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Esophageal adenocarcinoma (EAC) is a highly lethal malignancy. Due to its rising incidence, EAC has become a severe health challenge in Western countries. Current treatment strategies are mainly chosen based on disease stage and clinical features, whereas the biological background is hardly considered. In this study, we performed a comprehensive review of existing studies and discussed how etiology, genetics and epigenetic characteristics, together with the tumor microenvironment, contribute to the malignant behavior and dismal prognosis of EAC. During the development of EAC, several intestinal-type proteins and signaling cascades are induced. The anti-inflammatory and immunosuppressive microenvironment is associated with poor survival. The accumulation of somatic mutations at the early phase and chromosomal structural rearrangements at relatively later time points contribute to the dynamic and heterogeneous genetic landscape of EAC. EAC is also characterized by frequent DNA methylation and dysregulation of microRNAs. We summarize the findings of dysregulations of specific cytokines, chemokines and immune cells in the tumor microenvironment and conclude that DNA methylation and microRNAs vary with each different phase of BE, LGD, HGD, early EAC and invasive EAC. Furthermore, we discuss the suitability of the currently employed therapies in the clinic and possible new therapies in the future. The development of targeted and immune therapies has been hampered by the heterogeneous genetic characteristics of EAC. In view of this, the up-to-date knowledge revealed by this work is absolutely important for future EAC studies and the discovery of new therapeutics.

Despite innovations in cancer therapeutics, cancer remains associated with high mortality and is one of biggest health challenges worldwide. Therefore, developing precise cancer imaging and effective treatments is an unmet clinical need. A relatively novel type of therapeutics are heavy chain variable domain antibody fragments (VHHs) derived from llamas. Here, we explored the suitability of VHHs for cancer imaging and therapy through reviewing the existing literature. We searched the MEDLINE, EMBASE and Cochrane databases and identified 32 papers on molecular imaging and 41 papers on therapy that were suitable for comprehensive reviewing. We found that VHHs harbor a higher specificity and affinity compared to mAbs, which contributes to high-quality imaging and less side-effects on healthy cells. The employment of VHHs in cancer imaging showed remarkably shorter times between administration and imaging. Studies showed that 18 F and 99m Tc are two optimal radionuclides for imaging with VHHs and that site-specific labelling is the optimal conjugation modality for VHHs with radionuclide or fluorescent molecules. We found different solutions for reducing kidney retention and immunogenicity of VHHs. VHHs as anticancer therapeutics have been tested in photodynamic therapy, targeted radionuclide therapy, immunotherapy and molecular targeted therapy. These studies showed that VHHs target unique antigen epitopes, which are distinct from the ones recognized by mAbs. This advantage means that VHHs may be more effective for targeted anticancer therapy and can be combined with mAbs. We found that high cellular internalization and specificity of VHHs contributes to the effectiveness and safety of VHHs as anticancer therapeutics. Two clinical trials have confirmed that VHHs are effective and safe for cancer imaging and therapy. Together, VHHs seem to harbor several advantages compared to mAbs and show potential for application in personalized treatment for cancer patients. VHH-based imaging and therapy are promising options for improving outcomes of cancer patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Hoefnagel and Krishnadath.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Background and Aims: Barrett's esophagus (BE) is the precursor lesion for esophageal adenocarcinoma (EAC). To detect EAC in early stage, patients with BE undergo endoscopic surveillance. Surveillance cohorts largely consist of nondysplastic BE (NDBE) patients with a low annual progression risk (<0.5%). Predictive biomarkers for malignant progression of NDBE could improve efficacy of surveillance. Biomarker research has mostly focused on aberrant protein expression on BE epithelial cells. Moreover, insight in cell signaling driving malignant transformation is unknown. This study uses a data-driven approach to analyze tumor-stroma interaction in NDBE which progressed to high-grade dysplasia or EAC., Methods: In this case-control study, we performed RNA sequencing analysis on index NDBE biopsies from 6 patients who, during long-term follow-up, progressed and 7 who did not progress to high-grade dysplasia/EAC. For control samples, squamous and duodenum tissues from BE patients were analyzed. For validation, we used quantitative PCR., Results: Significant differences in BE transcriptomic profiles between progressors and nonprogressors were found by principal component and differential expression analyses. Ingenuity pathway analysis indicated that 8 cell signaling pathways were significantly upregulated in the progressors, and 14 pathways were significantly downregulated. The most interesting finding was the upregulation of the xenobiotic metabolism pregnane X receptor signaling pathway in the progressor cohort, while of the downregulated pathways in progressors, several were related to the immune system., Conclusion: These novel transcriptomic insights are fundamental for developing (chemo-)preventive therapies. These could be therapies, which protect against toxins, including biles, responsible for pregnane X receptor activation or which enhance protective immune mechanisms. The identified RNA markers are promising biomarkers for improving risk stratification in surveillance programs., (© 2023 The Authors.)

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., (© 2022. The Author(s).)

Radiofrequency ablation (RFA) is the first-line treatment for flat Barrett's esophagus (BE) with dysplasia, however its role for persistent Barrett's esophagus (PBE) is unclear. PBE requires additional RFA sessions or application of cryotherapy to improve therapeutic response. We performed a retrospective cohort study evaluating cases of PBE treated by endoscopic eradication programs, with and without spray cryotherapy, and evaluated their safety and efficacy. We retrospectively identified patients with PBE, defined as ≤50% BE reduction or unchanged dysplasia after at least two RFA sessions. PBE was treated either by continued RFA (RFA Group) or converting to spray cryotherapy (CRYO Group), both followed by surveillance period. The rate of adverse events (AE), complete response of intestinal metaplasia (CRIM) and complete response of dysplasia (CRD) were recorded. A total of 46 patients, 23 per group, underwent 622 endoscopic therapies. Circumferential BE length was longer in the CRYO Group, but other baseline characteristics were similar, including maximal BE length. Esophageal strictures accounted for 14/16 total AE, 71% of which were RFA related, compared with 14% related to spray cryotherapy (P = 0.02). Overall CRIM/CRD rates in CRYO (83%) and RFA (96%) groups were not statistically different (P = 0.16), however cases in the CRYO Group required more treatment encounters (Median 19 vs. 12, P ≤ 0.01). Multimodal endotherapy is effective for eradicating PBE. Treatment programs incorporating spray cryotherapy are associated with less esophageal strictures but may require more treatment sessions to achieve eradication., (© The Author(s) 2021. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Hodgkin lymphoma (HL) survivors are at increased risk of developing second primary esophageal squamous cell cancer (ESCC). We aimed to gain insight in the driving events of ESCC in HL survivors (hESCC) by using RNA sequencing and NanoString profiling. Objectives were to investigate differences in RNA signaling between hESCC and sporadic ESCC (sESCC), and to look for early malignant changes in non-neoplastic esophageal tissue of HL survivors (hNN-tissue). We analyzed material of 26 hESCC cases, identified via the Dutch pathology registry (PALGA) and 17 sESCC cases from one academic institute and RNA sequencing data of 44 sESCC cases from TCGA. Gene expression profiles for the NanoString panel PanCancer IO 360 were obtained from 16/26 hESCC and four hNN-tissue, while non-neoplastic squamous tissue of four sporadic cases (sNN-tissue) served as reference profile. Hierarchical clustering, differential expression and pathway analyses were performed. Overall, the molecular profiles of hESCC and sESCC were similar. There was increased immune, HMGB1 and ILK signaling compared to sNN-tissue. The profiles of hNN-tissue were distinct from sNN-tissue, indicating early field effects in the esophagus of HL survivors. The BRCA1 pathway was upregulated in hESCC tissue, compared to hNN tissue. Analysis of expression profiles reveals overlap between hESCC and sESCC, and differences between hESCC and its surrounding hNN-tissue. Further research is required to validate our results and to investigate whether the changes observed in hNN-tissue are already detectable before development of hESCC. In the future, our findings could be used to improve hESCC patient management., Competing Interests: The authors have declared that no competing interests exist.

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.

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., Competing Interests: KKK was supported by Abbott Molecular. This does not alter our adherence to PLOS ONE policies on sharing data and materials. This was an investigator-initiated study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)