The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

BACKGROUND: Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. METHODS: Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. RESULTS: Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. CONCLUSIONS: Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

Homologous Recombination Repair Gene Mutations to Predict Olaparib Plus Bevacizumab Efficacy in the First-Line Ovarian Cancer PAOLA-1/ENGOT-ov25 Trial.

PURPOSE: The PAOLA-1/ENGOT-ov25 trial of maintenance olaparib plus bevacizumab for newly diagnosed advanced high-grade ovarian cancer demonstrated a significant progression-free survival (PFS) benefit over placebo plus bevacizumab, particularly in patients with homologous recombination deficiency (HRD)-positive tumors. We explored whether mutations in non-BRCA1 or BRCA2 homologous recombination repair (non-BRCA HRRm) genes predicted benefit from olaparib plus bevacizumab in PAOLA-1. METHODS: Eight hundred and six patients were randomly assigned (2:1). Tumors were analyzed using the Myriad MyChoice HRD Plus assay to assess non-BRCA HRRm and HRD status; HRD was based on a genomic instability score (GIS) of ≥ 42. In this exploratory analysis, PFS was assessed in patients harboring deleterious mutations using six non-BRCA HRR gene panels, three devised for this analysis and three previously published. RESULTS: The non-BRCA HRRm prevalence ranged from 30 of 806 (3.7%) to 79 of 806 (9.8%) depending on the gene panel used, whereas 152 of 806 (18.9%) had non-BRCA1 or BRCA2 mutation HRD-positive tumors. The majority of tumors harboring non-BRCA HRRm had a low median GIS; however, a GIS of > 42 was observed for tumors with mutations in five HRR genes (BLM, BRIP1, RAD51C, PALB2, and RAD51D). Rates of gene-specific biallelic loss were variable (0% to 100%) in non-BRCA HRRm tumors relative to BRCA1-mutated (99%) or BRCA2-mutated (86%) tumors. Across all gene panels tested, hazard ratios for PFS (95% CI) ranged from 0.92 (0.51 to 1.73) to 1.83 (0.76 to 5.43). CONCLUSION: Acknowledging limitations of small subgroup sizes, non-BRCA HRRm gene panels were not predictive of PFS benefit with maintenance olaparib plus bevacizumab versus placebo plus bevacizumab in PAOLA-1, irrespective of the gene panel tested. Current gene panels exploring HRRm should not be considered a substitute for HRD determined by BRCA mutation status and genomic instability testing in first-line high-grade ovarian cancer.

Treatment for Barrett's oesophagus.

This review has been withdrawn because it has been split into the following reviews: 'Pharmaceutical interventions for Barrett's oesophagus' and 'Endoscopic interventions for Barrett's oesophagus'.

Lectins in Cervical Screening.

Cervical screening in low-resource settings remains an unmet need. Lectins are naturally occurring sugar-binding glycoproteins whose binding patterns change as cancer develops. Lectins discriminate between dysplasia and normal tissue in several precancerous conditions. We explored whether lectins could be developed for cervical screening via visual inspection. Discovery work comprised lectin histochemistry using a panel of candidate lectins on fixed-human cervix tissue (high-grade cervical intraepithelial neoplasia (CIN3, n = 20) or normal (n = 20)), followed by validation in a separate cohort (30 normal, 25 CIN1, 25 CIN3). Lectin binding was assessed visually according to staining intensity. To validate findings macroscopically, near-infra red fluorescence imaging was conducted on freshly-resected cervix (1 normal, 7 CIN3), incubated with topically applied fluorescently-labelled lectin. Fluorescence signal was compared for biopsies and whole specimens according to regions of interest, identified by the overlay of histopathology grids. Lectin histochemistry identified two lectins-wheat germ agglutinin (WGA) and Helix pomatia agglutinin (HPA)-with significantly decreased binding to CIN3 versus normal in both discovery and validation cohorts. Findings at the macroscopic level confirmed weaker WGA binding (lower signal intensity) in CIN3 vs. normal for biopsies (p = 0.0308) and within whole specimens (p = 0.0312). Our findings confirm proof-of-principle and indicate that WGA could potentially be developed further as a probe for high-grade cervical disease.

Genomic evidence supports a clonal diaspora model for metastases of esophageal adenocarcinoma.

The poor outcomes in esophageal adenocarcinoma (EAC) prompted us to interrogate the pattern and timing of metastatic spread. Whole-genome sequencing and phylogenetic analysis of 388 samples across 18 individuals with EAC showed, in 90% of patients, that multiple subclones from the primary tumor spread very rapidly from the primary site to form multiple metastases, including lymph nodes and distant tissues-a mode of dissemination that we term 'clonal diaspora'. Metastatic subclones at autopsy were present in tissue and blood samples from earlier time points. These findings have implications for our understanding and clinical evaluation of EAC.

Barrett's oESophagus trial 3 (BEST3): study protocol for a randomised controlled trial comparing the Cytosponge-TFF3 test with usual care to facilitate the diagnosis of oesophageal pre-cancer in primary care patients with chronic acid reflux.

BACKGROUND: Early detection of oesophageal cancer improves outcomes; however, the optimal strategy for identifying patients at increased risk from the pre-cancerous lesion Barrett's oesophagus (BE) is not clear. The Cytosponge, a novel non-endoscopic sponge device, combined with the biomarker Trefoil Factor 3 (TFF3) has been tested in four clinical studies. It was found to be safe, accurate and acceptable to patients. The aim of the BEST3 trial is to evaluate if the offer of a Cytosponge-TFF3 test in primary care for patients on long term acid suppressants leads to an increase in the number of patients diagnosed with BE. METHODS: The BEST3 trial is a pragmatic multi-site cluster-randomised controlled trial set in primary care in England. Approximately 120 practices will be randomised 1:1 to either the intervention arm, invitation to a Cytosponge-TFF3 test, or the control arm usual care. Inclusion criteria are men and women aged 50 or over with records of at least 6 months of prescriptions for acid-suppressants in the last year. Patients in the intervention arm will receive an invitation to have a Cytosponge-TFF3 test in their general practice. Patients with a positive TFF3 test will receive an invitation for an upper gastro-intestinal endoscopy at their local hospital-based endoscopy clinic to test for BE. The primary objective is to compare histologically confirmed BE diagnosis between the intervention and control arms to determine whether the offer of the Cytosponge-TFF3 test in primary care results in an increase in BE diagnosis within 12 months of study entry. DISCUSSION: The BEST3 trial is a well-powered pragmatic trial testing the use of the Cytosponge-TFF3 test in the same population that we envisage it being used in clinical practice. The data generated from this trial will enable NICE and other clinical bodies to decide whether this test is suitable for routine clinical use. TRIAL REGISTRATION: This trial was prospectively registered with the ISRCTN Registry on 19/01/2017, trial number ISRCTN68382401 .

Selection and Application of Tissue microRNAs for Nonendoscopic Diagnosis of Barrett's Esophagus.

BACKGROUND & AIMS: MicroRNA (miRNA) is highly stable in biospecimens and provides tissue-specific profiles, making it a useful biomarker of carcinogenesis. We aimed to discover a set of miRNAs that could accurately discriminate Barrett's esophagus (BE) from normal esophageal tissue and to test its diagnostic accuracy when applied to samples collected by a noninvasive esophageal cell sampling device. METHODS: We analyzed miRNA expression profiles of 2 independent sets of esophageal biopsy tissues collected during endoscopy from 38 patients with BE and 26 patients with normal esophagus (controls) using Agilent microarray and Nanostring nCounter assays. Consistently up-regulated miRNAs were quantified by real-time polymerase chain reaction in esophageal tissues collected by Cytosponge from patients with BE vs without BE. miRNAs were expressed from plasmids and antisense oligonucleotides were expressed in normal esophageal squamous cells; effects on proliferation and gene expression patterns were analyzed. RESULTS: We identified 15 miRNAs that were significantly up-regulated in BE vs control tissues. Of these, 11 (MIR215, MIR194, MIR 192, MIR196a, MIR199b, MIR10a, MIR145, MIR181a, MIR30a, MIR7, and MIR199a) were validated in Cytosponge samples. The miRNAs with the greatest increases in BE tissues (7.9-fold increase in expression or more, P < .0001: MIR196a, MIR192, MIR194, and MIR215) each identified BE vs control tissues with area under the curve (AUC) values of 0.82 or more. We developed an optimized multivariable logistic regression model, based on expression levels of 6 miRNAs (MIR7, MIR30a, MIR181a, MIR192, MIR196a, and MIR199a), that identified patients with BE with an AUC value of 0.89, 86.2% sensitivity, and 91.6% specificity. Expression level of MIR192, MIR196a, MIR199a, combined that of trefoil factor 3, identified patients with BE with an AUC of 0.93, 93.1% sensitivity, and 93.7% specificity. Hypomethylation was observed in the promoter region of the highly up-regulated cluster MIR192-MIR194. Overexpression of these miRNAs in normal esophageal squamous cells increased their proliferation, via GRHL3 and PTEN signaling. CONCLUSIONS: In analyses of miRNA expression patterns of BE vs non-BE tissues, we identified a profile that can identify Cytosponge samples from patients with BE with an AUC of 0.93. Expression of MIR194 is increased in BE samples via epigenetic mechanisms that might be involved in BE pathogenesis.

Glucose transporter 1 expression as a marker of prognosis in oesophageal adenocarcinoma.

BACKGROUND: The current TNM staging system for oesophageal adenocarcinoma (OAC) has limited ability to stratify patients and inform clinical management following neo-adjuvant chemotherapy and surgery. RESULTS: Functional genomic analysis of the gene expression data using Gene Set Enrichment Analysis (GSEA) identified GLUT1 as putative prognostic marker in OAC.In the discovery cohort GLUT1 positivity was observed in 114 patients (80.9%) and was associated with poor overall survival (HR 2.08, 95% CI 1.1-3.94; p=0.024) following multivariate analysis. A prognostic model incorporating GLUT1, CRM and nodal status stratified patients into good, intermediate and poor prognosis groups (p< 0.001) with a median overall survival of 16.6 months in the poorest group.In the validation set 182 patients (69.5%) were GLUT1 positive and the prognostic model separated patients treated with neo-adjuvant chemotherapy and surgery (p<0.001) and surgery alone (p<0.001) into three prognostic groups. PATIENTS AND METHODS: Transcriptional profiling of 60 formalin fixed paraffin-embedded (FFPE) biopsies was performed. GLUT1 immunohistochemical staining was assessed in a discovery cohort of 141 FFPE OAC samples treated with neo-adjuvant chemotherapy and surgery at the Northern Ireland Cancer Centre from 2004-2012. Validation was performed in 262 oesophageal adenocarcinomas collected at four OCCAMS consortium centres. The relationship between GLUT1 staining, T stage, N stage, lymphovascular invasion and circumferential resection margin (CRM) status was assessed and a prognostic model developed using Cox Proportional Hazards. CONCLUSIONS: GLUT1 staining combined with CRM and nodal status identifies a poor prognosis sub-group of OAC patients and is a novel prognostic marker following potentially curative surgical resection.

Risk stratification of Barrett's oesophagus using a non-endoscopic sampling method coupled with a biomarker panel: a cohort study.

BACKGROUND: Barrett's oesophagus predisposes to adenocarcinoma. However, most patients with Barrett's oesophagus will not progress and endoscopic surveillance is invasive, expensive, and fraught by issues of sampling bias and the subjective assessment of dysplasia. We investigated whether a non-endoscopic device, the Cytosponge, could be coupled with clinical and molecular biomarkers to identify a group of patients with low risk of progression suitable for non-endoscopic follow-up. METHODS: In this multicentre cohort study (BEST2), patients with Barrett's oesophagus underwent the Cytosponge test before their surveillance endoscopy. We collected clinical and demographic data and tested Cytosponge samples for a molecular biomarker panel including three protein biomarkers (P53, c-Myc, and Aurora kinase A), two methylation markers (MYOD1 and RUNX3), glandular atypia, and TP53 mutation status. We used a multivariable logistic regression model to compute the conditional probability of dysplasia status. We selected a simple model with high classification accuracy and applied it to an independent validation cohort. The BEST2 study is registered with ISRCTN, number 12730505. FINDINGS: The discovery cohort consisted of 468 patients with Barrett's oesophagus and intestinal metaplasia. Of these, 376 had no dysplasia and 22 had high-grade dysplasia or intramucosal adenocarcinoma. In the discovery cohort, a model with high classification accuracy consisted of glandular atypia, P53 abnormality, and Aurora kinase A positivity, and the interaction of age, waist-to-hip ratio, and length of the Barrett's oesophagus segment. 162 (35%) of 468 of patients fell into the low-risk category and the probability of being a true non-dysplastic patient was 100% (99% CI 96-100) and the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 0% (0-4). 238 (51%) of participants were classified as of moderate risk; the probability of having high-grade dysplasia was 14% (9-21). 58 (12%) of participants were classified as high-risk; the probability of having non-dysplastic endoscopic biopsies was 13% (5-27), whereas the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 87% (73-95). In the validation cohort (65 patients), 51 were non-dysplastic and 14 had high-grade dysplasia. In this cohort, 25 (38%) of 65 patients were classified as being low-risk, and the probability of being non-dysplastic was 96·0% (99% CI 73·80-99·99). The moderate-risk group comprised 27 non-dysplastic and eight high-grade dysplasia cases, whereas the high-risk group (8% of the cohort) had no non-dysplastic cases and five patients with high-grade dysplasia. INTERPRETATION: A combination of biomarker assays from a single Cytosponge sample can be used to determine a group of patients at low risk of progression, for whom endoscopy could be avoided. This strategy could help to avoid overdiagnosis and overtreatment in patients with Barrett's oesophagus. FUNDING: Cancer Research UK.

The Discovery and Validation of Biomarkers for the Diagnosis of Esophageal Squamous Dysplasia and Squamous Cell Carcinoma.

The 5-year survival rate of esophageal cancer is less than 10% in developing countries, where more than 90% of these cancers are esophageal squamous cell carcinomas (ESCC). Endoscopic screening is undertaken in high incidence areas. Biomarker analysis could reduce the subjectivity associated with histologic assessment of dysplasia and thus improve diagnostic accuracy. The aims of this study were therefore to identify biomarkers for esophageal squamous dysplasia and carcinoma. A publicly available dataset was used to identify genes with differential expression in ESCC compared with normal esophagus. Each gene was ranked by a support vector machine separation score. Expression profiles were examined, before validation by qPCR and IHC. We found that 800 genes were overexpressed in ESCC compared with normal esophagus (P < 10(-5)). Of the top 50 genes, 33 were expressed in ESCC epithelium and not in normal esophagus epithelium or stroma using the Protein Atlas website. These were taken to qPCR validation, and 20 genes were significantly overexpressed in ESCC compared with normal esophagus (P < 0.05). TNFAIP3 and CHN1 showed differential expression with IHC. TNFAIP3 expression increased gradually through normal esophagus, mild, moderate and severe dysplasia, and SCC (P < 0.0001). CHN1 staining was rarely present in the top third of normal esophagus epithelium and extended progressively towards the surface in mild, moderate, and severe dysplasia, and SCC (P < 0.0001). Two novel promising biomarkers for ESCC were identified, TNFAIP3 and CHN1. CHN1 and TNFAIP3 may improve diagnostic accuracy of screening methods for ESCC. Cancer Prev Res; 9(7); 558-66. ©2016 AACR.

Molecular effects of Lapatinib in the treatment of HER2 overexpressing oesophago-gastric adenocarcinoma.

BACKGROUND: Lapatinib, a dual EGFR and HER2 inhibitor has shown disappointing results in clinical trials of metastatic oesophago-gastric adenocarcinomas (OGAs), and in vitro studies suggest that MET, IGFR, and HER3 confer resistance. This trial applied Lapatinib in the curative neoadjuvant setting and investigated the feasibility and utility of additional endoscopy and biopsy for assessment of resistance mechanisms ex vivo and in vivo. METHODS: Patients with HER2 overexpressing OGA were treated for 10 days with Lapatinib monotherapy, and then in combination with three cycles of Oxaliplatin and Capecitabine before surgery. Endoscopic samples were taken for molecular analysis at: baseline including for ex vivo culture +/- Lapatinib to predict in vivo response, post-Lapatinib monotherapy and at surgery. Immunohistochemistry (IHC) and proteomic analysis was performed to assess cell kinetics and signalling activity. RESULTS: The trial closed early (n=10) due to an anastomotic leak in two patients for which a causative effect of Lapatinib could not be excluded. The reduction in Phosphorylated-HER2 (P-HER2) and P-EGFR in the ex vivo-treated biopsy demonstrated good correlation with the in vivo response at day 10. Proteomic analysis pre and post-Lapatinib demonstrated target inhibition (P-ERBB2, P-EGFR, P-PI3K, P-AKT, and P-ERK) that persisted until surgery. There was also significant correlation between the activation of MET with the level of P-Erk (P=0.0005) and P-PI3K : T-PI3K (total PI3K) ratio (P=0.0037). There was no significant correlation between the activation status of IGFR and HER3 with downstream signalling molecules. CONCLUSIONS: Additional endoscopy and biopsy sampling for multiple biomarker endpoints was feasible and confirmed in vitro data that MET is likely to be a significant mechanism of Lapatinib resistance in vivo.

Analysis of dysplasia in patients with Barrett's esophagus based on expression pattern of 90 genes.

BACKGROUND & AIMS: Diagnoses of dysplasia, based on histologic analyses, dictate management decisions for patients with Barrett's esophagus (BE). However, there is much intra- and inter-observer variation in identification of dysplasia-particularly low-grade dysplasia. We aimed to identify a biomarker that could be used to assign patients with low-grade dysplasia to a low- or high-risk group. METHODS: We performed a stringent histologic assessment of 150 frozen esophageal tissues samples collected from 4 centers in the United Kingdom (from 2000 through 2006). The following samples with homogeneous diagnoses were selected for gene expression profiling: 28 from patients with nondysplastic BE, 10 with low-grade dysplasia, 13 with high-grade dysplasia (HGD), and 8 from patients with esophageal adenocarcinoma. A leave-one-out cross-validation analysis was used identify a gene expression signature associated with HGD vs nondysplastic BE. Functional pathways associated with gene signature sets were identified using the MetaCore analysis. Gene expression signature sets were validated using gene expression data on BE and esophageal adenocarcinoma accessed through National Center for Biotechnology Information Gene Expression Omnibus, as well as a separate set of samples (n = 169) collected from patients who underwent endoscopy in the United Kingdom or the Netherlands and analyzed histologically. RESULTS: We identified an expression pattern of 90 genes that could separate nondysplastic BE tissues from those with HGD (P < .0001). Genes in a pathway regulated by retinoic acid-regulated nuclear protein made the largest contribution to this gene set (P < .0001); the transcription factor MYC regulated at least 30% of genes within the signature (P < .0001). In the National Center for Biotechnology Information Gene Expression Omnibus validation set, the signature separated nondysplastic BE samples from esophageal adenocarcinoma samples (P = .0012). In the UK and Netherlands validation cohort, the signature identified dysplastic tissues with an area under the curve value of 0.87 (95% confidence interval: 0.82-0.93). Of samples with low-grade dysplasia (LGD), 64% were considered high risk according to the 90-gene signature; these patients had a higher rate of disease progression than those with a signature categorized as low risk (P = .047). CONCLUSIONS: We identified an expression pattern of 90 genes in esophageal tissues of patients with BE that was associated with low- or high-risk for disease progression. This pattern might be used in combination with histologic analysis of biopsy samples to stratify patients for treatment. It would be most beneficial for analysis of patients without definitive evidence of HGD but for whom early endoscopic intervention is warranted.

Evaluation of a minimally invasive cell sampling device coupled with assessment of trefoil factor 3 expression for diagnosing Barrett's esophagus: a multi-center case-control study.

BACKGROUND: Barrett's esophagus (BE) is a commonly undiagnosed condition that predisposes to esophageal adenocarcinoma. Routine endoscopic screening for BE is not recommended because of the burden this would impose on the health care system. The objective of this study was to determine whether a novel approach using a minimally invasive cell sampling device, the Cytosponge, coupled with immunohistochemical staining for the biomarker Trefoil Factor 3 (TFF3), could be used to identify patients who warrant endoscopy to diagnose BE. METHODS AND FINDINGS: A case-control study was performed across 11 UK hospitals between July 2011 and December 2013. In total, 1,110 individuals comprising 463 controls with dyspepsia and reflux symptoms and 647 BE cases swallowed a Cytosponge prior to endoscopy. The primary outcome measures were to evaluate the safety, acceptability, and accuracy of the Cytosponge-TFF3 test compared with endoscopy and biopsy. In all, 1,042 (93.9%) patients successfully swallowed the Cytosponge, and no serious adverse events were attributed to the device. The Cytosponge was rated favorably, using a visual analogue scale, compared with endoscopy (p < 0.001), and patients who were not sedated for endoscopy were more likely to rate the Cytosponge higher than endoscopy (Mann-Whitney test, p < 0.001). The overall sensitivity of the test was 79.9% (95% CI 76.4%-83.0%), increasing to 87.2% (95% CI 83.0%-90.6%) for patients with ≥3 cm of circumferential BE, known to confer a higher cancer risk. The sensitivity increased to 89.7% (95% CI 82.3%-94.8%) in 107 patients who swallowed the device twice during the study course. There was no loss of sensitivity in patients with dysplasia. The specificity for diagnosing BE was 92.4% (95% CI 89.5%-94.7%). The case-control design of the study means that the results are not generalizable to a primary care population. Another limitation is that the acceptability data were limited to a single measure. CONCLUSIONS: The Cytosponge-TFF3 test is safe and acceptable, and has accuracy comparable to other screening tests. This test may be a simple and inexpensive approach to identify patients with reflux symptoms who warrant endoscopy to diagnose BE.

The combination of autofluorescence endoscopy and molecular biomarkers is a novel diagnostic tool for dysplasia in Barrett's oesophagus.

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<0.001). Compared with the standard protocol, this panel had equal sensitivity for HGD/EC, with a 4.5-fold reduction in the number of biopsies. In an independent cohort of patients, the panel had a sensitivity and specificity for HGD/EC of 100% and 85%, respectively. CONCLUSIONS: A three-biomarker panel on a small number of AFI-targeted biopsies provides an accurate and objective diagnosis of dysplasia in BO. The clinical implications have to be studied further.

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis.

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n=66) and high-grade dysplasia (HGD; n=43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett's esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies.

Gastro-esophageal reflux disease symptoms and demographic factors as a pre-screening tool for Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) occurs as consequence of reflux and is a risk factor for esophageal adenocarcinoma. The current "gold-standard" for diagnosing BE is endoscopy which remains prohibitively expensive and impractical as a population screening tool. We aimed to develop a pre-screening tool to aid decision making for diagnostic referrals. METHODOLOGY/PRINCIPAL FINDINGS: A prospective (training) cohort of 1603 patients attending for endoscopy was used for identification of risk factors to develop a risk prediction model. Factors associated with BE in the univariate analysis were selected to develop prediction models that were validated in an independent, external cohort of 477 non-BE patients referred for endoscopy with symptoms of reflux or dyspepsia. Two prediction models were developed separately for columnar lined epithelium (CLE) of any length and using a stricter definition of intestinal metaplasia (IM) with segments ≥ 2 cm with areas under the ROC curves (AUC) of 0.72 (95%CI: 0.67-0.77) and 0.81 (95%CI: 0.76-0.86), respectively. The two prediction models included demographics (age, sex), symptoms (heartburn, acid reflux, chest pain, abdominal pain) and medication for "stomach" symptoms. These two models were validated in the independent cohort with AUCs of 0.61 (95%CI: 0.54-0.68) and 0.64 (95%CI: 0.52-0.77) for CLE and IM ≥ 2 cm, respectively. CONCLUSIONS: We have identified and validated two prediction models for CLE and IM ≥ 2 cm. Both models have fair prediction accuracies and can select out around 20% of individuals unlikely to benefit from investigation for Barrett's esophagus. Such prediction models have the potential to generate useful cost-savings for BE screening among the symptomatic population.

A genome-wide association study identifies new susceptibility loci for esophageal adenocarcinoma and Barrett's esophagus.

Esophageal adenocarcinoma is a cancer with rising incidence and poor survival. Most such cancers arise in a specialized intestinal metaplastic epithelium, which is diagnostic of Barrett's esophagus. In a genome-wide association study, we compared esophageal adenocarcinoma cases (n = 2,390) and individuals with precancerous Barrett's esophagus (n = 3,175) with 10,120 controls in 2 phases. For the combined case group, we identified three new associations. The first is at 19p13 (rs10419226: P = 3.6 × 10(-10)) in CRTC1 (encoding CREB-regulated transcription coactivator), whose aberrant activation has been associated with oncogenic activity. A second is at 9q22 (rs11789015: P = 1.0 × 10(-9)) in BARX1, which encodes a transcription factor important in esophageal specification. A third is at 3p14 (rs2687201: P = 5.5 × 10(-9)) near the transcription factor FOXP1, which regulates esophageal development. We also refine a previously reported association with Barrett's esophagus near the putative tumor suppressor gene FOXF1 at 16q24 and extend our findings to now include esophageal adenocarcinoma.

A systematic approach to therapeutic target selection in oesophago-gastric cancer.

OBJECTIVE: The success of personalised therapy depends on identification and inhibition of the oncogene(s) on which that tumour is dependent. We aimed to determine whether a receptor tyrosine kinase (RTK) array could be used to select the most effective therapeutic strategies in molecularly heterogeneous oesophago-gastric adenocarcinomas. DESIGN: Gene expression profiling from oesophago-gastric tumours (n=75) and preinvasive stages (n=57) identified the active signalling pathways, which was confirmed using immunohistochemistry (n=434). RTK arrays on a cell line panel (n=14) determined therapeutic targets for in vitro cytotoxic testing. Feasibility of this personalised approach was tested in tumour samples (n=46). RESULTS: MAPK was the most frequently activated pathway (32/75 samples (42.7%)) with progressive enrichment in preinvasive disease stages (p<0.05) and ERK phosphorylation in 148/434 (34.3%) independent samples. Cell lines displayed a range of RTK activation profiles. When no RTKs were activated, tyrosine kinase inhibitors (TKIs) and a Mek inhibitor were not useful (MKN1). In lines with a dominant phosphorylated RTK (OE19, MKN45 and KATOIII), selection of this TKI or Mek in nM concentrations induced cytotoxicity and inhibited Erk and Akt phosphorylation. In cells lines with complex activation profiles (HSC39 and OE33), a combination of TKIs or Mek inhibition (in nM concentrations) was necessary for cytotoxicity and inhibition of Erk and Akt phosphorylation. Human tumours demonstrated diverse activation profiles and 65% of cases had two or more active RTKs. CONCLUSIONS: The MAPK pathway is commonly activated in oesophago-gastric cancer following activation of a variety of RTKs. Molecular phenotyping can inform a rational choice of targeted therapy.

Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus.

BACKGROUND & AIMS: The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS: We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia ≥ 6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS: Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS: Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Evidence for a functional role of epigenetically regulated midcluster HOXB genes in the development of Barrett esophagus.

Barrett esophagus (BE) is a human metaplastic condition that is the only known precursor to esophageal adenocarcinoma. BE is characterized by a posterior intestinal-like phenotype in an anterior organ and therefore it is reminiscent of homeotic transformations, which can occur in transgenic animal models during embryonic development as a consequence of mutations in HOX genes. In humans, acquired deregulation of HOX genes during adulthood has been linked to carcinogenesis; however, little is known about their role in the pathogenesis of premalignant conditions. We hypothesized that HOX genes may be implicated in the development of BE. We demonstrated that three midcluster HOXB genes (HOXB5, HOXB6, and HOXB7) are overexpressed in BE, compared with the anatomically adjacent normal esophagus and gastric cardia. The midcluster HOXB gene signature in BE is identical to that seen in normal colonic epithelium. Ectopic expression of these three genes in normal squamous esophageal cells in vitro induces markers of intestinal differentiation, such as KRT20, MUC2, and VILLIN. In BE-associated adenocarcinoma, the activation midcluster HOXB gene is associated with loss of H3K27me3 and gain of AcH3, compared with normal esophagus. These changes in histone posttranslational modifications correlate with specific chromatin decompaction at the HOXB locus. We suggest that epigenetically regulated alterations of HOX gene expression can trigger changes in the transcriptional program of adult esophageal cells, with implications for the early stages of carcinogenesis.