Inhibition of BMP2 and BMP4 Represses Barrett's Esophagus While Enhancing the Regeneration of Squamous Epithelium in Preclinical Models.

BACKGROUND & AIMS: Barrett's esophagus is considered to be a metaplastic lesion that predisposes for esophageal adenocarcinoma. Development of Barrett's esophagus is considered to be driven by sonic hedgehog mediated bone morphogenetic protein (BMP) signaling. We aimed to investigate in preclinical in vivo models whether targeting canonical BMP signaling could be an effective treatment for Barrett's esophagus. METHODS AND RESULTS: Selective inhibition of BMP2 and BMP4 within an in vivo organoid model of Barrett's esophagus inhibited development of columnar Barrett's cells, while favoring expansion of squamous cells. Silencing of noggin, a natural antagonist of BMP2, BMP4, and BMP7, in a conditional knockout mouse model induced expansion of a Barrett's-like neo-columnar epithelium from multi-lineage glands. Conversely, in this model specific inhibition of BMP2 and BMP4 led to the development of a neo-squamous lineage. In an ablation model, inhibition of BMP2 and BMP4 resulted in the regeneration of neo-squamous epithelium after the cryoablation of columnar epithelium at the squamocolumnar junction. Through lineage tracing the generation of the neo-squamous mucosa was found to originate from K5+ progenitor squamous cells. CONCLUSIONS: Here we demonstrate that specific inhibitors of BMP2 and BMP4 attenuate the development of Barrett's columnar epithelium, providing a novel potential strategy for the treatment of Barrett's esophagus and the prevention of esophageal adenocarcinoma.

Identification of Novel Molecular Subgroups in Esophageal Adenocarcinoma to Predict Response to Neo-Adjuvant Therapies.

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.

Selective targeting BMP2 and 4 in SMAD4 negative esophageal adenocarcinoma inhibits tumor growth and aggressiveness in preclinical models.

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.

Novel In Vivo Mouse Cryoablation Model to Explore Unique Therapeutic Approaches for Premalignant Columnar Lesions.

Patients with epithelial metaplasias have an increased risk of developing malignancies. In Barrett's esophagus, neo-columnar epithelium develops proximal to the squamous-columnar junction (SCJ) in the esophagus as the result of prolonged exposure to bile and acid reflux. Patients require lifetime periodic surveillance, due to lack of effective eradication therapies. The shortage of innovative treatment options is mostly attributable to the paucity of adequate in vivo models of neo-columnar epithelium regeneration. This protocol describes the generation of a cryoablation model to study regeneration of neo-epithelia at the SCJ. Cryoablation of the columnar and squamous mucosa at the SCJ was achieved through local application of liquid N2O in wild-type and reporter mice in combination with acid suppression. Acid suppression alone, showed restoration of the SCJ with normal histological features of both the neo-columnar and neo-squamous epithelium within 14 days. As a proof of principle, mice were treated with mNoggin, an inhibitor of bone morphogenetic proteins (BMPs), which are involved in the development of columnar epithelia. Local application of mNoggin to the ablated area at the SCJ significantly reduced the development of the neo-columnar mucosa. Although this model does not faithfully recapitulate the exact characteristics of Barrett's esophagus, it is a well-suited tool to study the mechanisms of therapeutic inhibition of neo-columnar regeneration. It therefore represents an efficient and easy platform to test novel pharmacological therapies for treatment of neo-epithelial lesions at the SCJ.

Detection of circulating BMP5 as a risk factor for Barrett's esophagus.

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.

Smokeless Tobacco and Cigar and/or Pipe Are Risk Factors for Barrett Esophagus in Male Patients With Gastroesophageal Reflux Disease.

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<.001). The odds of developing BE in patients who used CS were 1.7 times higher than that in patients who never smoked cigarettes (odds ratio [OR], 1.7; 95% CI, 1.3-2.2). It was observed that when CS use was combined with either ST or CP use, the odds of having BE significantly increased (OR, 2.5; 95% CI, 1.2-5.2; P=.01 and OR, 1.9; 95% CI, 1.03-3.58; P=.04) in comparison to CS alone. There were no significant differences in body mass index and alcohol consumption between BE and GERD groups. CONCLUSION: This study suggests that there is indeed an association between CS and BE. We believe that this is the first time that ST and CP were associated with an even higher odds of developing BE. Further studies are needed to investigate whether the use of ST and CP is also associated with an increased risk of developing BE-associated adenocarcinoma.

microRNA 125a Regulates MHC-I Expression on Esophageal Adenocarcinoma Cells, Associated With Suppression of Antitumor Immune Response and Poor Outcomes of Patients.

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.

Dynamic clonal equilibrium and predetermined cancer risk in Barrett's oesophagus.

Surveillance of Barrett's oesophagus allows us to study the evolutionary dynamics of a human neoplasm over time. Here we use multicolour fluorescence in situ hybridization on brush cytology specimens, from two time points with a median interval of 37 months in 195 non-dysplastic Barrett's patients, and a third time point in a subset of 90 patients at a median interval of 36 months, to study clonal evolution at single-cell resolution. Baseline genetic diversity predicts progression and remains in a stable dynamic equilibrium over time. Clonal expansions are rare, being detected once every 36.8 patient years, and growing at an average rate of 1.58 cm(2) (95% CI: 0.09-4.06) per year, often involving the p16 locus. This suggests a lack of strong clonal selection in Barrett's and that the malignant potential of 'benign' Barrett's lesions is predetermined, with important implications for surveillance programs.

Genetic Abnormalities in Biliary Brush Samples for Distinguishing Cholangiocarcinoma from Benign Strictures in Primary Sclerosing Cholangitis.

Background. Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and is strongly associated with cholangiocarcinoma (CCA). The lack of efficient diagnostic methods for CCA is a major problem. Testing for genetic abnormalities may increase the diagnostic value of cytology. Methods. We assessed genetic abnormalities for CDKN2A, TP53, ERBB2, 20q, MYC, and chromosomes 7 and 17 and measures of genetic clonal diversity in brush samples from 29 PSC patients with benign biliary strictures and 12 patients with sporadic CCA or PSC-associated CCA. Diagnostic performance of cytology alone and in combination with genetic markers was evaluated by sensitivity, specificity, and area under the curve analysis. Results. The presence of MYC gain and CDKN2A loss as well as a higher clonal diversity was significantly associated with malignancy. MYC gain increased the sensitivity of cytology from 50% to 83%. However, the specificity decreased from 97% to 76%. The diagnostic accuracy of the best performing measures of clonal diversity was similar to the combination of cytology and MYC. Adding CDKN2A loss to the panel had no additional benefit. Conclusion. Evaluation of MYC abnormalities and measures of clonal diversity in brush cytology specimens may be of clinical value in distinguishing CCA from benign biliary strictures in PSC.

Derivation of genetic biomarkers for cancer risk stratification in Barrett's oesophagus: a prospective cohort study.

OBJECTIVE: The risk of developing adenocarcinoma in non-dysplastic Barrett's oesophagus is low and difficult to predict. Accurate tools for risk stratification are needed to increase the efficiency of surveillance. We aimed to develop a prediction model for progression using clinical variables and genetic markers. METHODS: In a prospective cohort of patients with non-dysplastic Barrett's oesophagus, we evaluated six molecular markers: p16, p53, Her-2/neu, 20q, MYC and aneusomy by DNA fluorescence in situ hybridisation on brush cytology specimens. Primary study outcomes were the development of high-grade dysplasia or oesophageal adenocarcinoma. The most predictive clinical variables and markers were determined using Cox proportional-hazards models, receiver operating characteristic curves and a leave-one-out analysis. RESULTS: A total of 428 patients participated (345 men; median age 60 years) with a cumulative follow-up of 2019 patient-years (median 45 months per patient). Of these patients, 22 progressed; nine developed high-grade dysplasia and 13 oesophageal adenocarcinoma. The clinical variables, age and circumferential Barrett's length, and the markers, p16 loss, MYC gain and aneusomy, were significantly associated with progression on univariate analysis. We defined an 'Abnormal Marker Count' that counted abnormalities in p16, MYC and aneusomy, which significantly improved risk prediction beyond using just age and Barrett's length. In multivariate analysis, these three factors identified a high-risk group with an 8.7-fold (95% CI 2.6 to 29.8) increased HR when compared with the low-risk group, with an area under the curve of 0.76 (95% CI 0.66 to 0.86). CONCLUSIONS: A prediction model based on age, Barrett's length and the markers p16, MYC and aneusomy determines progression risk in non-dysplastic Barrett's oesophagus.

Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

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.

Aberrant TP53 detected by combining immunohistochemistry and DNA-FISH improves Barrett's esophagus progression prediction: a prospective follow-up study.

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 < 0.001, Chi(2) -test). Combining the techniques detected TP53 abnormalities in 100% of patients with LGD, HGD, and EAC. Multivariate analysis showed that IHC (hazard ratio: 17, 95% CI: 3.2-96, P = 0.001) and FISH (hazard ratio: 7.3, 95% CI: 1.3-41, P = 0.02) were both independent significant predictors of progression. Combining FISH and IHC in assessing TP53 abnormalities leads to an increased detection rate of TP53 aberrations and improved accuracy for predicting BE progression.

Immune signaling and regulation in esophageal cancer.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on signaling pathways that can be targeted with immunotherapy; the role of micro-RNAs in the immune response to esophageal cancer; and the association between obesity, the immune system, and esophageal adenocarcinoma.

Nano-curcumin inhibits proliferation of esophageal adenocarcinoma cells and enhances the T cell mediated immune response.

In Western countries the incidence of the esophageal adenocarcinoma (EAC) has risen at a more rapid rate than that of any other malignancy. Despite intensive therapies this cancer is associated with extreme high morbidity and mortality. For this reason, novel effective therapeutic strategies are urgently required. Dendritic Cell (DC)-based immunotherapy is a promising novel treatment strategy, which combined with other anti-cancer strategies has been proven to be beneficial for cancer patients. Curcumin (diferuloylmethane), is a natural polyphenol that is known for its anti-cancer effects however, in it's free form, curcumin has poor bioavailability. The aim of this study was to investigate whether using a highly absorptive form of curcumin, dispersed with colloidal nano-particles, named Theracurmin would be more effective against EAC cells and to analyze if this new compound affects DC-induced T cell response. As a result, we show efficient uptake of nano-curcumin by the EAC cell lines, OE33, and OE19. Moreover, nano-curcumin significantly decreased the proliferation of the EAC cells, while did not affect the normal esophageal cell line HET-1A. We also found that nano-curcumin significantly up-regulated the expression of the co-stimulatory molecule CD86 in DCs and significantly decreased the secretion of pro-inflammatory cytokines from in vitro activated T cells. When we combined T cells with nano-curcumin treatment in OE19 and OE33, we found that the basic levels of T cell induced cytotoxicity of 6.4 and 4.1%, increased to 15 and 13%, respectively. In conclusion, we found that nano-curcumin is effective against EAC, sensitizes EAC cells to T cell induced cytotoxicity and decreases the pro-inflammatory signals from T cells. Combining DC immunotherapy with nano-curcumin is potentially a promising approach for future treatment of EAC.

Cutting edge: The adapters EAT-2A and -2B are positive regulators of CD244- and CD84-dependent NK cell functions in the C57BL/6 mouse.

EWS/FLI1-activated transcript 2 (EAT-2)A and EAT-2B are single SH2-domain proteins, which bind to phosphorylated tyrosines of signaling lymphocyte activation molecule family receptors in murine NK cells. While EAT-2 is a positive regulator in human cells, a negative regulatory role was attributed to the adapter in NK cells derived from EAT-2A-deficient 129Sv mice. To evaluate whether the genetic background or the presence of a selection marker in the mutant mice could influence the regulatory mode of these adapters, we generated EAT-2A-, EAT-2B-, and EAT-2A/B-deficient mice using C57BL/6 embryonic stem cells. We found that NK cells from EAT-2A- and EAT-2A/B-deficient mice were unable to kill tumor cells in a CD244- or CD84-dependent manner. Furthermore, EAT-2A/B positively regulate phosphorylation of Vav-1, which is known to be implicated in NK cell killing. Thus, as in humans, the EAT-2 adapters act as positive regulators of signaling lymphocyte activation molecule family receptor-specific NK cell functions in C57BL/6 mice.

The SLAM and SAP gene families control innate and adaptive immune responses.

The nine SLAM-family genes, SLAMF1-9, a subfamily of the immunoglobulin superfamily, encode differentially expressed cell-surface receptors of hematopoietic cells. Engagement with their ligands, which are predominantly homotypic, leads to distinct signal transduction events, for instance those that occur in the T or NK cell immune synapse. Upon phosphorylation of one or more copies of a unique tyrosine-based signaling motif in their cytoplasmic tails, six of the SLAM receptors recruit the highly specific single SH2-domain adapters SLAM-associated protein (SAP), EAT-2A, and/or EAT-2B. These adapters in turn bind to the tyrosine kinase Fyn and/or other protein tyrosine kinases connecting the receptors to signal transduction networks. Individuals deficient in the SAP gene, SH2D1A, develop an immunodeficiency syndrome: X-linked lympho-proliferative disease. In addition to operating in the immune synapse, SLAM receptors initiate or partake in multiple effector functions of hematopoietic cells, for example, neutrophil and macrophage killing and platelet aggregation. Here we discuss the current understanding of the structure and function of these recently discovered receptors and adapter molecules in the regulation of adaptive and innate immune responses.