Ashten N. Omstead

Primary tumor microRNA signature predicts recurrence and survival in patients with locally advanced esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is an aggressive cancer necessitating the development of improved risk stratification tools for personalized care. Previously, microRNAs have been shown to correlate with the progression and prognosis of various cancer types; however, the value in EAC remains largely unexplored. We performed global microRNA profiling on 32 formalin-fixed, paraffin-embedded EAC specimens to identify microRNAs associated with progression. Literature search and pathway analysis further refined output to five significantly deregulated candidate biomarkers. Four of the five microRNAs (miR-652-5p, miR-7-2-3p, miR-3925-3p, and miR-219-3p) were validated by qRT-PCR. Survival outcomes were evaluated in testing set of 26 stage II/III EAC patients to determine the prognostic relevance of the selected microRNAs. In the testing set, miR-652-5p and miR-7-2-3p expressions were significantly associated with progression-free survival (p-value = .00771 and p-value = .00293). The highest area under receiver operating characteristic (ROC) curve was 0.8212 for the combination of miR-652-5p and miR-7-2-3p. Collectively, our findings demonstrated that the miR-652-5p/miR-7-2-3p signature may serve as a promising prognostic marker in patients with locally advanced EAC.

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-&agr; (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, Hsp90&bgr;, 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.

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

BackgroundImproved 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.MethodsThis 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.ResultsOf 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.ConclusionsThe 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.

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

Abstract 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.

High yield reproducible rat model recapitulating human Barrett's carcinogenesis

AIM To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma (EAC) using the modified Levrat model of end-to-side esophagojejunostomy. METHODS End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10 (n = 6), 17 (n = 8), 24 (n = 9), 31 (n = 6), 38 (n = 6), and 40 (n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation was completed to determine respective rates of carcinogenic development. Quantitative reverse transcription-polymerase chain reaction was performed to determine gene expression levels of MUC2, CK19, and CK20, and results were compared to determine significant differences throughout disease progression stages. RESULTS The overall study mortality was 15%. Causes of mortality included anastomotic leak, gastrointestinal hemorrhage, stomach ulcer perforation, respiratory infection secondary to aspiration, and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery, 100% of animals presented with esophagitis. Barrett’s esophagus (BE) was first observed at 10 wk, and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk, and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition, two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine, respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. CONCLUSION Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden, which may facilitate broader adoption to study EAC development, progression, and therapeutics.

CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic option for a deadly disease

Esophageal adenocarcinoma (EAC) is a deadly disease with limited therapeutic options. In the present study, we determined the preclinical efficacy of CDK4/6 inhibitor abemaciclib for treatment of EAC. In vitro, apoptosis, proliferation, and pathway regulation were evaluated in OE19, OE33, and FLO1 EAC cell lines. In vivo, esophagojejunostomy was performed on rats to induce EAC. At 36 weeks post-surgery, MRI and endoscopic biopsy established baseline tumor volume and molecular correlates, respectively. Next, the study animals were randomized to 26mg/kg intraperitoneal abemaciclib treatment or vehicle control for 28 days. Pre and post treatment MRIs, histopathology, and qRT-PCR were utilized to determine response. Our results demonstrated treatment with abemaciclib lead to increased apoptosis, and decreased proliferation in OE19 (p=0.185), OE33 (p=0.048), and FLO1 (p=0.043) with anticipated downstream molecular inhibition. In vivo, 78.9% of treatment animals demonstrated >20% tumor volume decrease (placebo 0%). Mean tumor volume changed in the treatment arm by -65.5% (placebo +133.5%) (p<0.01), and prevalence changed by -37.5% (placebo +16.7%) (p<0.01). Pre vs post treatment qRT-PCR demonstrated significant inhibition of all downstream molecular correlates. Overall our findings suggest potent antitumor efficacy of abemaciclib against EAC with evident molecular pathway inhibition and reasonable safety, establishing the rationale for future clinical development.