Sitravatinib combined with PD-1 blockade enhances cytotoxic T-cell infiltration by M2 to M1 tumor macrophage repolarization in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer-related mortality. Sitravatinib is a novel multi-gene tyrosine kinase inhibitor (TKI) that targets tumor-associated macrophage (TAM) receptors, VEGF, PDGF and c-Kit. Currently, sitravatinib is actively being studied in clinical trials across solid tumors and other TKIs have shown efficacy in combination with immune checkpoint inhibitors (ICI) in cancer models. In this study, we investigated the anti-tumor activity of sitravatinib alone and in combination with PD-1 blockade in an EAC rat model. Treatment response was evaluated by mortality, pre- and post-treatment MRI, gene expression, immunofluorescence and immunohistochemistry. Our results demonstrated adequate safety and significant tumor shrinkage in animals treated with sitravatinib, and more profoundly, sitravatinib and PD-1 inhibitor, AUNP-12 (P < 0.01). Suppression of TAM receptors resulted in increased gene expression of pro-inflammatory cytokines and decreased expression of anti-inflammatory cytokines, enhanced infiltration of CD8+ T cells, and M2 to M1 macrophage phenotype repolarization in the tumor microenvironment of treated animals (P < 0.01). Moreover, endpoint immunohistochemistry staining corroborated the anti-tumor activity by downregulation of Ki67 and upregulation of Caspase-3 in the treated animals. Additionally, pretreatment gene expression of TAM receptors and PD-L1 were significantly higher in major responders compared with the non-responders, in animals that received sitravatinib and AUNP-12 (P < 0.02), confirming that TAM suppression enhances the efficacy of PD-1 blockade. In conclusion, this study proposes a promising immunomodulatory strategy using a multi-gene TKI to overcome developed resistance to an ICI in EAC, establishing rationale for future clinical development.

Targeting interleukin-6 as a treatment approach for peritoneal carcinomatosis.

Peritoneal carcinomatosis (PC) is a complex manifestation of abdominal cancers, with a poor prognosis and limited treatment options. Recent work identifying high concentrations of the cytokine interleukin-6 (IL-6) and its soluble receptor (sIL-6-Rα) in the peritoneal cavity of patients with PC has highlighted this pathway as an emerging potential therapeutic target. This review article provides a comprehensive overview of the current understanding of the potential role of IL-6 in the development and progression of PC. We discuss mechansims by which the IL-6 pathway may contribute to peritoneal tumor dissemination, mesothelial adhesion and invasion, stromal invasion and proliferation, and immune response modulation. Finally, we review the prospects for targeting the IL-6 pathway in the treatment of PC, focusing on common sites of origin, including ovarian, gastric, pancreatic, colorectal and appendiceal cancer, and mesothelioma.

CSF-1R inhibitor, pexidartinib, sensitizes esophageal adenocarcinoma to PD-1 immune checkpoint blockade in a rat model.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, a multi-gene tyrosine kinase inhibitor, through targeting colony-stimulating factor 1 (CSF-1) receptor (CSF-1R), down modulates macrophage-mediated pro-survival tumor signaling. Previously, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in solid tumors. In this study, we investigated the antitumor activity of pexidartinib alone or in combination with blockade of PD-1 in a de novo EAC rat model. Here, we showed limited toxicity with significant tumor shrinkage in pexidartinib treated animals compared to controls, single agent and in combination with a PD-1 inhibitor, AUNP-12. Suppression of CSF-1/CSF-1R axis resulted in enhanced infiltration of CD3 + CD8 + T cells with reduced M2 macrophage polarization, in the tumor microenvironment (TME). Endpoint tissue gene expression in pexidartinib treated animals demonstrated upregulation of BAX, Cas3, TNFα, IFNγ and IL6 and downregulation of Ki67, IL13, IL10, TGFß and Arg1 (P < 0.05). Additionally, among the pexidartinib treated animals responders compared to nonresponders demonstrated a significant upregulation of pretreatment CSF-1 gene, confirming that tumor-associated macrophage suppression directly translates to clinical benefit. Moreover, a posttreatment serum cytokine assay exhibited similar systemic trends as the gene expression in the TME, depicting increases in proinflammatory cytokines and decreases in anti-inflammatory cytokines. In conclusion, our study established a promising combinatorial strategy using a CSF-1R inhibitor to overcome resistance to PD-1/PD-L1 axis blockade in an EAC model, providing the rationale for future clinical strategies.

A microRNA-based liquid biopsy signature for the early detection of esophageal squamous cell carcinoma: a retrospective, prospective and multicenter study.

BACKGROUND: Currently, there is no clinically relevant non-invasive biomarker for early detection of esophageal squamous cell carcinoma (ESCC). Herein, we established and evaluated a circulating microRNA (miRNA)-based signature for the early detection of ESCC using a systematic genome-wide miRNA expression profiling analysis. METHODS: We performed miRNA candidate discovery using three ESCC tissue miRNA datasets (n = 108, 238, and 216) and the candidate miRNAs were confirmed in tissue specimens (n = 64) by qRT-PCR. Using a serum training cohort (n = 408), we conducted multivariate logistic regression analysis to develop an ESCC circulating miRNA signature and the signature was subsequently validated in two independent retrospective and two prospective cohorts. RESULTS: We identified eighteen initial miRNA candidates from three miRNA expression datasets (n = 108, 238, and 216) and subsequently validated their expression in ESCC tissues. We thereafter confirmed the overexpression of 8 miRNAs (miR-103, miR-106b, miR-151, miR-17, miR-181a, miR-21, miR-25, and miR-93) in serum specimens. Using a serum training cohort, we developed a circulating miRNA signature (AUC:0.83 [95%CI:0.79-0.87]) and the diagnostic performance of the miRNA signature was confirmed in two independent validation cohorts (n = 126, AUC:0.80 [95%CI:0.69-0.91]; and n = 165, AUC:0.89 [95%CI:0.83-0.94]). Finally, we demonstrated the diagnostic performance of the 8-miRNA signature in two prospective cohorts (n = 185, AUC:0.92, [95%CI:0.87-0.96]); and (n = 188, AUC:0.93, [95%CI:0.88-0.97]). Importantly, the 8-miRNA signature was superior to current clinical serological markers in discriminating early stage ESCC patients from healthy controls (p < 0.001). CONCLUSIONS: We have developed a novel and robust circulating miRNA-based signature for early detection of ESCC, which was successfully validated in multiple retrospective and prospective multinational, multicenter cohorts.

A blood-based circulating microbial metagenomic panel for early diagnosis and prognosis of oesophageal adenocarcinoma.

BACKGROUND: Emerging evidence indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in oesophageal adenocarcinoma (EAC) patients, particularly as novel non-invasive, early detection, surveillance and prognostic classifiers. METHODS: Metagenome sequencing was performed on 81 serum specimens collected across EAC spectrum, with sequencing reads classified using Bracken and MetaPhlAn3. Followed by the Linear Discriminant Analysis effect size (LEfSe) method to identify microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build classifiers. RESULTS: A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI: 0.78-0.95; P < 0.001) and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 (95% CI: 0.66-0.92; P = 0.006)). CONCLUSION: This study firstly describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC. TRANSLATIONAL RELEVANCE: Accumulating data indicates the clinical relevance of specific microbial signatures as diagnostic and prognostic biomarkers, in multiple cancers. However, to date, no studies have systematically interrogated circulating metagenome profiling in patients with oesophageal adenocarcinoma (EAC). Herein, we performed metagenome sequencing in serum specimens from EAC patients 81 collected across EAC spectrum and observed a significant loss of alpha and beta diversity, with a shift in microbial taxa between each group-at the phylum, genus, and species level-with Lactobacillus sakei as the most prominent species in gastroesophageal reflux (GERD) vs other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus (L. Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 and this panel, in conjunction with the TNM stage, was a robust predictor of overall survival. This study for the first time describes unique blood-based microbial profiles in patients across EAC carcinogenesis, that are further utilised to establish a novel circulating diagnostic and prognostic metagenomic signature for EAC.

Measurement of outflow resistance imposed by magnetic sphincter augmentation: defining normal values and clinical implication.

INTRODUCTION: No manometric criteria have been defined to select patients for magnetic sphincter augmentation (MSA). The first step to establish such criteria is to measure the outflow resistance at esophagogastric junction (EGJ) imposed by MSA. This resistance needs to be overcome by the esophageal contraction in order for the esophagus to empty and to avoid postoperative dysphagia. This study was designed to measure the outflow resistance caused by MSA in patients free of postoperative dysphagia. METHODS: Records of the patients who underwent MSA in our institution were reviewed. A group of MSA patients with excellent functional outcome, who were free of clinically significant postoperative dysphagia, were selected. These patients then underwent high-resolution impedance manometry (HRIM) at a target date of 1 year after surgery. The outflow resistance was measured by the esophageal intrabolus pressure (iBP) recorded 2 cm proximal to the lower esophageal sphincter (LES). RESULTS: The study population consisted of 43 patients. HRIM was performed at mean of 20.4 (10.4) months after surgery. The mean (SD) amplitude of the iBP was 13.5 (4.3) before surgery and increased to 19.1 (5.6) after MSA (p < 0.0001). Patients with a smaller size LINX device (≤ 14 beads) had a similar iBP when compared to those with a larger device (> 15 beads) [19.7 (4.5) vs. 18.4 (5.9), p = 0.35]. There was a significant correlation between the iBP and % incomplete bolus clearance [Spearman R: 0.44 (95% CI 0.15-0.66), p = 0.0032]. The 95th percentile value for iBP after MSA was 30.4 mmHg. CONCLUSION: The EGJ outflow resistance measured by iBP is increased after MSA. The upper limit of normal for iBP is 30 mmHg in this cohort of patients who were free of dysphagia after MSA. This degree of resistance needs to be overcome by distal esophageal contraction and will likely be requisite to prevent persistent postoperative dysphagia.

Magnetic sphincter augmentation (MSA) in patients with hiatal hernia: clinical outcome and patterns of recurrence.

INTRODUCTION: Magnetic sphincter augmentation (MSA) is an effective treatment for patients with gastroesophageal reflux disease. In early studies, patients with a hiatal hernia (HH) ≥ 3 cm were excluded from consideration for implantation and initially the FDA considered its use as "precautionary" in this context. This early approach has led to an attitude of hesitance among some surgeons to offer this therapy to patients with HH. This study was designed to evaluate the impact of HH status on the outcome of MSA and to report the rate of HH recurrence after MSA. METHODS AND PROCEDURES: This is a retrospective review of prospectively collected data of patients who underwent MSA between June 2013 and August 2017. Baseline clinical and objective data were collected. Patients were divided into four groups based on HH status: no HH, small HH (< 3 cm), large HH (≥ 3 cm), and paraesophageal hernia (PEH). Patient satisfaction, GERD-HRQL and RSI data, freedom from PPI, need for postoperative dilation, length of hospitalization, 90-day readmission rate, need for device removal, and HH recurrence was compared between groups. RESULTS: There were 350 patients [60% female, mean (SD) age: 53.5 (13.8)] who underwent MSA. There were 65 (18.6%) with no HH, 205 (58.6%) with small HH (< 3 cm), 58 (16.6%) with large HH (≥ 3 cm) and 22 (6.2%) with PEH. At a mean follow-up of 13.6 (10.4) months, the rate of outcome satisfaction was similar between the groups (86%, 87.9%, 92.2% and 93.8%, p = 0.72). This was also true for GERD-HRQL total score clinical improvement (79.1%, 77.8%, 82% and 87.5%, p = 0.77). The rate of postoperative dysphagia (p = 0.33) and freedom from PPIs (p = 0.96) were similar among the four groups. Duration of hospitalization was higher among those with a large HH or PEH, and only PEH patients had a higher 90-day readmission rate (p = 0.0004). There was no difference between the need for dilation among groups (p = 0.13). The need for device removal (5% overall) was similar between the four groups (p = 0.28). HH recurrence was 10% in all groups combined, and only 7 of 240 (2.9%) patients required reoperation; the majority of these patients underwent a minimal dissection approach (no hernia repair) at the index operation. The incidence of recurrent HH increased in direct correlation with the preoperative HH size (0%, 10.1%, 16.6 and 20%, p = 0.032). CONCLUSION: In the largest series of MSA implantation, we demonstrate that the excellent outcomes and high degree of satisfaction after MSA are independent of the presence or size of HH. Despite higher rates of hernia recurrence in large HH and PEH patients, the rates of postoperative endoscopic intervention, and device removal is similar to those with no or small HH. The minimal dissection approach to MSA should be abandoned.

Comparison of surgical payer costs and implication on the healthcare expenses between laparoscopic magnetic sphincter augmentation (MSA) and laparoscopic Nissen fundoplication (LNF) in a large healthcare system.

INTRODUCTION: Magnetic sphincter augmentation (MSA) is a promising antireflux surgical treatment. The cost associated with the device may be perceived as a drawback by payers, which may limit the adoption of this technique. There are limited data regarding the cost of MSA in the management of reflux disease. The aims of the study were to report the clinical outcome and quality of life measures in patients after MSA and to compare the pharmaceutical and procedure payer costs and the disease-related and overall expense of MSA compared to laparoscopic Nissen fundoplication (LNF) from a payer perspective. METHODS AND PROCEDURES: This prospective observational study was performed in conjunction with the region's largest health insurance company. Data were collected on patients who underwent MSA over a 2-year period beginning in September 2015 at the study network hospitals. The LNF comparison group was procured from members' claims data of the payer. Inclusion was predicated by patients having continuous coverage during study period. The total procedural reimbursement and the disease-related and overall medical claims submitted up to 12 months prior to surgery and up to 12 months following surgery were obtained. The payer reimbursement data are presented as allowed cost per member per month (PMPM). These values were then compared between groups. RESULTS: There were 195 patients who underwent MSA and 1131 that had LNF. MSA results in comparable symptom control, PPI elimination rate, and quality of life measures compared to values reported for LNF in the literature. The median (IQR) reimbursement of surgery was $13,522 (13,195-14,439) for those who underwent MSA and $13,388 (9951-16,261) for patients with LNF, p = 0.02. In patients who underwent MSA, the median reimbursement related to the upper gastrointestinal disease was $ 305 PMPM, at 12 months prior to surgery and $ 104 at 12 months after surgery, representing 66% decrease in cost. These values were $ 233 PMPM and $126 PMPM for patients who underwent LNF, representing a 46% decrease (p = 0.0001). At 12 months following surgery, the reimbursement for overall medical expenses had decreased by 10.7% in the MSA group and 1.4% in the LNF group when compared to the preoperative baseline reimbursement. The reimbursement for PPI use after surgery showed a 95% decrease in the MSA group and 90% among LNF group when compared to the preoperative baseline (p = 0.10). CONCLUSION: When compared with LNF, MSA results in a reduction of disease-related expenses for the payer in the year following surgery. While MSA is associated with a higher procedural payer cost compared to LNF, payer costs may offset due to reduction in the expenses after surgery.

The Dynamic and Transient Immune Microenvironment in Locally Advanced Esophageal Adenocarcinoma Post Chemoradiation.

OBJECTIVE: The aim of this study was to assess the impact of chemoradiation on the immune microenvironment to influence and optimally design future neoadjuvant clinical trials. SUMMARY BACKGROUND DATA: Programmed death (PD)-1 inhibitors in metastatic gastroesophageal cancer have demonstrated response rates of approximately 25% in programmed death ligand-1 (PD-L1+) tumors. Unfortunately, the majority of patients do not respond. Therefore, a rationale strategy of combining immunotherapeutic agents with chemoradiation in earlier stage esophageal cancer may prevent metastatic disease in patients. METHODS: To determine the effects of chemoradiation on resected esophageal adenocarcinomas, we examined the immune microenvironment pre- and post-chemoradiation using immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and functional analysis of tumor-infiltrating lymphocytes. Additionally, to assess the duration and dependency of radiation-induced PD-L1 upregulation, a surgical rat reflux model of esophageal adenocarcinoma is used. First, tumor-bearing animals were dosed with single-fraction 13Gy or 16Gy radiation to determine safety, dose correlation, and PD-L1 upregulation using qRT-PCR post-radiation. Next, longitudinal PD-L1 expression levels within individual animals were determined using serial endoscopic biopsies at baseline, 1, 5, and 9 weeks post 16Gy radiation. RESULTS: The majority of cancers displayed enhanced interferon γ and activated CD8+ T lymphocytes at the tumor stroma interface. These tumors also demonstrated enhanced upregulation of PD-L1 and multiple other immune checkpoints including TIM3, GITR, IDO1, LAG3, OX40, and KIR. The animal model results indicated PD-L1 upregulation is dose-dependent and transiently elevated post radiation exposure. CONCLUSIONS: Collectively, these findings provide insights into the evolving immune landscape after chemoradiation and have significant implications for neoadjuvant trial designs that will combine radiotherapy with immune checkpoint inhibitors.

Phase II Study of Irinotecan Plus Panitumumab as Second-Line Therapy for Patients with Advanced Esophageal Adenocarcinoma.

LESSON LEARNED: Panitumumab plus irinotecan is not active for the treatment of esophageal adenocarcinoma. BACKGROUND: Esophageal adenocarcinoma (EAC) is a lethal cancer with increasing incidence. Panitumumab (Pa) is a fully humanized IgG2 monoclonal antibody against human EGFR. Cetuximab (Cx) combined with irinotecan (Ir) is active for second-line treatment of colorectal cancer. This phase II study was designed to evaluate Pa plus Ir as second-line therapy for advanced EAC. METHODS: The primary endpoint was response rate (RR). Patients with one prior treatment were given Pa 9 mg/m2 on day 1 and Ir 125 mg/m2 on days 1 and 8 of each 21-day cycle. Inclusion criteria were confirmed EAC, measurable disease, no prior Ir or Pa, performance status <2, and normal organ function. RESULTS: Twenty-four patients were enrolled; 18 were eligible and evaluable. These patients were all white, with a median age of 62.5 years (range, 33-79 years), and included 15 men and 3 women. The median number of cycles was 3.5. The most common grade 1-2 adverse events were fatigue, diarrhea, anemia, leukopenia, and hypoalbuminemia. Grade 3-4 adverse events included hematologic, gastrointestinal, electrolyte, rash, fatigue, and weight loss. The median follow-up was 7.2 months (range, 2.3-14 months). There were no complete remissions. The partial response rate was 6% (1/18; 95% confidence interval [CI], 0.01-0.26). The clinical benefit (partial response [PR] plus stable disease [SD]) rate was 50%. The median overall survival was 7.2 months (95% CI, 4.1-8.9) with an 11.1% 1-year survival rate. The median progression-free survival was 2.9 months (95% CI, 1.6-5.3). CONCLUSION: Irinotecan and panitumumab as second-line treatment for advanced EAC are not active.

Serial Endoscopic Evaluation of Esophageal Disease in a Cancer Model: A Paradigm Shift for Esophageal Adenocarcinoma (EAC) Drug Discovery and Development.

A rat model of surgically induced reflux recapitulates the development and progression of human esophageal adenocarcinoma (EAC). In this study, reflux was induced in rats followed by postoperative endoscopy with biopsy, to diagnose and monitor disease progression. Overall, percentage agreement between visual endoscopy and gold standard histology was 95%, with disease-specific classification accuracies of 100% and 75% for Barrett's with dysplasia and EAC, respectively. Additionally, the percentage agreement for biopsy in tumors >4 mm was 75%. Thereby, establishing endoscopic evaluation as a reliable tool to assess disease progression and provide biopsies for downstream correlates in a de novo EAC model.

Sep70/Pepsin expression in hypopharynx combined with hypopharyngeal multichannel intraluminal impedance increases diagnostic sensitivity of laryngopharyngeal reflux.

BACKGROUND: Improved methods of diagnosis of laryngopharyngeal reflux (LPR) would enable surgeons to better identify patients who may benefit from antireflux surgery (ARS). The objective of the present study was to assess if hypopharyngeal Pepsin and Sep70 expression combined with hypopharyngeal multichannel intraluminal impedance (HMII) has the potential to increase diagnostic sensitivity of LPR. METHODS: This study was performed on patients who underwent unsedated transnasal endoscopy with hypopharyngeal biopsy and 24-h HMII to determine abnormal proximal exposure (APE) and DeMeester score (DMS) from 2013 to 2016. Pepsin and Sep70 protein expression was assessed by Western blots of biopsy specimens. The outcomes of ARS were assessed using reflux symptom index (RSI). HMII APE classification, Sep 70, and Pepsin protein levels were compared in normative and symptomatic LPR patients and further analyzed alongside quality of life changes following ARS. RESULTS: Of 30 subjects enrolled, 23 were excluded for abnormal HMII results or endoscopic evidence of esophagitis. Seven subjects and 105 patients were included in the normative and symptomatic groups, respectively. Compared to the normative group, only Pepsin expression was significantly higher in the symptomatic group [APE+/LPR+ (p = 0.000), APE+/LPR- (p = 0.001), and APE- (p = 0.047)]. Further, the ratio of Sep70/Pepsin was significantly lower in the symptomatic group [APE+/LPR+ (p = 0.008), APE+/LPR- (p = 0.000), and APE- (p = 0.050)], and a cutoff ratio for a diagnosis of LPR was established as < 158. Of 105 symptomatic patients, 48 patients underwent ARS. Of these, 17 patients had complete pre- and post-RSI questionnaires. LPR symptoms improved in 15 (88%), of whom 2 were APE- but met criteria for a diagnosis of LPR based on the Sep70/Pepsin cutoff. CONCLUSIONS: The identified Sep70/Pepsin ratio may serve as a reliable biomarker for the diagnosis of LPR. As a result, this may help identify additional patients who have a false-negative HMII result due to the 24-h testing window.

PI3K/mTOR Dual Inhibitor, LY3023414, Demonstrates Potent Antitumor Efficacy Against Esophageal Adenocarcinoma in a Rat Model.

OBJECTIVE: The purpose of the current study is to determine the efficacy of a PI3K/mTOR dual inhibitor, LY3023414, on established EAC in an in vivo model. BACKGROUND: Esophageal adenocarcinoma (EAC) is a highly lethal cancer with limited treatment options. The PI3K/mTOR pathway is upregulated in EAC and may be a target for novel therapies. METHODS: Esophagojejunostomy was performed on Sprague-Dawley rats to induce carcinogenesis, and LY3023414 was cyclically administered intraperitoneally between 32 and 40 weeks postsurgery to treatment animals. Magnetic resonance imaging (MRI) and histology were used to determine clinical response. Immunohistochemistry, immunofluorescence, and Western blot were used to validate apoptosis by cleaved caspase-3, proliferation by Ki67, and pathway inhibition, respectively. RESULTS: Mean MRI tumor volume increased by 109.2% in controls (n = 32) and decreased by 56.8% in treatment animals (n=17) (P < 0.01). Treatment with LY3023414 demonstrated tumor volume increase in 0% (control = 46.4%) (P < 0.01), decrease in 58.8% (control = 7.1%) (P < 0.01), and stable volume in 41.2% (control = 46.4%) (P = 0.77). EAC prevalence in controls increased by 25%; whereas, prevalence in treatment animals decreased by 29.4% (P < 0.01). Approximately, 75% of treatment animals presenting with residual masses on MRI had a histological response >50%. Increased apoptosis by cleaved caspase-3 (P = 0.03) and decreased proliferation by Ki67 (P < 0.01) were demonstrated in the treatment arm, when compared with the control arm. On Western blot analysis of pathway checkpoints, p-mTOR (p=0.03) and PI3K-α (P = 0.04) were downregulated in treatment responsive residual tumors, when compared with controls. CONCLUSIONS: LY3023414 demonstrates efficacy against EAC in a preclinical model, establishing the rationale for clinical testing.

Preclinical Study of AUY922, a Novel Hsp90 Inhibitor, in the Treatment of Esophageal Adenocarcinoma.

OBJECTIVE: To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo. BACKGROUND: EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy. METHODS: In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression. RESULTS: In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90ß, and CDK4, and upregulation of Hsp72. CONCLUSIONS: AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

Associations of microbiota and toll-like receptor signaling pathway in esophageal adenocarcinoma.

BACKGROUND: Toll-like receptors (TLRs) recognize known molecules from microbes and have an established role in tumorigenesis. Using a rat model of esophageal adenocarcinoma, and human clinical samples, we investigated genes central to TLR-mediated signal transduction and characterized the esophageal microbiome across the spectrum of esophageal adenocarcinoma carcinogenesis. METHODS: We surgically induced bile/acid reflux in rats and their esophagi were harvested at 40 weeks post-surgery. Tissue samples from the model were selected for gene expression profiling. Additionally, for rat and human samples microbiome analysis was performed using PCR-ESI-MS-TOF technology with validation by fluorescence in situ hybridization. RESULTS: Gene expression results in the rat model indicated a significant upregulation of TLRs 1-3, 6, 7 and 9 in EAC compared to normal epithelium. PCR-ESI-MS-TOF analysis revealed a prevalence of Escherichia coli in Barrett's esophagus (60%) and esophageal adenocarcinoma (100%), which was validated by fluorescence in situ hybridization. In the human clinical samples, Streptococcus pneumonia was detected in high abundance in gastroesophageal reflux disease and Barrett's esophagus (50-70%) in comparison to tumor adjacent normal epithelium, dysplasia, and esophageal adenocarcinoma (20-30%). E. coli was detected in the Barrett's esophagus and esophageal adenocarcinoma groups but was absent in the tumor adjacent normal epithelium, dysplasia, and the gastroesophageal reflux disease groups. CONCLUSIONS: We demonstrated an association between the TLR signaling pathway and E. coli hinting towards possible early molecular changes being mediated by microbes in the rat model of esophageal adenocarcinoma carcinogenesis. Studies on human clinical samples also corroborate results to some extent; however, a study with larger sample size is needed to further explore this association.

A utility of peroral endoscopic myotomy (POEM) across the spectrum of esophageal motility disorders.

BACKGROUND: Peroral endoscopic myotomy (POEM) has been performed as a novel endoscopic procedure to treat achalasia with favorable outcome. The objective of this study was to assess the outcome of POEM in our initial series and to assess the safety and efficacy of POEM in a variety of esophageal motility-related clinical problems. METHODS: This is a retrospective cross-sectional study involving all patients with esophageal motility disorders defined by the Chicago classification, who had undergone consideration for POEM at our institution. Validated questionnaires such as gastroesophageal reflux disease health-related quality of life (GERD-HRQL), reflux symptom index (RSI) and achalasia disease-specific health-related quality of life were obtained pre- and postoperatively. RESULTS: From January 2013 to October 2014, a total of 35 POEMs (achalasia n = 25, non-achalasia n = 10) were performed on 33 patients (female n = 20, male n = 13, mean age 56.9 years). There was no mortality. The rate of inadvertent mucosotomy was 17.1%. The rate of complications requiring interventions was 5.7%. During a mean follow-up period of 7 months (range 0.5-17), 92% of patients with achalasia and 75% of those with non-achalasia motility disorders had a symptomatic improvement in dysphagia. Chest pain was completely resolved in all patients with achalasia (8/8) and 80% of patients with non-achalasia (4/5). The GERD-HRQL, RSI and dysphagia scores significantly improved after POEM in patients with achalasia. There was a significant improvement in GERD-HRQL and RSI scores, and a trend toward lower dysphagia score in patients with non-achalasia. CONCLUSIONS: The outcome of POEM to treat achalasia and non-achalasia motility disorders is consistent with previous studies. Potential benefit of POEM includes not only its flexibility to adjust the length and location of myotomy but also the ability to extend myotomy proximally without thoracoscopy or thoracotomy. POEM can be combined with laparoscopic procedures and used as "salvage" for localized esophageal dysmotility.

Hypopharyngeal pepsin and Sep70 as diagnostic markers of laryngopharyngeal reflux: preliminary study.

INTRODUCTION: The management of laryngopharyngeal reflux (LPR) has been challenging. Hypopharyngeal multichannel intraluminal impedance (HMII) has shown to increase the sensitivity in diagnosing LPR. The objective of this study is to investigate the potential use of pepsin and Sep70 as diagnostic tools for detection of LPR in combination with HMII. MATERIALS AND METHODS: Tissue samples of hypopharynx, distal esophagus, and gastric cardia were collected from patients with LPR symptoms regardless of gastroesophageal reflux (GERD) diagnosis and underwent HMII to detect LPR and high esophageal reflux (HER: reflux 2 cm distal to upper esophageal sphincter) events. Patients were classified into two groups based on the presence of abnormal proximal exposure (APE), which was defined as LPR ≥1/day and/or HER ≥5/day: (1) positive-APE and (2) negative-APE. Patients with typical GERD symptoms without LPR symptoms who did not undergo HMII were used as a "control" GERD group. Protein was isolated from tissue samples and Western blot analysis of pepsin and Sep70 was performed. Pepsinogen was used as a control to differentiate pepsin from pepsinogen. Relative quantitation was performed using Image Studio Lite Software with normalization against the internal actin of each blot. RESULTS: From October 2012 to September 2013, 55 patients underwent HMII. Of 55, 20 patients underwent biopsies from hypopharynx (17 positive-APE and 3 negative-APE). Ten patients with typical GERD symptoms were identified from tissue bank as a "control" GERD group. Pepsin was detected in distal esophagus and hypopharynx in all groups without significant difference among groups. However, Sep70 in distal esophagus and hypopharynx was significantly depleted in the positive-APE group compared to the other groups (p = 0.032 and 0.002, respectively). CONCLUSION: Depletion of Sep70 with the presence of pepsin in the hypopharynx may indicate cellular injury in laryngopharynx due to constant proximal reflux. However, the normative data for these markers have to be validated.

Evaluation of a 4-protein serum biomarker panel-biglycan, annexin-A6, myeloperoxidase, and protein S100-A9 (B-AMP)-for the detection of esophageal adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC. METHODS: A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease. RESULTS: With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P<.05). Eleven proteins from this data set were then tested using enzyme-linked immunosorbent assays in serum samples, of which 5 proteins were significantly elevated in abundance among patients who had EAC compared with normal controls, which mirrored trends across the disease spectrum present in the tissue data. By using serum data, a Bayesian rule-learning predictive model with 4 biomarkers was developed to accurately classify disease class; the cross-validation results for the merged data set yielded accuracy of 87% and an area under the receiver operating characteristic curve of 93%. CONCLUSIONS: Serum biomarkers hold significant promise for the early, noninvasive detection of EAC.

The Evolving Landscape of Neoadjuvant Immunotherapy in Gastroesophageal Cancer.

Despite advances in treatment strategies and surgical approaches in recent years, improving survival outcomes in esophagogastric cancer (EGC) patients treated with curative intent remains a significant area of unmet need. The recent emergence of adjuvant immunotherapy as the standard of care for resected EGC demonstrates the impact of immunotherapy in improving recurrence-free survival. Neoadjuvant and perioperative immunotherapies represent another promising approach with potential advantages over adjuvant therapy. Despite the promising results of early neoadjuvant immunotherapy studies, there are several challenges and future research needs. The optimal timing, duration and number of doses in relation to surgery and the optimal combination of immunotherapies are still unclear. In addition, rigorous correlative studies need to be performed to identify biomarkers for patient selection and treatment response prediction to maximize the benefits of neoadjuvant immunotherapy. In this review, we provide a concise summary of the current standard of care for resectable EGC and discuss the rationale for the use of immune checkpoint inhibitors in this setting and the pre-clinical and early clinical data of these novel therapies. Finally, we will examine the potential role and future direction of immunotherapy in the treatment paradigm and the perceived challenges and opportunities that lay ahead.

Elucidating the Heterogeneity of Immunotherapy Response and Immune-Related Toxicities by Longitudinal ctDNA and Immune Cell Compartment Tracking in Lung Cancer.

PURPOSE: Although immunotherapy is the mainstay of therapy for advanced non-small cell lung cancer (NSCLC), robust biomarkers of clinical response are lacking. The heterogeneity of clinical responses together with the limited value of radiographic response assessments to timely and accurately predict therapeutic effect-especially in the setting of stable disease-calls for the development of molecularly informed real-time minimally invasive approaches. In addition to capturing tumor regression, liquid biopsies may be informative in capturing immune-related adverse events (irAE). EXPERIMENTAL DESIGN: We investigated longitudinal changes in circulating tumor DNA (ctDNA) in patients with metastatic NSCLC who received immunotherapy-based regimens. Using ctDNA targeted error-correction sequencing together with matched sequencing of white blood cells and tumor tissue, we tracked serial changes in cell-free tumor load (cfTL) and determined molecular response. Peripheral T-cell repertoire dynamics were serially assessed and evaluated together with plasma protein expression profiles. RESULTS: Molecular response, defined as complete clearance of cfTL, was significantly associated with progression-free (log-rank P = 0.0003) and overall survival (log-rank P = 0.01) and was particularly informative in capturing differential survival outcomes among patients with radiographically stable disease. For patients who developed irAEs, on-treatment peripheral blood T-cell repertoire reshaping, assessed by significant T-cell receptor (TCR) clonotypic expansions and regressions, was identified on average 5 months prior to clinical diagnosis of an irAE. CONCLUSIONS: Molecular responses assist with the interpretation of heterogeneous clinical responses, especially for patients with stable disease. Our complementary assessment of the peripheral tumor and immune compartments provides an approach for monitoring of clinical benefits and irAEs during immunotherapy.