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Epitope mapping using BMP heterodimers (BMP4/7, BMP2/6 and BMP2/7).

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4+ malignancies. Mol Cancer Ther; 14(11); 2527–40. ©2015 AACR.

Supplementary file containing Supplementary materials and methods, supplementary figure legends and supplementary references.

Summary of epitope binning experiments of the VHHs and controls

The precursor metaplastic mucosal lesion that predisposes for esophageal adenocarcinoma is Barrett's esophagus. Because the signal transduction events that occur in Barrett's esophagus are poorly understood, this study aimed at generating a comprehensive description of cellular kinase activity in Barrett's esophagus, normal squamous esophagus, and gastric cardia to gain more insight into the pathogenesis of Barrett's esophagus. Peptide arrays, exhibiting 1,176 specific consensus sequences for protein kinases, were used to produce a global analysis of cellular kinase activity in biopsies of Barrett's esophagus, and results were compared with the neighboring cardia and squamous epithelia. Several differences in kinase activity using immunoblot analysis and enzyme activity assays were validated in biopsies of 27 Barrett's esophagus patients. Three unique kinome profiles are described and compared. We identified cascades of activated kinases showing that mitogen-activated protein kinase and epidermal growth factor receptor activity are both significantly altered in Barrett's esophagus compared with squamous and gastric cardia epithelia. Another novel finding is that the glycolysis pathway is significantly up-regulated in Barrett's esophagus, which is illustrated by an up-regulated pyruvate kinase activity. Here, the unique kinome profile of Barrett's esophagus is made available as a comprehensive database. Several signaling pathways are revealed as specifically expressed in Barrett's esophagus when compared with the adjacent normal epithelia. These unique findings provide novel insight in the pathogenesis of Barrett's esophagus that will ultimately help to resolve the increasing problem of Barrett's esophagus and prevention of esophageal adenocarcinoma. (Cancer Res 2006; 66(24): 11605-12)

Supplementary Tables 1-2 from Comparison of Kinome Profiles of Barrett's Esophagus with Normal Squamous Esophagus and Normal Gastric Cardia

Simple Summary Gene expression of esophageal adenocarcinoma is highly heterogeneous. In general, these cancers have poor prognosis and unpredictable responses to chemo- and radiotherapy. Investigating expression profiles from RNA from pre-treatment biopsies are highly attractive to investigate the existence of diverse biological groups and signatures associated with the clinical response to current treatment strategies. We identified and validated three distinct biological esophageal adenocarcinoma subgroups and identified immune signatures with association to therapy response using RNA sequencing. These findings aid in understanding biological mechanisms' underlying response to neo-adjuvant treatment. Esophageal adenocarcinoma (EAC) is a highly aggressive cancer and its response to chemo- and radiotherapy is unpredictable. EACs are highly heterogeneous at the molecular level. The aim of this study was to perform gene expression analysis of EACs to identify distinct molecular subgroups and to investigate expression signatures in relation to treatment response. In this prospective observational study, RNA sequencing was performed on pre-treatment endoscopic EAC biopsies from a discovery cohort included between 2012 and 2017 in one Dutch Academic Center. Four additional cohorts were analyzed for validation purposes. Unsupervised clustering was performed on 107 patients to identify biological EAC subgroups. Specific cell signaling profiles were identified and evaluated with respect to predicting response to neo-adjuvant chemo(radio)therapy. We identified and validated three distinct biological EAC subgroups, characterized by (1) p38 MAPK/Toll-like receptor signaling; (2) activated immune system; and (3) impaired cell adhesion. Subgroup 1 was associated with poor response to chemo-radiotherapy. Moreover, an immune signature with activated T-cell signaling, and increased number of activated CD4 T memory cells, neutrophils and dendritic cells, and decreased M1 and M2 macrophages and plasma cells, was associated with complete histopathological response. This study provides a novel molecular classification for EACs. EAC subgroup 1 proved to be more therapy-resistant, while immune signaling was increased in patients with complete response to chemo-radiotherapy. Our findings give insight into the biology of EACs and in cellular signaling mechanisms underlying response to neo-adjuvant treatment. Future implementation of this classification will improve patient stratification and enhance the development of targeted therapies.

In a previous study on esophageal adenocarcinoma (EAC) cases submitted to surgery (without neoadjuvant treatment), an algorithm (EACGSE classification) 1 based on morphologic distinctions provided significant prognostic impact. Tumor budding is a specific type of invasive growth pattern and was found to be an important prognostic parameter in gastrointestinal cancers. 2–3 We aimed to evaluate the prevalence of budding in glandular adenocarcinomas and its impact on survival. The EAC cohort classified according to EACGSE 1 was included in the study: among the different histotypes, 215 cases, classified as well (WD) and poorly (PD) differentiated glandular adenocarcinomas, were considered. Tumor budding was defined as single cells or clusters of up to four cells at the invasive margin. Budding was grouped according to ITBCC 2 into Bd1 (0-4 buds), Bd2 (5-9 buds), and Bd3 (10 or more buds). Furthermore, the most prognostically relevant cut off for bud number in EAC was defined by ROC analysis. Cancer specific survival analyses were performed using the Kaplan–Meier method and Cox regression analyses. Tumor budding was assessed in one hotspot (a field measuring 0.785 mm2) at the invasive front and ROC analysis identified 8 buds per field cut off as the most informative in relation to survival. By using this cut off, frequency of budding was higher in glandular PD (84/124,68%) cases than in WD ones (7/91,8%, p Tumor budding showed a prognostically negative impact in glandular EACs. The 8 buds per field cut off appears to provide the best prognostic discriminant. Tumor budding assessment should be added to the identification of other prognostic factors already described in the same series (stage, histotype and vascular invasion). 1

Purpose Abnormalities within the Sonic Hedgehog (SHH), Bone Morphogenetic Protein (BMP) and SMAD4 signalling pathways have been associated with the malignant behavior of esophageal adenocarcinoma (EAC). We recently developed two specific llama-derived antibodies (VHHs), C4C4 and C8C8, which target BMP4 and BMP2/4, respectively. Here we aimed to demonstrate the feasibility of the VHHs for the treatment of EAC and to elucidate its underlying mechanism. Methods Gene Set Enrichment Analysis (GSEA) was performed on a TCGA dataset, while expression of SHH, BMP2/4 and SMAD4 was validated in a cohort of EAC patients. The effects of the VHHs were tested on the recently established SMAD4(-) ISO76A primary EAC cell line and its counterpart SMAD4(+) ISO76A. In a patient-derived xenograft (PDX) model, the VHHs were evaluated for their ability to selectively target tumor cells and for their effects on tumor growth and survival. Results High expression of BMP2/4 was detected in all SMAD4 negative EACs. SHH upregulated BMP2/4 expression and induced p38 MAPK signaling in the SMAD4(-) ISO76A cells. Inhibition of BMP2/4 by VHHs decreased the aggressive and chemo-resistant phenotype of the SMAD4(-) ISO76A but not of the SMAD4(+) ISO76A cells. In the PDX model, in vivo imaging indicated that VHHs effectively targeted tumor cells. Both VHHs significantly inhibited tumor growth and acted synergistically with cisplatin. Furthermore, we found that C8C8 significantly improved survival of the mice. Conclusions Our data indicate that increased BMP2/4 expression triggers aggressive non-canonical BMP signaling in SMAD4 negative EAC. Inhibiting BMP2/4 decreases malignant behavior and improves survival. Therefore, VHHs directed against BMP2/4 hold promise for the treatment of SMAD4 negative EAC.

Summary Introduction Esophageal adenocarcinoma (EAC) is an aggressive cancer, associated with reflux esophagitis and intestinal metaplasia (IM). One underlying biological mechanism, which possibly drives the development of EAC, is the dysregulated expression of Bone Morphogenetic Proteins (BMPs). Aim To investigate if local delivery of Noggin, a BMP antagonist, reduced EAC. Methods After obtaining proof of principal on local delivery of a Noggin/Sucralfate substance, a randomized controlled trial to test the effects of Noggin on EAC development was performed in a surgical rat model. In the model, an esophago-jejunostomy leads to development of reflux-esophagitis, IM and eventually EAC. Rats were treated by Noggin/Sucralfate or Sucralfate alone. Treatment was administered from 26 to 29 weeks after the operation. Results Of the 112 operated rats, 52 survived beyond 26 weeks. Finally, 25 rats treated with Noggin/Sucralfate and 21 with Sucralfate, were evaluated. At the end, 39 (85%) of the animals had IM while 28 (61%) developed cancer. There were significantly more cancers in the Noggin/Sucralfate arm (50%) versus the Sucralfate group (73%) (Chi square, P Conclusion In this study, we demonstrated that inhibition of BMPs by Noggin reduced development of EAC in a surgical esophagitis-IM-EAC rat model. In future, effective targeting of the BMP pathway with selective BMP-inhibitors could become an important asset to improve EAC patient outcome.

Objective:This study compared outcomes of patients with esophageal cancer and clinically complete response (cCR) after neoadjuvant chemoradiotherapy (nCRT) undergoing active surveillance or immediate surgery. Background:Since nearly one-third of patients with esophageal cancer show pathologically complete response after nCRT according to CROSS regimen, the oncological benefit of immediate surgery in cCR is topic of debate. Methods:Patients with cCR based on endoscopic biopsies and endoscopic ultrasonography with fine-needle aspiration initially declining or accepting immediate surgery after nCRT were identified between 2011 and 2018. Primary endpoint was overall survival (OS). The secondary endpoints were progression-free survival (PFS), rate and timing of distant dissemination, and postoperative outcomes. Results:Some 98 patients with cCR were identified: 31 in the active surveillance- and 67 in the immediate surgery group with median followup of survivors of 27.7 and 34.8 months, respectively. Propensity score matching resulted in 2 comparable groups (n = 29 in both groups). Patients undergoing active surveillance or immediate surgery had a 3-year OS of 77% and 55% (HR 0.41; 95% CI 0.14-1.20, P = 0.104), respectively. The 3-year PFS was 60% and 54% (HR 1.08; 95% CI 0.44-2.67, P = 0.871), respectively. Patients undergoing active surveillance or immediate surgery had a comparable distant dissemination rate (both groups 28%), radical resection rate (both groups 100%), and severity of postoperative complications (Clav- ien-Dindo grade ≥ 3: 43% vs 45%, respectively). Conclusion:In this retrospective study, OS and PFS in patients with cCR undergoing active surveillance or immediate surgery were not significantly different. Active surveillance with postponed surgery for recurrent disease was not associated with a higher distant dissemination rate or more severe adverse postoperative outcomes.

Simple Summary: Esophageal adenocarcinoma (EAC) is one of the two major subtypes of esophageal cancer. In early disease stage, many EAC patients are asymptomatic. Most patients will present with late-stage disease in case of dysphagia and/or weight loss. Patients who undergo treatment with curative intent, have 5-year survival rates rarely exceeding 30%. Currently, curative treatment consists of chemo- and radiotherapy combined with surgical resection. Despite differences between tumors at the molecular level, all patients receive similar treatment, which results in heterogeneous therapeutic response. The aim of this review is to discuss the current research on molecular characteristics in EAC, which may predict tumor response. Moreover, we also discuss the rationale and research on adjusted regimens for EAC with for instance chemoradiotherapy and surveillance instead of (immediate) surgical resection. In future, these findings will lead to more personalized treatment approaches for EAC. Esophageal cancers confer a major health challenge and are highly aggressive malignancies with poor prognosis. Esophageal adenocarcinoma (EAC) is one of the two major histopathological subtypes of esophageal cancer. Despite advances in treatment modalities, the prognosis of patients with EAC remains poor, with a 5-year survival rate that rarely exceeds 30% in patients treated with curative intent. Chemoradiotherapy followed by resection is the treatment of choice for EAC patients, which are deemed to be curable. Current patient stratification and treatments are based on outcomes from clinical trials. Unfortunately, the molecular heterogeneity of EAC which determines the chemo- and radiosensitivity of these cancers are not taken into account. A more personalized approach in the treatment of EAC could improve patient outcomes. This review aims at summarizing literature on translational and clinical research in the field of EAC which could be of importance to develop personalized approaches. As suggested by the TCGA, expression data features molecular classifications by different platforms, including miRNA, genomic mutations and reverse-phase protein arrays. Here, we summarize literature on transcriptomic, data-driven approaches to identify distinct subtypes of EAC associated with molecular features. These novel classifications may determine the responsiveness to chemo(radio)therapy and help to identify novel molecular targets within cell signaling pathways. Moreover, we discuss the current clinical research efforts on tailored treatment regimens for patients with EAC taking into account the heterogeneous response to chemoradiotherapy. We summarize the evidence regarding active surveillance instead of immediate surgical resection after application of neoadjuvant chemo(radio)therapy in EAC. We consider that in future patients with complete response to chemo(radio)therapy, predicted by (transcriptomic) biomarkers, might benefit most from this approach. Finally, challenges to overcome for current findings to be implemented in clinical practice and move the field forward are being discussed.

Significance We found that cancer-associated fibroblasts, the most abundant noncancer cell type in esophageal cancer tissue, contribute to the resistance of tumors against currently applicable treatments. These cancer-associated fibroblasts do so by producing and secreting IL-6. IL-6 induces a mesenchymal, resistant tumor cell state. Inhibition of IL-6 reverted the mesenchymal cell state and resensitized tumor cells to therapy. Serum-borne proxies for the presence of IL-6–producing fibroblasts were identified and found to be predictive for the response to neoadjuvant therapy., Esophageal adenocarcinoma (EAC) has a dismal prognosis, and survival benefits of recent multimodality treatments remain small. Cancer-associated fibroblasts (CAFs) are known to contribute to poor outcome by conferring therapy resistance to various cancer types, but this has not been explored in EAC. Importantly, a targeted strategy to circumvent CAF-induced resistance has yet to be identified. By using EAC patient-derived CAFs, organoid cultures, and xenograft models we identified IL-6 as the stromal driver of therapy resistance in EAC. IL-6 activated epithelial-to-mesenchymal transition in cancer cells, which was accompanied by enhanced treatment resistance, migratory capacity, and clonogenicity. Inhibition of IL-6 restored drug sensitivity in patient-derived organoid cultures and cell lines. Analysis of patient gene expression profiles identified ADAM12 as a noninflammation-related serum-borne marker for IL-6–producing CAFs, and serum levels of this marker predicted unfavorable responses to neoadjuvant chemoradiation in EAC patients. These results demonstrate a stromal contribution to therapy resistance in EAC. This signaling can be targeted to resensitize EAC to therapy, and its activity can be measured using serum-borne markers.

Background Active surveillance has been proposed for patients with oesophageal cancer in whom there is a complete clinical response after neoadjuvant chemoradiotherapy (nCRT). However, endoscopic biopsies have limited negative predictive value in detecting residual disease. This study determined the location of residual tumour following surgery to improve surveillance and endoscopic strategies. Methods The present study was based on patients who participated in the prospective preSANO trial with adenocarcinoma or squamous cell carcinoma of the oesophagus or oesophagogastric junction treated in four Dutch hospitals between 2013 and 2016. Resection specimens and endoscopic biopsies taken during clinical response evaluations after nCRT were reviewed by two expert gastrointestinal pathologists. The exact location of residual disease in the oesophageal wall was determined in resection specimens. Endoscopic biopsies were assessed for the presence of structures representing the submucosal layer of the oesophageal wall. Results In total, 119 eligible patients underwent clinical response evaluations after nCRT followed by standard surgery. Residual tumour was present in endoscopic biopsies from 70 patients, confirmed on histological analysis of the resected organ. Residual tumour was present in the resection specimen from 27 of the other 49 patients, despite endoscopic biopsies being negative. Of these 27 patients, residual tumour was located in the mucosa in 18, and in the submucosa beneath tumour‐free mucosa in eight. One patient had tumour in muscle beneath tumour‐free mucosa and submucosa. Conclusion Most residual disease after nCRT missed by endoscopic biopsies was located in the mucosa. Active surveillance could be improved by more sampling and considering submucosal biopsies., Remnant cancer missed by endoscopic biopsies was located in the mucosa in two‐thirds of patients with residual disease after neoadjuvant chemoradiotherapy (nCRT). One‐third of patients had residual disease in the submucosa underneath a tumour‐free mucosa. The yield of biopsies in active surveillance after nCRT could be improved by sampling larger areas of oesophageal mucosa and using biopsies targeting the submucosa. What lies beneath

Background Endoscopic evaluation of the esophageal mucosa may play a role in an active surveillance strategy after neoadjuvant chemoradiotherapy (nCRT) for esophageal cancer. This study investigated the yield of endoscopic findings for detection of residual disease. Methods Patients from the multicenter preSANO cohort, who underwent nCRT followed by surgery for esophageal or junctional cancer, were included. Upper endoscopy was performed 6 and 12 weeks after nCRT. Patients with residual disease at 6 weeks underwent immediate surgery. Endoscopic records were reviewed for presence of stenosis, suspicion of residual tumor, scar tissue, and ulceration. Presence and type of endoscopic findings were compared with outcome of the resection specimen. Results 118 of 156 patients (76 %) had residual disease in the resection specimen. Endoscopic suspicion of residual tumor was significantly associated with presence of residual disease. At 6 weeks, 40/112 patients with residual disease and 4/33 patients with complete response had endoscopic suspicion of residual tumor (36 % vs. 12 %; P = 0.01), while this was reported in 16/73 and 0/28 patients, respectively, at 12 weeks (22 % vs. 0 %; P Conclusions Endoscopic suspicion of residual tumor was the only endoscopic finding associated with residual disease. Based on its positive predictive value, this endoscopic finding may contribute to the diagnostic strategy used in active surveillance.

Stage significantly affects survival of esophageal and esophago-gastric junction adenocarcinomas (EA/EGJAs), however, limited evidence for the prognostic role of histologic subtypes is available. The aim of the study was to describe a morphologic approach to EA/EGJAs and assess its discriminating prognostic power. Histologic slides from 299 neoadjuvant treatment-naïve EA/EGJAs, resected in five European Centers, were retrospectively reviewed. Morphologic features were re-assessed and correlated with survival. In glandular adenocarcinomas (240/299 cases—80%), WHO grade and tumors with a poorly differentiated component ≥6% were the most discriminant factors for survival (both p &lt, 0.0001), distinguishing glandular well-differentiated from poorly differentiated adenocarcinomas. Two prognostically different histologic groups were identified: the lower risk group, comprising glandular well-differentiated (34.4%) and rare variants, such as mucinous muconodular carcinoma (2.7%) and diffuse desmoplastic carcinoma (1.7%), versus the higher risk group, comprising the glandular poorly differentiated subtype (45.8%), including invasive mucinous carcinoma (5.7%), diffuse anaplastic carcinoma (3%), mixed carcinoma (6.7%) (CSS p &lt, 0.0001, DFS p = 0.001). Stage (p &lt, 0.0001), histologic groups (p = 0.001), age &gt, 72 years (p = 0.008), and vascular invasion (p = 0.015) were prognostically significant in the multivariate analysis. The combined evaluation of stage/histologic group identified 5-year cancer-specific survival ranging from 87.6% (stage II, lower risk) to 14% (stage IVA, higher risk). Detailed characterization of histologic subtypes contributes to EA/EGJA prognostic prediction.

Barrett’s esophagus (BE) predisposes for the malignant condition of esophageal adenocarcinoma (EAC). Since BE patients have few or no symptoms, most of these patients are not identified and not included in surveillance programs. These BE patients are at risk of developing advanced-stage EAC. At present, non-invasive tests to identify BE patients from the general population are lacking. We and others showed that Bone Morphogenetic Protein 4 (BMP4), and other BMPs are upregulated in BE. We aimed to determine if circulating BMPs can be identified and used as blood biomarkers to identify BE patients at high risk in the general population. In this study, we could detect the different BMPs in the blood of 112 BE patients and 134 age- and sex-matched controls. Concentration levels of BMP2, BMP4, and BMP5 were elevated in BE patients, with BMP2 and BMP5 significantly increased. BMP5 remained significant after multivariate analysis and was associated with an increased risk for BE with an OR of 1.49 (p value 0.01). Per log (pg/mL) of BMP5, the odds of having BE increased by 50%. Future optimization and validation studies might be needed to prove its utility as a non-invasive method for the detection of BE in high-risk populations and screening programs.

INTRODUCTION: Our study aimed at investigating tumor heterogeneity in esophageal adenocarcinoma (EAC) cells regarding clinical outcomes. METHODS: Thirty-eight surgical EAC cases who underwent gastroesophageal resection with lymph node dissection in 3 university centers were included. Archival material was analyzed via high-throughput cell sorting technology and targeted sequencing of 63 cancer-related genes. Low-pass sequencing and immunohistochemistry (IHC) were used to validate the results. Results Thirty-five of 38 EACs carried at least one somatic mutation that was absent in the stromal cells; 73.7%, 10.5%, and 10.5% carried mutations in tumor protein 53, cyclin dependent kinase inhibitor 2A, and SMAD family member 4, respectively. In addition, 2 novel mutations were found for hepatocyte nuclear factor-1 alpha in 2 of 38 cases. Tumor protein 53 gene abnormalities were more informative than p53 IHC. Conversely, loss of SMAD4 was more frequently noted with IHC (53%) and was associated with a higher recurrence rate (P = 0.015). Only through cell sorting we were able to detect the presence of hyperdiploid and pseudodiploid subclones in 7 EACs that exhibited different mutational loads and/or additional copy number amplifications, indicating the high genetic heterogeneity of these cancers. DISCUSSION: Selective cell sorting allowed the characterization of multiple molecular defects in EAC subclones that were missed in a significant number of cases when whole-tumor samples were analyzed. Therefore, this approach can reveal subtle differences in cancer cell subpopulations. Future studies are required to investigate whether these subclones are responsible for treatment response and disease recurrence.

PURPOSE Approximately 15% to 43% of esophageal adenocarcinomas (EACs) are human epidermal growth factor receptor 2 (HER2) positive. Because dual-agent HER2 blockade demonstrated a survival benefit in breast cancer, we conducted a phase II feasibility study of trastuzumab and pertuzumab added to neoadjuvant chemoradiotherapy (nCRT) in patients with EAC. PATIENTS AND METHODS Patients with resectable HER2-positive EAC received standard nCRT with carboplatin and paclitaxel and 41.4 Gy of radiotherapy, with 4 mg/kg of trastuzumab on day 1, 2 mg/kg per week during weeks 2 to 6, and 6 mg/kg per week during weeks 7, 10, and 13 and 840 mg of pertuzumab every 3 weeks. The primary end point was feasibility, defined as ≥ 80% completion of treatment with both trastuzumab and pertuzumab. An exploratory comparison of survival with a propensity score–matched cohort receiving standard nCRT was performed, as were exploratory pharmacokinetic and biomarker analyses. RESULTS Of the 40 enrolled patients (78% men; median age, 63 years), 33 (83%) completed treatment with trastuzumab and pertuzumab. No unexpected safety events were observed. R0 resection was achieved in all patients undergoing surgery, with pathologic complete response in 13 patients (34%). Three-year progression-free and overall survival (OS) were 57% and 71%, respectively (median follow-up, 32.1 months). Compared with the propensity score–matched cohort, a significantly longer OS was observed with HER2 blockade (hazard ratio, 0.58; 95% CI, 0.34 to 0.97). Results of pharmacokinetic analysis and activity on [18F]fluorodeoxyglucose positron emission tomography scans did not correlate with survival or pathologic response. Patients with HER2 3+ overexpression or growth factor receptor–bound protein 7 (Grb7) –positive tumors at baseline demonstrated significantly better survival ( P = .007) or treatment response ( P = .016), respectively. CONCLUSION Addition of trastuzumab and pertuzumab to nCRT in patients with HER2-positive EAC is feasible and demonstrates potentially promising activity compared with historical controls. HER2 3+ overexpression and Grb7 positivity are potentially predictive for survival and treatment response, respectively.

Trastuzumab, a monoclonal antibody against HER2, has become standard of care for metastatic HER2-overexpressing esophagogastric adenocarcinoma and is currently investigated as (neo)adjuvant treatment option in HER2-positive esophagogastric adenocarcinoma. The HER2 status is commonly determined on archived material of the primary tumor. However, this status may change over the course of treatment or disease progression. The aim of this study was to assess the dynamics of HER2 status in esophageal adenocarcinoma (EAC) in patients with resectable and recurrent disease, and to determine the associations of these changes with clinical outcome. Discordance, defined as any change in HER2 status between matched biopsy and post-neoadjuvant chemoradiation therapy resection specimen (N = 170), or between matched resection specimen and recurrence of patients not eligible for curative treatment (N = 61), was determined using the standardized HER2 status scoring system. Clinically relevant positive discordance was defined as a change to HER2 positive status, as this would imply eligibility for HER2-targeted therapy. A difference in HER2 status between biopsy and resection specimen and resection specimen and metachronous recurrence was observed in 2.1% (n = 3) and 3.3% (n = 2) of the paired cases, respectively. Clinically relevant discordance was detected in 1.4% (n = 2) of the resectable patients and 1.6% (n = 1) of the patients with recurrent disease. Patients with HER2-positive status tumors before start of neoadjuvant treatment showed better overall survival, but not statistically significant. No association between HER2 status discordance and survival was found. Clinically relevant HER2 status discordance was observed and in order to prevent under-treatment of patients, the assessment of HER2 status in the metastatic setting should preferably be performed on the most recently developed lesions if the previous HER2 assessment on archival material of the primary tumor was negative.

Barrett's esophagus is the only known mucosal precursor for the highly malignant esophageal adenocarcinoma. Malignant degeneration of non-dysplastic Barrett's esophagus occurs in < 0.6% per year in Dutch surveillance cohorts. Therefore, it has been proposed to increase the surveillance intervals from 3 to 5 years, potentially increasing development of advanced stage interval cancers. To prevent such cases robust biomarkers for more optimal stratification over longer follow up periods for non-dysplastic Barrett's patients are required. In this multi-center study, aberrations for chromosomes 7, 17, and structural abnormalities for c-MYC, CDKN2A, TP53, Her-2/neu and 20q assessed by DNA fluorescence in situ hybridization on brush cytology specimens, were used to determine marker scores and to perform clonal diversity measurements, as described previously. In this study, these genetic biomarkers were combined with clinical variables and analyzed to obtain the most efficient cancer prediction model after an extended period of follow-up (median time of 7 years) by applying Cox regression modeling, bootstrapping and leave-one-out analyses. A total of 334 patients with Barrett's esophagus without dysplasia from 6 community hospitals (n = 220) and one academic center (n = 114) were included. The annual progression rate to high grade dysplasia and/or esophageal adenocarcinoma was 1.3%, and to adenocarcinoma alone 0.85%. A prediction model including age, Barrett circumferential length, and a clonicity score over the genomic set including chromosomes 7, 17, 20q and c-MYC, resulted in an area under the curve of 0.88. The sensitivity and specificity of this model were 0.91 and 0.38. The positive and negative predictive values were 0.13 (95% CI 0.09 to 0.19) and 0.97 (95% CI 0.93 to 0.99). We propose the implementation of the model to identify non-dysplastic Barrett's patients, who are required to remain in surveillance programs with 3-yearly surveillance intervals from those that can benefit from less frequent or no surveillance.

Introduction The preSANO-trial aimed to determine accuracy of clinical response evaluations (CREs) after neoadjuvant chemoradiotherapy (nCRT) in patients with locally-advanced esophageal cancer. After introduction of the ‘bite-on-bite’ biopsy-technique, most residual tumors were detected. Aim of this study was to determine the location of residual tumors that were not detected during CREs and whether or not endoscopic (bite-on-bite) biopsies had the theoretical potential to detect these tumors. Methods In this side-study of the prospective preSANO trial, biopsies and resection specimens were independently revised by two GI-pathologists. All patients were included that had residual tumor in the resection specimen that was not detected during two clinical response evaluations, 6 and 12 weeks after completion of nCRT. In the resection specimen, the tumor regression grade was defined for each esophageal wall layer. It was determined how often submucosal tumors under a tumor-free mucosal layer were missed during CREs. Biopsies taken during CREs were revised for the presence of submucosal tissue. This was defined as presence of submucosal structures, i.e. submucosal glands and/or thick-walled vessel-structures. Results Some 103 of 207 patients underwent CREs followed by surgery. Residual tumor was not detected during CREs in 33 patients. Resection specimens of 28 of these patients were available for revision. Missed residual tumors were located in the mucosal layer of the esophageal wall in 64% of these patients. Residual tumors were located in the submucosal layer, under a tumor-free mucosal layer, in 29% of patients. One patient still had tumor under a tumor-free mucosal- and submucosal layer. Submucosal structures were detected in two patients and it was uncertain whether submucosal tissue was present in six patients, while no specific submucosal structures were detected in 21 patients. Conclusion The majority of patients in whom residual tumor remained undetected during clinical response evaluations had tumor cells in the mucosal layer of the esophageal wall. Nearly one third of the patients had tumor in the submucosal layer under a tumor-free mucosa. Whether these submucosal tumors can be detected using endoscopic biopsies is uncertain. Further improvement of the accuracy of CREs should focus on sampling of larger mucosal areas, for example by using brush techniques.

Aim Our study is aimed to evaluate the impact of SMAD4 expression on the clinical outcome of therapy for esophageal adenocarcinoma (EAC). Background & Methods Esophageal adenocarcinoma is characterized by a high genetic heterogeneity and a low survival rate, despite the adoption of aggressive therapies. We evaluated the expression of SMAD4, a tumor suppressor gene frequently mutated in cancer. by immunohistochemistry in 67 formalin-embedded (FFPE) EAC surgical specimens (patients were primarily treated with surgical resection-naïve cases), 34 of which were analyzed for SMAD4 gene by Next Generation Sequencing (NGS) with a dedicated target panel. Loss of SMAD4 protein was defined by a complete loss of expression in least 30% of cancer cells. Survival curves were determined using Kaplan–Meier methods. Results Loss of SMAD4 immunoreactivity was found in 33 out of 67 EAC cases (49.3%). Among a subset of 34 cases assessed in NGS, SMAD4 mutations were found in 3 cases (8.8%), all associated with protein loss. Loss of SMAD4 expression was also found in several cases with no mutations in SMAD4 gene (17/34, 50%). Loss of SMAD4 immunoreactivity was associated with poor overall survival (p=0.013) and higher risk of recurrence (p=0.001). Conclusion Loss of SMAD4 expression was a recurrent event in EAC, linked with genetic mutations in few cases, whereas in the majority of cases it might be related to epigenetic mechanisms, such as promoter hypermethylation. SMAD4 loss was strongly associated with recurrence and poor survival in patients, suggesting SMAD4 expression as potential prognostic biomarker in EAC. Further studies are required to develop strategic therapeutic options directly targeting SMAD4 and/or its regulators.

Background Endoscopic ultrasound (EUS) measurements of residual thickness and residual area have been suggested to correlate with histopathological residual tumor after neoadjuvant chemoradiotherapy (nCRT) for esophageal cancer. This study assessed the predictive value of EUS-based measurements using tumor thickness and tumor area before nCRT, and residual thickness and residual area 6 and 12 weeks after completion of nCRT for detection of residual disease. Methods This was a substudy of the diagnostic multicenter preSANO trial. The primary end point of the current study was the percentage of tumor regression grade (TRG) 3 – 4 (> 10 % vital tumor cells) residual disease that was detected using EUS-based measurements. Associations of absolute measurements of residual thickness/area and proportional change compared with baseline were evaluated. In the case of a statistically significant association, optimal cut-offs to distinguish TRG3 – 4 residual disease from TRG1 (no vital tumor cells) were determined using Youden’s J index. Results 138 patients were included. Residual thickness and residual area were statistically significantly associated with TRG3 – 4 residual disease 12 weeks after completion of nCRT (odds ratio 1.36, P Conclusions EUS measurements of residual thickness and residual area adequately detected TRG3 – 4 residual disease with a sensitivity of almost 90 % 12 weeks after completion of nCRT. Hence, residual thickness and residual area may aid in the restaging of esophageal cancer after nCRT.

Esophageal adenocarcinoma (EAC) has a dismal prognosis, and survival benefits of recent multimodality treatments remain small. Cancer-associated fibroblasts (CAFs) are known to contribute to poor outcome by conferring therapy resistance to various cancer types, but this has not been explored in EAC. Importantly, a targeted strategy to circumvent CAF-induced resistance has yet to be identified. By using EAC patient-derived CAFs, organoid cultures, and xenograft models we identified IL-6 as the stromal driver of therapy resistance in EAC. IL-6 activated epithelial-to-mesenchymal transition in cancer cells, which was accompanied by enhanced treatment resistance, migratory capacity, and clonogenicity. Inhibition of IL-6 restored drug sensitivity in patient-derived organoid cultures and cell lines. Analysis of patient gene expression profiles identified ADAM12 as a noninflammation-related serum-borne marker for IL-6-producing CAFs, and serum levels of this marker predicted unfavorable responses to neoadjuvant chemoradiation in EAC patients. These results demonstrate a stromal contribution to therapy resistance in EAC. This signaling can be targeted to resensitize EAC to therapy, and its activity can be measured using serum-borne markers.

Targeted therapy is lagging behind in esophageal adenocarcinoma (EAC). To guide the development of new treatment strategies, we provide an overview of the prognostic biomarkers in resectable EAC treated with curative intent. The Medline, Cochrane and EMBASE databases were systematically searched, focusing on overall survival (OS). The quality of the studies was assessed using a scoring system ranging from 0–7 points based on modified REMARK criteria. To evaluate all identified prognostic biomarkers, the hallmarks of cancer were adapted to fit all biomarkers based on their biological function in EAC, resulting in the features angiogenesis, cell adhesion and extra-cellular matrix remodeling, cell cycle, immune, invasion and metastasis, proliferation, and self-renewal. Pooled hazard ratios (HR) and 95% confidence intervals (CI) were derived by random effects meta-analyses performed on each hallmarks of cancer feature. Of the 3298 unique articles identified, 84 were included, with a mean quality of 5.9 points (range 3.5–7). The hallmarks of cancer feature ‘immune’ was most significantly associated with worse OS (HR 1.88, (95%CI 1.20–2.93)). Of the 82 unique prognostic biomarkers identified, meta-analyses showed prominent biomarkers, including COX-2, PAK-1, p14ARF, PD-L1, MET, LC3B, IGFBP7 and LGR5, associated to each hallmark of cancer.

Technological progress within the last 15–20 years has significantly increased our knowledge about the molecular basis of cancer development, tumor progression, and treatment response. As a consequence, a vast number of biomarkers have been proposed, but only a small fraction of them have found their way into clinical use. The aim of this paper is to describe the specific demands a clinically relevant biomarker should meet and how biomarkers can be tested stepwise. We name this procedure the “triple-R principle”: robustness, reproducibility, and relevance. The usefulness of this principle is illustrated with the marker TP53. Since it is mutated in a broad spectrum of cancer entities, TP53 can be considered a very promising marker. Thus, TP53 has been studied in detail but there is still no explicit consensus about its clinical value. By considering our own experience and reviewing the literature, we demonstrate that a major problem of current biomarker research is disregard of whether the biomarker is prognostic or predictive. As an example, it is demonstrated that TP53 is not a prognostic marker, but rather a purely predictive marker, and that disregard of this fact has made this otherwise strong biomarker appear as not being clinically useful so far.

Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications.

Barrett's esophagus (BE) is clinically significant, as it is the only known precursor lesion for esophageal adenocarcinoma. To develop improved therapies for the treatment of BE, a greater understanding of the disease process at the molecular genetic level is needed. However, achieving a greater understanding will require improved preclinical models so that the disease process can be more closely studied and novel therapies can be tested. Our concise review highlights progress in the development of preclinical models for the study of BE and identifies the most suitable model in which to test novel therapies.

Objective: To investigate the effect of smokeless tobacco (ST), cigar and/or pipe smoking (CP) on the development of Barrett esophagus (BE) in white male patients with gastroesophageal reflux disease (GERD). Patients and Methods: A total of 1015 records of white male adults with BE (cases; n=508) or GERD (controls, n=507) were reviewed for lifestyle factors. Logistic regression analyses were performed after adjusting for lifestyle factors to assess the effects of ST and CP on the risk of developing BE. Differences between patients with BE and those with GERD were compared using chi-square and t tests. Results: Patients with BE were significantly older than patients with GERD (mean age, 66±12 years for patients with BE and 55±15 years for patients with GERD; P

Summary Background After neoadjuvant chemoradiotherapy for oesophageal cancer, roughly half of the patients with squamous cell carcinoma and a quarter of those with adenocarcinoma have a pathological complete response of the primary tumour before surgery. Thus, the necessity of standard oesophagectomy after neoadjuvant chemoradiotherapy should be reconsidered for patients who respond sufficiently to neoadjuvant treatment. In this study, we aimed to establish the accuracy of detection of residual disease after neoadjuvant chemoradiotherapy with different diagnostic approaches, and the optimal combination of diagnostic techniques for clinical response evaluations. Methods The preSANO trial was a prospective, multicentre, diagnostic cohort study at six centres in the Netherlands. Eligible patients were aged 18 years or older, had histologically proven, resectable, squamous cell carcinoma or adenocarcinoma of the oesophagus or oesophagogastric junction, and were eligible for potential curative therapy with neoadjuvant chemoradiotherapy (five weekly cycles of carboplatin [area under the curve 2 mg/mL per min] plus paclitaxel [50 mg/m 2 of body-surface area] combined with 41·4 Gy radiotherapy in 23 fractions) followed by oesophagectomy. 4–6 weeks after completion of neoadjuvant chemoradiotherapy, patients had oesophagogastroduodenoscopy with biopsies and endoscopic ultrasonography with measurement of maximum tumour thickness. Patients with histologically proven locoregional residual disease or no-pass during endoscopy and without distant metastases underwent immediate surgical resection. In the remaining patients a second clinical response evaluation was done (PET–CT, oesophagogastroduodenoscopy with biopsies, endoscopic ultrasonography with measurement of maximum tumour thickness, and fine-needle aspiration of suspicious lymph nodes), followed by surgery 12–14 weeks after completion of neoadjuvant chemoradiotherapy. The primary endpoint was the correlation between clinical response during clinical response evaluations and the final pathological response in resection specimens, as shown by the proportion of tumour regression grade (TRG) 3 or 4 (>10% residual carcinoma in the resection specimen) residual tumours that was missed during clinical response evaluations. This study was registered with the Netherlands Trial Register (NTR4834), and has been completed. Findings Between July 22, 2013, and Dec 28, 2016, 219 patients were included, 207 of whom were included in the analyses. Eight of 26 TRG3 or TRG4 tumours (31% [95% CI 17–50]) were missed by endoscopy with regular biopsies and fine-needle aspiration. Four of 41 TRG3 or TRG4 tumours (10% [95% CI 4–23]) were missed with bite-on-bite biopsies and fine-needle aspiration. Endoscopic ultrasonography with maximum tumour thickness measurement missed TRG3 or TRG4 residual tumours in 11 of 39 patients (28% [95% CI 17–44]). PET–CT missed six of 41 TRG3 or TRG4 tumours (15% [95% CI 7–28]). PET–CT detected interval distant histologically proven metastases in 18 (9%) of 190 patients (one squamous cell carcinoma, 17 adenocarcinomas). Interpretation After neoadjuvant chemoradiotherapy for oesophageal cancer, clinical response evaluation with endoscopic ultrasonography, bite-on-bite biopsies, and fine-needle aspiration of suspicious lymph nodes was adequate for detection of locoregional residual disease, with PET–CT for detection of interval metastases. Active surveillance with this combination of diagnostic modalities is now being assessed in a phase 3 randomised controlled trial (SANO trial; Netherlands Trial Register NTR6803). Funding Dutch Cancer Society.

Objective. Molecular processes underlying Barrett's malignant development are poorly understood. Matrix metalloproteases (MMPs) are enzymes involved in inflammation, tissue remodeling, and malignant development. Therefore, active MMPs may have a role in early metaplasia development and Barrett's esophagus' malignant progression. We desired to gain more insight into the role of MMPs during the Barrett's esophagus pathogenesis sequence. Material and methods. In a surgical Barrett's mouse model, and in nonmalignant Barrett's and malignant esophageal cell lines, the activity of MMPs was investigated using a MMP activatable probe. MMP activity was further validated in Barrett's esophagus and esophageal adenocarcinoma patient biopsies and was further differentiated by investigating MMP9 and MMP13 expressions. Results. The mouse model showed probe activation in stromal cells early on in the esophagitis and metaplasia stages. MMP probe activation was higher in the Barrett's and cancer cell lines and biopsies as compared to normal cells and tissues. Co-immunostainings confirmed that, at the tissue level, the probe activation was mostly confined to CD45-positive stromal cells. MMP13 expression was highest in Barrett's metaplasia, whereas MMP9 was highest in the esophageal adenocarcinomas. Conclusion. During the Barrett's pathogenesis process, MMP activity is increased early on in the inflamed esophagus and remains high in metaplasia and esophageal adenocarcinoma. However, there is a switch of MMP13 to MMP9 expression once neoplasia develops. In the future, detecting specific MMP subtypes could be used for distinguishing nonmalignant from neoplastic Barrett's esophagus

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4+ malignancies. Mol Cancer Ther; 14(11); 2527–40. ©2015 AACR.

Background & Aims Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples. Methods We performed quantitative polymerase chain reaction array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs (miRNAs) that regulate their expression. We performed luciferase assays to validate interactions between miRNAs and potential targets. We overexpressed candidate miRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses to identify changes in messenger RNA (mRNA) and protein expression; we studied the effects of cytotoxic T cells. We performed miRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC in the Netherlands. Clinical and survival data were collected for patients, and EAC subtypes were determined. Results We found OE19 cells to have increased levels of 7 miRNAs. Of these, we found binding sites for miRNA 125a (MIR125a)-5p in the 3′ untranslated region of the TAP2 mRNA and binding sites for MIR148a-3p in 3′ untranslated regions of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these miRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and nontumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved in antigen presentation also expressed high levels of genes that regulate the adaptive immune response, PD-L1, PD-L2, and IDO1; these EACs had a poor response to neoadjuvant chemoradiotherapy and associated with shorter overall survival times of patients. Conclusions In studies of EAC cell lines and tumor tissues, we found increased levels of MIR125a-5p and MIR148a-3p to reduce levels of TAP2 and MHC-I, required for antigen presentation. High expression of MHC-I molecules by EAC correlated with markers of an adaptive immune response and significantly shorter overall survival times of patients.

Barrett's esophagus (BE) goes through a sequence of low grade dysplasia (LGD) and high grade dysplasia (HGD) to esophageal adenocarcinoma (EAC). The current gold standard for BE outcome prediction, histopathological staging, can be unreliable. TP53 abnormalities may serve as prognostic biomarkers. TP53 protein accumulation detected by immunohistochemistry (IHC) indirectly assesses TP53 mutations. DNA fluorescent in situ hybridization (FISH) on brush cytology specimens directly evaluates gene locus loss. We evaluated if IHC and FISH are complementary tools to assess TP53 abnormalities and tested their prognostic value in a long-term prospective follow-up of a BE cohort. TP53 IHC on tissue sections and FISH on brush cytology specimens were evaluated for 116 BE patients with respect to the different histological stages. The TP53 abnormalities were further studied in a panel of cell lines representative of the Barrett's carcinogenic sequence. For 91patients, the predictive value of TP53 abnormalities with respect to progression to HGD/EAC was tested after long term follow-up. The frequency of IHC and FISH TP53 abnormalities increased significantly with increasing histological stage (P

Bile acid reflux is known to be associated with the development of Barrett's esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient's refluxates induced expansion of multi-layered glandular structures and development of columnar metaplasia at the squamo-columnar junction. The glycine conjugated bile acids induced an intestinal type of metaplasia more typical for Barrett's esophagus. Through lineage tracing, we excluded the involvement of K5+, DCLK1+, and LGR5+ progenitor cells as the primary source in the development of the glandular metaplastic epithelium. We show that the mechanism behind development of metaplasia involves crypt fission and may be independent of stem cell proliferation. Our findings support the hypothesis that in humans, BE arises from non-squamous cells residing in submucosal gland ducts and that induction of intestinal type of metaplasia is most effectively induced by glycine-conjugated bile acids. These novel insights may lead to more effective strategies to prevent development of Barrett's esophagus and esophageal adenocarcinoma.

The following paper on the molecular biology of Barrett's esophagus (BE) includes commentaries on signaling pathways central to the development of BE including Hh, NF-κB, and IL-6/STAT3; surgical approaches for esophagectomy and classification of lesions by appropriate therapy; the debate over the merits of minimally invasive esophagectomy versus open surgery; outcomes for patients with pharyngolaryngoesophagectomy; the applications of neoadjuvant chemotherapy and chemoradiotherapy; animal models examining the surgical models of BE and esophageal adenocarcinoma; the roles of various morphogens and Cdx2 in BE; and the use of in vitro BE models for chemoprevention studies.