Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation.

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

Patients unfit for neoadjuvant therapy may still undergo resection of locally advanced esophageal or esophagogastric junctional cancer with acceptable oncological results.

Neoadjuvant chemotherapy (NA) is routinely offered to patients undergoing resection for locally advanced (≥cT3Nx or cTxN+) esophageal or esophagogastric junctional (EGJ) cancer in the United Kingdom. Patients with comorbidity precluding the use of NA can be considered for resection yet the effect of omitting NA on survival is unclear. METHODS: Retrospective review of prospectively collected clinical data from patients undergoing attempted curative therapy for ≥cT3Nx or cTxN+ esophageal or EGJ (Siewert type I-III) cancer between 2001 and 2013. RESULTS: NA was commenced in 289 patients and primarily comprised 2 cycles of cisplatin and 5-fluorouracil (264 patients, 91%). Surgery alone was planned for 82 patients with NA omitted due to comorbidity. Patients undergoing surgery alone were matched for clinical variables and stage with those undergoing NA but were significantly older (mean=8 y, P<0.001). NA was associated with an improved median overall survival of 28.7 months, compared with 20.9 months for patients undergoing surgery alone (P=0.008). Patients undergoing surgery alone had a 90-day postoperative mortality rate of 10% compared with 3% for those undergoing NA (P=0.011). In patients discharged postoperatively, the median overall survival benefit of NA was 2.7 months (P=0.048). Those 19% of patients experiencing a significant histologic response to NA demonstrated further improved survival. CONCLUSIONS: NA improves survival in patients undergoing resection for locally advanced esophageal or EGJ cancer; however, the median benefit is <3 months in patients discharged postoperatively. Patients precluded from NA achieve acceptable oncological results but experience a higher risk of perioperative mortality.

Quantitative Shotgun Proteomics Unveils Candidate Novel Esophageal Adenocarcinoma (EAC)-specific Proteins.

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches.

A rare case of primary seminoma of the small bowel with lymph node involvement.

INTRODUCTION: One thousand four hundred new cases of testicular cancer are diagnosed annually in the UK, with peak incidence in men aged 25-35 years old. Seminomas account for over 40% of cases. The involvement of the gastrointestinal tract with seminoma is unusual. CASE DESCRIPTION: We present a rare case of primary seminoma of the small bowel with lymph node involvement in a 30-year-old man who presented with iron deficiency anaemia and non-specific postprandial abdominal pain. A computed tomographic scan revealed small bowel wall thickening and mesenteric lymphadenopathy. Laparoscopic jejunal resection was performed and subsequent histology confirmed the diagnosis of primary seminoma of the small bowel. The patient also received three courses of cisplastin-based chemotherapy with good therapeutic outcome. DISCUSSION: The possible anatomical origins of the seminoma include: the small bowel itself, a result of anomalies during embryogenesis, location of the lymphatic drainage of the testis, an occult testicular metastasis or a viable metastasis from a primary testicular lesion which had already regressed. CONCLUSION: The investigation of unexplained iron deficiency anaemia in a young male patient requires full investigation of the entire gastrointestinal tract.

A 4-gene signature predicts survival of patients with resected adenocarcinoma of the esophagus, junction, and gastric cardia.

BACKGROUND & AIMS: The incidence of esophageal and junctional adenocarcinoma has increased 6-fold in the past 30 years and 5-year survival remains approximately 20%. Current staging is limited in its ability to predict survival which has ramifications for treatment choices. The aim of this study was to generate and validate a molecular prognostic signature for esophageal adenocarcinoma. METHODS: Gene expression profiling was performed and the resulting 42,000 gene signatures correlated with clinical and pathologic features for 75 snap-frozen esophageal and junctional resection specimens. External validation of selected targets was performed on 371 independent cases using immunohistochemistry to maximize clinical applicability. RESULTS: A total of 119 genes were associated significantly with survival and 270 genes with the number of involved lymph nodes. Filtering of these lists resulted in a shortlist of 10 genes taken forward to validation. Four genes proved to be prognostic at the protein level (deoxycytidine kinase [DCK], 3'-phosphoadenosine 5'-phosphosulfate synthase 2 [PAPSS2], sirtuin 2 [SIRT2], and tripartite motif-containing 44 [TRIM44]) and were combined to create a molecular prognostic signature. This 4-gene signature was highly predictive of survival in the independent external validation cohort (0/4 genes dysregulated 5-year survival, 58%; 95% confidence interval [CI], 36%-80%; 1-2/4 genes dysregulated 5-year survival, 26%; 95% CI, 20%-32%; and 3-4/4 genes dysregulated 5-year survival, 14%; 95% CI, 4%-24% (P = .001). Furthermore, this 4-gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (P = .013). CONCLUSIONS: This study has generated a clinically applicable prognostic gene signature that independently predicts survival in an external validation cohort and may inform management decisions.

Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers.

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett's esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPalpha, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine-cytokine receptor interactions and TGF-beta. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

Pathology findings and validation of gastric and esophageal cancer cases in a European cohort (EPIC/EUR-GAST).

OBJECTIVE: Cardia, non-cardia and intestinal and diffuse subtypes of gastric cancer may have different trends and etiological factors. However, the available information is not always collected in population cancer registries, and heterogeneous criteria have been applied for the histopathological classification of tumors. We describe the pathological features of incident gastric and esophageal cancers identified within the European Prospective Investigation into Cancer and Nutrition (EPIC). MATERIAL AND METHODS: In an investigation on gastric and esophageal cancer (EUR-GAST) in the EPIC project, a validation study of diagnoses reported by EPIC centers was conducted by a European panel of pathologists. Original pathology reports, stained slides of tumors and the respective paraffin blocks were requested from the centers. RESULTS: The whole series encompassed 467 cancer cases (gastric and esophageal cancers). Material was available for histopathological validation in 263 cases (56%); in the remaining cases, information was retrieved from the original reports (n=110; 24%) or codes provided by the EPIC centers (n=94; 20%). Among cases submitted to histopathological validation reported originally as unknown histotype or unknown site, a specific diagnosis was made in 95% and 74% of the cases, respectively. In cases for which only the original reports were available, the respective percentages were 46% and 67%. Gastric adenocarcinomas were classified according to site (cardia (29.4%), non-cardia (48.2%) and unknown (22.4%)) and histological type (intestinal (33.4%), diffuse (33.7%) and mixed, unclassified or unknown (32.9%)). Frequency of cardia was higher in Northern countries (35%) than in Mediterranean countries (18%). CONCLUSIONS: In addition to providing epidemiological data within the EPIC cohort on gastric and esophageal adenocarcinomas, the results reported here confirm the relevance of a validation study, notably for multicenter studies.

Retinoic acid-induced glandular differentiation of the oesophagus.

BACKGROUND: Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus. METHODS: RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 muM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. RESULTS: RA biosynthesis was increased in Barrett's oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. CONCLUSION: RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.

In vivo and in vitro evidence for transforming growth factor-beta1-mediated epithelial to mesenchymal transition in esophageal adenocarcinoma.

There is increasing evidence that epithelial to mesenchymal transition (EMT) is involved in cancer progression. Because local invasion and metastasis occurs early in the pathogenesis of esophageal adenocarcinoma, we hypothesized that EMT may be important in this disease. Using immunohistochemistry in a well-characterized set of adenocarcinoma tissues, we showed down-regulation of epithelial markers (E-cadherin and cytokeratin 18) and up-regulation of mesenchymal markers (vimentin and alpha-smooth muscle actin) with concomitant transforming growth factor-beta1 (TGF-beta1) expression at the invasive margin compared with the central tumor. A panel of esophageal cell lines was examined for the ability of TGF-beta1 to induce EMT in vitro. TE7 cells were selected as a model because TGF-beta1 (0-5 ng/mL) treatment induced morphologic and molecular expression changes suggestive of EMT. In TE7 cells, these TGF-beta1-induced changes were reversed by 100 ng/mL of bone morphogenetic protein 7 (BMP7), another member of the TGF-beta1 superfamily. EMT was mediated via canonical TGF-beta1 signaling with concomitant up-regulation of SMAD-interacting protein 1. Alterations in functional variables (aggregation, wounding, motility, and invasion) following TGF-beta1 treatment were consistent with a more invasive phenotype. These functional changes were reversed by BMP7 and SMAD4 RNA interference in vitro. These data suggest that TGF-beta1-mediated EMT may be relevant in esophageal carcinogenesis.

Detecting and quantifying apoptosis in tissue sections.

Various techniques exist for the identification of apoptosis in tissue sections or intact cells. The use of simple morphology, electron microscopy, DNA-end labeling techniques, and immunochemical methods are reviewed, with a particular emphasis on in situ end-labeling. The analysis of apoptotic cells and methods for their quantification are also discussed.

Surface expression of minichromosome maintenance proteins provides a novel method for detecting patients at risk for developing adenocarcinoma in Barrett's esophagus.

PURPOSE: The purpose is to determine whether a novel cell cycle marker, minichromosome maintenance protein 2 (Mcm2), predicted esophageal adenocarcinoma (AC) risk in Barrett's esophagus (BE) and whether this could be used in combination with a surface sampling method. EXPERIMENTAL DESIGN: Archival specimens [30 normal squamous esophagus (NE), 20 gastric antrum (GA), 13 duodenum (D2), 62 BE +/- dysplasia, and 16 (AC)] were stained for Mcm2. Sequential biopsies from nine patients who developed AC during surveillance were compared with 18 matched controls who did not progress. Prospective endoscopic cytological brushings (61 NE, 90 BE +/- dysplasia, and 11 AC) were scored as Mcm2 positive or negative. RESULTS: Mcm2 was not expressed on the luminal surface of NE, GA, and D2. In BE, the percentage of surface cells expressing Mcm2 correlated highly with the degree of dysplasia (P < 0.0001). In patients who developed AC, biopsies before dysplasia had higher Mcm2 expression than the matched control patients (mean, 28.4 and 3.4% positive cells, respectively, P < 0.0001). In the prospective cohort, the histopathological diagnosis of dysplasia or AC and the Mcm2-positive brushings were concordant in 91% of the patients (P < 0.005), and the results correlated with the frequency of cases with surface expression of Mcm2 in the retrospective study (P < 0.0001). CONCLUSIONS: Surface expression of Mcm2 can be used to detect dysplasia and AC, as well as patients with BE at risk for subsequent development of dysplasia and AC. A brushing technique combined with Mcm2 staining has the potential to be exploited in surveillance and screening protocols.