Forkhead box F1 induces columnar phenotype and epithelial-to-mesenchymal transition in esophageal squamous cells to initiate Barrett's like metaplasia.

Multiple genome-wide association studies (GWAS) have linked Forkhead Box F1 (FOXF1) to Barrett's esophagus (BE). Understanding whether FOXF1 is involved in initiation of Barrett's metaplasia could allow FOXF1 to be used for risk stratification and for therapy. Two-dimensional cell cultures and three-dimensional organoid cultures and well-annotated human biopsies were used to determine the role of FOXF1 in BE pathogenesis. Multiple established esophageal squamous and BE cell lines were tested in gain- and loss-of-function studies. Initiation of a BE-like metaplastic change was evaluated by measuring characteristic cytokeratins and global gene expression profiling and by culturing organoids. Epithelial-mesenchymal transition (EMT) was evaluated by immunostaining for E-cadherin, vimentin and Snail, and by cell motility assay. Columnar esophageal epithelium of BE patients exhibited higher expression of FOXF1 compared to normal squamous esophageal epithelium of GERD patients (P < 0.001). Acidic bile salts induced nuclear FOXF1 in esophageal squamous cells. FOXF1 overexpression in normal esophageal squamous cells: (a) increased columnar cytokeratins and decreased squamous cytokeratins, (b) converted squamous organoids to glandular organoids, and (c) switched global gene profiles to resemble that of human BE epithelium (P = 2.1685e - 06 for upregulated genes and P = 8.3378e - 09 for downregulated genes). FOXF1 inhibition in BE cell lines led to loss of BE differentiation markers, CK7, and mucin 2. Also, FOXF1 induced EMT and promoted cell motility in normal esophageal squamous epithelial cells. FOXF1-induced genes mapped to pathways such as Cancer, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair. In conclusion, FOXF1 promotes a BE-like columnar phenotype and cell motility in esophageal squamous epithelial cells, which may have a critical role in BE development. FOXF1 should be studied further as a biomarker for BE and as a target for BE treatment.

Discordance Among Pathologists in the United States and Europe in Diagnosis of Low-Grade Dysplasia for Patients With Barrett's Esophagus.

BACKGROUND & AIMS: There is suboptimal inter-observer agreement, even among expert gastrointestinal pathologists, in the diagnosis of low-grade dysplasia (LGD) in patients with Barrett's esophagus (BE). We analyzed histopathologic criteria required for a diagnosis of LGD using the new subcategories of LGD with inflammatory and dysplastic features. We categorized each diagnosis based on the level of confidence and assessed inter-observer agreement among gastrointestinal pathologists from 5 tertiary centers in the United States and Europe. METHODS: In the first phase of the study, 3 pathologists held a consensus conference at which they discussed the diagnostic criteria for LGD. In the second phase, 79 slides from patients with BE (23 samples of non-dysplastic BE, 22 samples of LGD, and 34 samples of high-grade dysplasia) were identified, randomly assigned to 7 pathologists (4 from the United States and 3 from Europe), and interpreted in a blinded fashion. κ Values were calculated for inter-observer agreement. We performed multinomial logistic regression analysis to assess the weighting of histologic features with the diagnosis. RESULTS: The overall κ value for diagnosis was 0.43 (95% confidence interval [CI], 0.42-0.48). When categorized based on degree of dysplasia, the κ value was 0.22 (95% CI, 0.11-0.29) for non-dysplastic BE, 0.11 (95% CI, 0.004-0.15) for LGD, and 0.43 (95% CI, 0.36-0.46) for high-grade dysplasia. When all pathologists made a diagnosis with high confidence, the inter-observer agreement was substantial among the US pathologists (κ, 0.63; 95% CI, 0.61-0.66) and European pathologists (κ, 0.80; 95% CI, 0.74-0.97). The κ values for all diagnoses made by European pathologists were higher than those made by US pathologists. CONCLUSIONS: In an analysis of criteria used in histopathologic diagnosis of LGD, we did not observe improvement in level of agreement among experienced pathologists, even after accounting for inflammation. The level of inter-observer agreement increased with level of pathologist confidence. There was also a difference in reading of histopathology samples of BE tissues between US and European pathologists.

A Multi-Institutional Prospective Trial Confirms Noninvasive Blood Test Maintains Predictive Value in African American Men.

PURPOSE: The 4Kscore® test accurately detects aggressive prostate cancer and reduces unnecessary biopsies. However, its performance in African American men has been unknown. We assessed test performance in a cohort of men with a large African American representation. MATERIALS AND METHODS: Men referred for prostate biopsy at 8 Veterans Affairs medical centers were prospectively enrolled in the study. All men underwent phlebotomy for 4Kscore test assessment prior to prostate biopsy. The primary outcome was the detection of Grade Group 2 or higher cancer on biopsy. We assessed the discrimination, calibration and clinical usefulness of 4Kscore to predict Grade Group 2 or higher prostate cancer and compared it to a base model consisting of age, digital rectal examination and prostate specific antigen. Additionally, we compared test performance in African American and nonAfrican American men. RESULTS: Of the 366 enrolled men 205 (56%) were African American and 131 (36%) had Grade Group 2 or higher prostate cancer. The 4Kscore test showed better discrimination (AUC 0.81 vs 0.74, p <0.01) and higher clinical usefulness on decision curve analysis than the base model. Test prediction closely approximated the observed risk of Grade Group 2 or higher prostate cancer. There was no difference in test performance in African American and nonAfrican American men (0.80 vs 0.84, p = 0.32), The test outperformed the base model in each group. CONCLUSIONS: The 4Kscore test accurately predicts aggressive prostate cancer for biopsy decision making in African American and nonAfrican American men.

In-class didactic versus self-directed teaching of the probe-based confocal laser endomicroscopy (pCLE) criteria for Barrett's esophagus.

BACKGROUND AND AIMS: Optimal teaching methods for disease recognition using probe-based confocal laser endomicroscopy (pCLE) have not been developed. Our aim was to compare in-class didactic teaching vs. self-directed teaching of Barrett's neoplasia diagnosis using pCLE. METHODS: This randomized controlled trial was conducted at a tertiary academic center. Study participants with no prior pCLE experience were randomized to in-class didactic (group 1) or self-directed teaching groups (group 2). For group 1, an expert conducted a classroom teaching session using standardized educational material. Participants in group 2 were provided with the same material on an audio PowerPoint. After initial training, all participants graded an initial set of 20 pCLE videos and reviewed correct responses with the expert (group 1) or on audio PowerPoint (group 2). Finally, all participants completed interpretations of a further 40 videos. RESULTS: Eighteen trainees (8 medical students, 10 gastroenterology trainees) participated in the study. Overall diagnostic accuracy for neoplasia prediction by pCLE was 77 % (95 % confidence interval [CI] 74.0 % - 79.2 %); of predictions made with high confidence (53 %), the accuracy was 85 % (95 %CI 81.8 % - 87.8 %). The overall accuracy and interobserver agreement was significantly higher in group 1 than in group 2 for all predictions (80.4 % vs. 73 %; P = 0.005) and for high confidence predictions (90 % vs. 80 %; P < 0.001). Following feedback (after the initial 20 videos), the overall accuracy improved from 73 % to 79 % (P = 0.04), mainly driven by a significant improvement in group 1 (74 % to 84 %; P < 0.01). Accuracy of prediction significantly improved with time in endoscopy training (72 % students, 77 % FY1, 82 % FY2, and 85 % FY3; P = 0.003). CONCLUSION: For novice trainees, in-class didactic teaching enables significantly better recognition of the pCLE features of Barrett's esophagus than self-directed teaching. The in-class didactic group had a shorter learning curve and were able to achieve 90 % accuracy for their high confidence predictions.

Clinical outcomes in patients with a diagnosis of "indefinite for dysplasia" in Barrett's esophagus: a multicenter cohort study.

BACKGROUND AND STUDY AIM: Data are limited on the natural history of patients with Barrett's esophagus with a diagnosis of "indefinite for dysplasia" (IND). The aims of this study were to: (i) determine rates of progression to high grade dysplasia (HGD) or esophageal adenocarcinoma, and compare these with rates for low grade dysplasia (LGD); and (ii) determine the proportion of patients whose histological IND diagnosis changed on follow-up endoscopy. PATIENTS AND METHODS: Demographic, endoscopic, and histologic information of patients with diagnoses of IND and LGD and at least 12 months of follow-up were extracted from the database of a multicenter Barrett's esophagus study. Rates and times for progression to HGD and esophageal adenocarcinoma and regression to nondysplastic epithelium were calculated. Proportions of diagnoses upgraded to HGD/esophageal adenocarcinoma or downgraded to nondysplastic epithelium at first follow-up endoscopy were evaluated. RESULTS: Amongst 2264 patients, 83 with a diagnosis of IND (mean age 60 years, 95 % men, 95 % white; mean follow-up 5.6 years) and 79 with diagnosis of LGD were identified. In the IND group, annual incidences of esophageal adenocarcinoma and HGD were 0.21 % and 0.64 %, respectively, representing a combined incidence of 0.8 %. Mean time to progression was 4.72 years. Within the IND group 55 % patients showed regression to nondysplastic epithelium at first follow-up endoscopy and the overall regression rate was 80 %. Corresponding rates in LGD patients were similar. CONCLUSIONS: Lesions diagnosed as IND and LGD show similar biological behavior and can be treated as a single category with respect to surveillance and follow-up.

Persistence of nondysplastic Barrett's esophagus identifies patients at lower risk for esophageal adenocarcinoma: results from a large multicenter cohort.

BACKGROUND & AIMS: Recent population-based studies have shown a low risk of esophageal adenocarcinoma (EAC) in patients with nondysplastic Barrett's esophagus (NDBE). We evaluated whether persistence of NDBE over multiple consecutive surveillance endoscopic examinations could be used in risk stratification of patients with Barrett's esophagus (BE). METHODS: We performed a multicenter outcomes study of a large cohort of patients with BE. Based on the number of consecutive surveillance endoscopies showing NDBE, we identified 5 groups of patients. Patients in group 1 were found to have NDBE at their first esophagogastroduodenoscopy (EGD). Patients in group 2 were found to have NDBE on their first 2 consecutive EGDs. Similarly, patients in groups 3, 4, and 5 were found to have NDBE on 3, 4, and 5 consecutive surveillance EGDs. A logistic regression model was built to determine whether persistence of NDBE independently protected against development of cancer. RESULTS: Of a total of 3515 patients with BE, 1401 patients met the inclusion criteria (93.3% white; 87.5% men; median age, 60 ±17 years). The median follow-up period was 5 ± 3.9 years (7846 patient-years). The annual risk of EAC in groups 1 to 5 was 0.32%, 0.27%, 0.16%, 0.2%, and 0.11%, respectively (P for trend = .03). After adjusting for age, sex, and length of BE, persistence of NDBE, based on multiple surveillance endoscopies, was associated with a gradually lower likelihood of progression to EAC. CONCLUSIONS: Persistence of NDBE over several endoscopic examinations identifies patients who are at low risk for development of EAC. These findings support lengthening surveillance intervals or discontinuing surveillance of patients with persistent NDBE.

Standard endoscopy with random biopsies versus narrow band imaging targeted biopsies in Barrett's oesophagus: a prospective, international, randomised controlled trial.

BACKGROUND: White light endoscopy with random biopsies is the standard for detection of intestinal metaplasia (IM) and neoplasia in patients with Barrett's oesophagus (BO). Narrow band imaging (NBI) highlights surface patterns that correlate with IM and neoplasia in BO. OBJECTIVE: To compare high-definition white light (HD-WLE) and NBI for detection of IM and neoplasia in BO. DESIGN: International, randomised, crossover trial comparing HD-WLE and NBI. Patients referred for BO screening/surveillance at three tertiary referral centres were prospectively enrolled and randomised to HD-WLE or NBI followed by other procedures in 3-8 weeks. During HD-WLE, four quadrant biopsies every 2 cm, together with targeted biopsies of visible lesions (Seattle protocol), were obtained. During NBI examination, mucosal and vascular patterns were noted and targeted biopsies were obtained. All biopsies were read by a single expert gastrointestinal pathologist in a blinded fashion. RESULTS: 123 patients with BO (mean age 61; 93% male; 97% Caucasian) with mean circumferential and maximal extents of 1.8 and 3.6 cm, respectively, were enrolled. Both HD-WLE and NBI detected 104/113 (92%) patients with IM, but NBI required fewer biopsies per patient (3.6 vs 7.6, p<0.0001). NBI detected a higher proportion of areas with dysplasia (30% vs 21%, p=0.01). During examination with NBI, all areas of high-grade dysplasia and cancer had an irregular mucosal or vascular pattern. CONCLUSIONS: NBI targeted biopsies can have the same IM detection rate as an HD-WLE examination with the Seattle protocol while requiring fewer biopsies. In addition, NBI targeted biopsies can detect more areas with dysplasia. Regular appearing NBI surface patterns did not harbour high-grade dysplasia/cancer, suggesting that biopsies could be avoided in these areas.

Clinical utility and interobserver agreement of autofluorescence imaging and magnification narrow-band imaging for the evaluation of Barrett's esophagus: a prospective tandem study.

BACKGROUND: New endoscopic imaging techniques, such as autofluorescence imaging (AFI) and narrow-band imaging (NBI), have been developed to improve the detection of neoplastic lesions in Barrett's esophagus (BE). OBJECTIVE: To evaluate the clinical utility of AFI and magnification NBI to detect high-grade dysplasia (HGD) and early esophageal adenocarcinoma (EAC) and the interobserver agreement. DESIGN: Prospective tandem study of eligible patients. SETTING: Single, academic tertiary care center. PATIENTS: Forty-two patients with a history of confirmed BE were prospectively enrolled. INTERVENTIONS: The BE segment was examined under high-definition white-light endoscopy, and the presence of visible lesions was recorded. Subsequently, AFI and magnification NBI were performed in tandem on areas of the BE segment away from visible lesions; images obtained by these 2 systems were graded according to the color of reflected light and surface patterns, respectively. Biopsy specimens were obtained at the end of the procedure. MAIN OUTCOME MEASUREMENTS: The sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of the AFI and NBI patterns for the detection of HGD/EAC and interobserver agreement. RESULTS: Of the 42 patients enrolled, 14 (33%) had HGD/EAC. On patient-based analysis, AFI alone had a sensitivity, specificity, and NPV of 50%, 61%, and 71%, respectively, and the overall accuracy for the detection of HGD/EAC patients was 57%. By using magnification NBI in tandem fashion, the sensitivity and NPV improved to 71% and 76%, respectively, with a decrease in specificity to 46% and in overall accuracy to 55%. The 2 techniques had moderate interobserver agreement for both the patterns and prediction of histology. LIMITATIONS: Uncontrolled study performed at an academic center by expert endoscopists in a high-risk population. CONCLUSIONS: By using a multimodality endoscope, both AFI and magnification NBI had limited clinical accuracy and moderate overall interobserver agreement. AFI does not appear to be useful as a broad-based technique for the detection of neoplasia in patients with BE.

Risk factors for progression of low-grade dysplasia in patients with Barrett's esophagus.

BACKGROUND & AIMS: Data vary on the progression of low-grade dysplasia (LGD) in patients with Barrett's esophagus (BE); in patients with LGD, we investigated the incidence of high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) and compared progression in patients with different forms of LGD (prevalent vs incident and multifocal vs unifocal). We assessed the effects of consensus diagnosis of LGD on progression rates to HGD and EAC among expert pathologists. METHODS: In a multicenter outcomes project, 210 patients with BE and LGD (classified as incident, prevalent, or persistent) were included. Patients were followed up for an average of 6.2 years (959.6 patient-years). Persistent LGD was defined as detection of LGD on ≥2 consecutive occasions during the follow-up period and extent as either unifocal (LGD at one level of BE segment) or multifocal (>1 level). Histology specimens were reviewed by 2 blinded pathologists. RESULTS: Six patients developed EAC (incidence of 0.44%/year), and 21 developed HGD (incidence of 1.6%/year). The incidence of the combination of HGD and EAC was 1.83%/year. There were no associations between presence of prevalent, incident, or persistent LGD and the extent of LGD with progression rates. Based on consensus diagnosis of 88 reviewed specimens, there was no difference in the progression of LGD to either EAC (the incidence based on analyses by the local pathologist was 0.18%/year, the incidence when there was agreement between the local and one central pathologist was 0.21%/year, and the incidence when all 3 pathologists were in agreement was 0.39%/year) or combined HGD and EAC (0.94%/year, 0.87%/year, and 0.84%/year, respectively). CONCLUSIONS: Overall, patients with BE and LGD have a low annual incidence of EAC, similar to nondysplastic BE. There are no risk factors for progression and there is significant interobserver variation in diagnosis, even among expert pathologists.

Adequacy of esophageal squamous mucosa specimens obtained during endoscopy: are standard biopsies sufficient for postablation surveillance in Barrett's esophagus?

BACKGROUND: After endoscopic eradication therapy (EET) for Barrett's esophagus (BE), surveillance of residual/recurrent intestinal metaplasia/dysplasia including subsquamous tissue is performed by using biopsy forceps. OBJECTIVE: The goal of this study was to compare the adequacy of biopsy specimens obtained from neo-squamous (post-EET patients) and native (non-BE patients) squamous mucosa. DESIGN: A case-control study using squamous biopsy specimens obtained at 2 tertiary referral centers was conducted. INTERVENTIONS: Two experienced GI pathologists reviewed specimens from patients with neo- (post-EET patients) and native (non-BE patients) squamous mucosa in a blinded fashion after developing standardized criteria to assess tissue depth. MAIN OUTCOME MEASUREMENTS: The primary outcome was the proportion of biopsy specimens that contained any amount of lamina propria. RESULTS: A total of 193 biopsy specimens (1692 tissue pieces) from 104 patients were reviewed: 163 neo- and 30 native squamous. Of all biopsy specimens, only 37% contained any amount of lamina propria, and, furthermore, fewer than 4% of specimens had sufficient lamina propria (ie, more than two thirds of the entire squamous tissue present). When examining individual squamous tissue pieces, fewer than 11% contained lamina propria. No statistically significant differences in the presence of lamina propria were detected between neo- and native squamous mucosa. CONCLUSION: The majority of esophageal squamous biopsy specimens obtained during endoscopy do not demonstrate lamina propria and subepithelial structures. This is true for both neo- and native squamous mucosa. Biopsy specimens of neo-squamous mucosa obtained after EET appear to be inadequate to exclude subsquamous intestinal metaplasia/dysplasia because lamina propria is not present in more than 60% of specimens. This has larger implications in the clinical management of BE patients after EET.