13 BE with high grade dysplasia (HGD) and 8 EA]. Subsequent validation was performed on a BE tissue microarray (TMA) (60 NDBE, 19 LGD and 29 HGD), and EA TMA (n = 278). Results Seventy eight genes were differentially expressed between NDBE and HGD. c-MYC was selected as a top target as the expression levels progressively increased with dysplasia stage and cancer with a fold change of 2.46 between NDBE and HGD. mRNA expression levels were significantly higher in HGD and EA compared to NDBE (p Table 1. Immunohistochemistry for c-MYC in oesophageal samples. NDBE: Non-dysplastic Barrett’s oesophagus, LGD: Low-grade dysplasia, HGD: High grade dysplasia. Conclusion Immunohistochemistry for c-MYC is a promising molecular biomarker in Barrett’s oesophagus. It could also enable better risk stratification in patients with samples in the LGD and indefinite for dysplasia categories. Disclosure of Interest None Declared.

Introduction Biomarkers have been proposed to improve risk stratification in Barrett9s oesophagus (BO), however molecular heterogeneity of BO can hamper detection of molecular changes in random biopsies. Use of Autofluorescence Imaging (AFI) within endoscopic Trimodal Imaging (ETMI) can improve dysplasia detection, but has high false positive rate. Aims of study were (a) validate biomarkers previously published in separate patient cohorts in single study (b) assess whether AFI can increase detection of biomarkers (c) combine ETMI and biomarkers to improve risk stratification of patients with BO. Methods Prospective European multicentre study. Each patient underwent ETMI with targeted biopsies on AFI positive (AFI+) areas and one AFI negative (AFI−) area, as well as random quadrantic biopsies. DNA content abnormalities (aneuploidy/tetraploidy); loss of heterozygosity (LOH) at 9p and 17p loci; RUNX3, HPP1 and p16 methylation; immunohistochemistry (IHC) for p53 and Cyclin A were tested on targeted biopsies. Each biomarker was correlated with the dysplasia and AFI status. Results 111 patients with 210 biopsy areas were included in the analysis (AFI+, n=120; AFI-, n=90). Univariate per-biopsy analysis showed that all biomarkers correlated with dysplasia (p Conclusion AFI increases detection rate for molecular biomarkers. A panel of 3 molecular biomarkers on a small number of AFI targeted biopsies can efficiently predict the dysplasia status and potentially inform therapeutic management of patients with BE. Competing interests None declared.

Introduction A less expensive and safer alternative to standard sedated endoscopy (SE) needs to be considered as a screening method to detect Barrett9s oesophagus (BE) in the population, with the aim of reducing the mortality associated with oesophageal adenocarcinoma. The Endosheath® transnasal oesophagoscope (TNE) can potentially offer a new alternative to conventional standard endoscopy in diagnosing Barrett9s oesophagus. The Endosheath® technology uses a sterile, disposable sheath which covers the ultra thin flexible oesophagoscope and isolates it from the patient. The oesophagoscope is placed in a new sheath prior to each procedure which obviates the need for machine washing and permits a quick turnaround. Aim: A pilot study to evaluate the efficacy of TNE in diagnosing BE compared with SE and to assess patient acceptability of TNE. Methods Patients referred for surveillance endoscopy for BE or a clinically indicated routine endoscopy were recruited to both TNE and SE in a randomised cross-over design. The interval between the procedures was at least 6 weeks. TNE findings of endoscopic BE, and presence of intestinal metaplasia (IM) on the biopsy samples were compared against SE, which was used as gold standard. A 10-point visual analogue scale (0 represented the worst experience and 10 the best experience) to assess the post-endoscopy experience and a single question addressing preference for endoscopy type were used to measure patient acceptability of the procedures. Results 15 patients completed the study, 10 males and 5 females with a mean age of 62.85 years (range 50–76 years). Nine of which were BE surveillance patients and six were referred for a clinically indicated routine endoscopy. Eight patients were randomised to the SE as the first procedure. All the 11 patients with an endoscopic diagnosis of BE on SE were accurately identified with the TNE (sensitivity 100%; specificity 100%). Biopsies were taken in all the 11 Barrett9s segments except in one Conclusion Endosheath® transnasal oesophagoscope is accurate in diagnosing endoscopic BE and can detect IM. It is better tolerated and preferred by patients, making it a useful screening tool for BE with potential for use in primary care. Competing interests None declared.

Introduction In order to detect all cases of Barrett9s oesophagus (BE) in the population, with the aim of reducing adenocarcinoma associated mortality, screening needs to be considered. Unsedated transnasal endoscopy (TNE) can potentially offer a safer and less expensive alternative to conventional sedated endoscopy (CE) for detecting BE. Aims (1) To evaluate the sensitivity and specificity of TNE in diagnosing BE compared with CE. (2) To assess patient acceptability of TNE for a future multicentre screening trial. Methods Patients with known BE under surveillance and those referred for routine endoscopy for other reasons underwent both TNE and CE in a randomised cross-over design. TNE findings of endoscopic BE, biopsy size and presence of intestinal metaplasia (IM) were compared against CE, which was used as gold standard. Pre and post endoscopy short-form spielberger state-trait questionnaires (SF6), a visual analogue scale and a single question addressing preference for endoscopy type were used to measure patient acceptability of the procedures. Results Of 57 patients randomised, 50 completed the study. All 22 BE cases with columnar lined epithelium (CLE) on CE were correctly diagnosed by TNE. Although the biopsy size of TNE was significantly smaller compared to CE (mean size 2.59 vs 4.74 p Conclusion TNE is accurate in diagnosing BE and detecting IM. Despite the smaller biopsy size, it detected an equal number of IM cases and showed potential to detect dysplasia. It was well tolerated and preferred by patients and may be an efficient screening method for BE.

The paper by C H Hawkes (Is multiple sclerosis a sexually transmitted infection?1) has caused predictable distress to people with multiple sclerosis (MS) and their families. Living with MS is a difficult enough experience without such sudden and avoidable alarm. The UK Multiple …

Twenty five patients with oliguric renal failure were treated by a combination of continuous arteriovenous haemofiltration and intermittent haemodialysis over 18 months. Haemofiltration was given for a mean of 6.6 days and the mean filtration volume was 6.0 1/day. Fourteen of the 25 patients survived beyond two months after the period of oliguria. Haemofiltration proved to be a simple and effective method of fluid removal; it allowed maintenance of stable fluid balance and permitted optimum nutrition during prolonged oliguria.