The Amsterdam ReBus progressor cohort: identification of 165 Barrett's surveillance patients who progressed to early neoplasia and 723 nonprogressor patients

The Amsterdam ReBus progressor cohort: identification of 165 Barrett's surveillance patients who... SUMMARY Patient selection is suboptimal in most studies focused on identifying biological markers for neoplastic progression in Barrett's esophagus (BE). This study aims to describe a stringently selected community-based case-control cohort of non-dysplastic BE (NDBE) patients who progressed to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) and BE patients who never progressed to be used for future biomarker studies. We identified all patients referred for endoscopic work-up of BE neoplasia at three tertiary referral centers for treatment of BE neoplasia between 2000 and 2013. We performed a detailed registration of any endoscopic surveillance history before neoplastic progression. Controls were selected from a retrospective BE surveillance registration in 10 community hospitals. A total of 887 patients were referred for endoscopic work-up of BE neoplasia. Based on predefined selection criteria, we identified 165 progressor patients (82% men; mean age 55 years ± 10.4) with a baseline endoscopy demonstrating NDBE > 2 years before neoplastic progression. Using the same predefined selection criteria, 723 nonprogressor patients (67% men; mean age 57 years ± 11.3) with >2 years of endoscopic surveillance were identified. Median length of the BE segment was 5 cm (IQR 4–7) in progressors and 4 cm (IQR 2–6) in controls. Median duration of surveillance was 89 months (IQR 54–139) in progressors and 76 months (IQR 47–116) in nonprogressors. Paraffin embedded biopsies are available for biomarker research in all patients. Ethical approval was obtained and material transfer agreements were signed with all 58 contributing pathology labs. This is the largest community-based case-control cohort of BE patients with and without progression to early neoplasia. The stringent selection criteria and the availability of paraffin embedded biopsy specimens make this a unique cohort for biomarker studies. ABBREVIATIONS ABBREVIATIONS BE Barrett's esophagus NDBE non-dysplastic BE LGD low-grade dysplasia HGD high-grade dysplasia EAC esophageal adenocarcinoma LOH loss of heterozygosity AOL Aspergillus oryzae lectin AUC area under the receiver-operating characteristic curve INTRODUCTION In Barrett's esophagus (BE), the squamous epithelial lining of the distal esophagus has been replaced by columnar epithelium containing intestinal metaplasia upon histological investigation of biopsies.1,2 International guidelines recommend endoscopic surveillance with biopsies in patients with BE to detect early neoplasia at a curable stage.2,3 However, endoscopic surveillance has several limitations, such as questionable cost-effectiveness, difficulties with endoscopic identification of early neoplasia, biopsy sampling error, and considerable interobserver variation among pathologists for diagnosing dysplasia.4–7 Despite these known shortcomings, management of BE patients is still based on the subjective histological evaluation of biopsy specimens obtained during surveillance. Patients with non-dysplastic BE (NDBE) are offered lifelong endoscopic surveillance with a 3–5 year interval, whereas patients with low-grade dysplasia (LGD) undergo intensified endoscopic surveillance or may be considered for endoscopic therapy.8,9 A finding of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) warrants therapy.2,3 To optimize management of BE patients, much research is focused on identifying biological markers. Such markers may enable objective evaluation of the presence of neoplasia during BE surveillance and may be used for risk-stratification to identify patients with an increased risk to develop HGD/EAC over time. High-risk patients can then undergo intensified endoscopic surveillance or prophylactic ablation of their BE segment. On the other hand, surveillance may be discontinued or the interval may be prolonged in patients with a low risk profile. Several promising biological markers have been identified such as loss of heterozygosity (LOH) at 17p (p53 gene) or 9p (CDKN2A or p16 gene), chromosomal aneuploidy, hypermethylation of promoter sites for genes such as p16, RUNX3 and HPP1, markers of cell proliferation such as cyclin A and MCM2, and decreased binding of a specific glycan-binding protein Aspergillus oryzae lectin (AOL).10–18 These biological markers have generally been identified in retrospectively identified cohorts of patients who progressed to HGD/EAC (‘progressors’) and in whom biopsy material of their prior surveillance history was available. These progressors were matched for age and sex with a comparable group of BE patients without progression to HGD/EAC during follow-up (‘non-progressors’). All published biomarker studies, however, have significant limitations relating to the selection of patients and the selection of samples. First, many studies have included progressors in whom the diagnosis of HGD/EAC may be questioned: in these patients the diagnosis was based only on a single biopsy, obtained at a single endoscopy, and evaluated by a single pathologist.10–12 Given the interobserver variability in histological diagnosis of HGD/EAC, this may imply that the group of progressors may have been diluted by inclusion of patients without real progression.7 Second, most studies have included progressors with only a short interval between the index BE diagnosis and the diagnosis of HGD/EAC: many studies only excluded cases that progressed within 6 months and in the majority of progressors the interval between the baseline endoscopy used for biomarker testing and the date of progression was less than 2 years.10–15,17,18 With such a relatively short interval between the baseline diagnosis of NDBE and the detection of HGD/EAC, the possibility of prevalent HGD/EAC at baseline cannot be excluded. In order to exclude the presence of HGD/EAC at baseline, the quality of the index endoscopy and the extent of random biopsy sampling at baseline is also important. None of the published biomarker studies have used the quality of baseline sampling as a selection criterion. Apart from the time interval between baseline and HGD/EAC diagnosis and the quality of random biopsy sampling at the baseline endoscopy, the stage of disease at the time of progression is also relevant to exclude prevalent HGD/EAC. Most published cohorts have included all patients with HGD/EAC irrespective of the stage of disease at the time of diagnosis.10–12,14,15,17,18 However, in patients with advanced cancer, even a long interval (e.g. 5 years) between the baseline BE diagnosis and cancer diagnosis may not suffice to exclude the possibility of prevalent neoplasia at baseline. Finally, another limitation of many published biomarker studies is that these have used fresh-frozen biopsy specimens, which is not a practical and readily available source of material outside the academic setting.10–15 In our opinion, for a biomarker panel to be applicable in the setting of community BE surveillance, the assays should perform well on paraffin embedded biopsy specimens. The main objective of this project (the Predict neoplastic progREssion in Barrett's esophagUS, or Amsterdam ReBus project) was to construct a case-control cohort that can be used to identify and validate biological markers for neoplastic progression in BE. We first aimed to identify a cohort of BE patients who progressed to HGD/EAC using strict criteria for the diagnosis and stage of neoplastic progression, the interval between baseline sampling and progression diagnosis, and the quality of the baseline endoscopy. Second, we aimed to identify a cohort of community-based BE patients who did not manifest HGD/EAC using the same strict selection criteria to allow for matching with progressors for age, gender, length of BE, and duration of follow-up. Third, we aimed to collect all relevant patient data and tissue blocks with informed consent of patients and legal and ethical transfer of all rights to our unit. The identification of this cohort of progressors and nonprogressors is anticipated to be the starting point for multiple biomarker studies in this field. This publication describes the selection process and characteristics of this cohort. METHODS Setting and source population BE surveillance patients who progressed to HGD/EAC were identified at three centers in the Netherlands: the Academic Medical Center (AMC) Amsterdam, the St Antonius Hospital in Nieuwegein, and the Catharina Hospital in Eindhoven. These three centers have a tertiary referral function for endoscopic work-up and treatment of early Barrett's neoplasia, with approximately 120 BE patients undergoing endoscopic work-up for HGD/EAC per year. In approximately 50% of these patients, the early neoplasia is a coincidental finding at the initial endoscopy. The other 50% of patients, however, have been diagnosed with BE at an earlier stage and have a BE surveillance history prior to the diagnosis of HGD/EAC.19 Controls without progression were identified from a retrospective Barrett's surveillance registration cohort that contains approximately 2500 BE patients from 10 community hospitals in the Amsterdam region.20 Identification, data collection, and inclusion criteria for progressors Two researchers independently performed a chart evaluation of all patients who underwent endoscopic work-up for BE neoplasia at the three participating centers between January 2000 and December 2013. Endoscopic imaging and treatment of early neoplasia were performed at the three participating sites, but the prior surveillance history in community hospitals was only superficially summarized on referral. For the purpose of this project, all surveillance endoscopies with biopsies were identified in the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA database). The PALGA database has nationwide coverage since 1991, archiving the reports of all pathology laboratories in the Netherlands.21 Subsequently, all original surveillance endoscopy and pathology reports were retrieved from the referring hospitals. Irrespective of the final histological diagnosis or presence of a surveillance history, standardized case report forms were completed for all patients. This included information on baseline characteristics (e.g. relevant medical history, esophageal landmarks), surveillance history (e.g. baseline BE diagnosis, quality of baseline biopsy sampling, number and outcome of surveillance endoscopies, interval between baseline BE diagnosis and first HGD/EAC diagnosis), the endpoint of progression (e.g. worst histological diagnosis, number of endoscopies with HGD/EAC, and specimen type in which HGD/EAC was diagnosed) and treatment modality and outcome of treatment. We only included patients with maximum T1-disease at time of progression based on either an endoscopic resection specimen, esophagectomy specimen, or biopsy specimens obtained at two separate endoscopies. All histological diagnoses of progression were made by at least two pathologists. The minimum required surveillance interval before neoplastic progression was 2 years. The baseline endoscopy demonstrating NDBE was required to be of sufficient quality (i.e. > 50% of required number of biopsies according to Seattle protocol were obtained). We only included patients with a baseline diagnosis of NDBE. For this a histological diagnosis by a single pathologist sufficed. In case of a baseline diagnosis of LGD, slides were reviewed by two expert pathologists. Such patients were only included if this baseline diagnosis was unanimously downstaged to NDBE. Identification, data collection and inclusion criteria for nonprogressors In 2003, the Amsterdam Gastroenterological Association (also known as the ‘Amsterdam Gutclub’) initiated a retrospective registration of all Barrett's patients in 16 hospitals in the Amsterdam region. At that time, the PALGA database of each participating hospital was searched with the search strings ‘Barrett’ and ‘intestinal metaplasia and esophagus.’ Two research nurses, each with at least 3 years of experience with Barrett's surveillance, reviewed all endoscopy and pathology reports in the participating hospitals for verification of the diagnosis of BE, defined as columnar lined epithelium on endoscopy and intestinal metaplasia on histology. After verification of the diagnosis, patient demographics, history, and information from endoscopy and pathology reports were entered in a dedicated registration database. Following this retrospective analysis, a prospective BE surveillance registry was initiated in 6 of these 16 hospitals. In the remaining 10 hospitals, the retrospective analysis was updated in 2012, which enabled us to identify newly diagnosed BE patients and retrieve long-term follow-up information for the patients that were identified in the first retrospective analysis. All patients who did not eventually develop HGD or EAC and who underwent at least 2 endoscopies with a minimum surveillance interval of 2 years were eligible for inclusion as nonprogressor. We performed central expert pathology review of all baseline biopsies with an original diagnosis of LGD and only included patients in whom this diagnosis was downstaged to NDBE. The prospective surveillance cohort in the remaining 6 hospitals includes progressor and nonprogressor patients and remains available as an independent validation cohort for phase 4 biomarker studies. The relation between the respective patient cohorts is depicted in Figure 1. Fig. 1 View largeDownload slide The Amsterdam ReBus project: a graphical representation of the source population for the different patient cohorts. Fig. 1 View largeDownload slide The Amsterdam ReBus project: a graphical representation of the source population for the different patient cohorts. Endpoints and duration of surveillance The endpoint of the study was the first diagnosis of HGD/EAC (progressors) or the most recent endoscopic procedure with a histological diagnosis (nonprogressors). Duration of surveillance was calculated as the interval between the endoscopy date on which intestinal metaplasia was first detected and the endpoint of the study. The minimum duration of surveillance was calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed at least 2 years before the endpoint. Patient material, ethical and legal considerations All selected patients were contacted to obtain written informed consent for transferring medical data and paraffin embedded tissue, obtained during surveillance endoscopies in referring hospitals, to the AMC for biomarker research. The Dutch Medical Research Involving Human Subjects Act (Wet Medisch-wetenschappelijk Onderzoek met mensen, WMO) does not apply to this project and the ethics committee of the AMC has, therefore, exempted this study from formal ethical review. With respect to the processing of personal (medical) data, the Dutch Data Protection Act (Wet Bescherming Persoonsgegevens) and the Dutch Civil Code on Agreements with Regard to Medical Treatment (de Wet Geneeskundige BehandelingsOvereenkomst) apply. The principals of these laws regarding the use of data for research purposes are structured in the Code of Conduct ‘Use of Data in Health Research’ (Gedragscode Gezondheidsonderzoek - 2004). All human tissue samples that were collected under this protocol, are samples obtained for diagnosis and medical treatment purposes. These samples are considered residual human tissue. To this type of tissue, the Dutch Code of Conduct ‘Proper Secondary Use of Human Tissue’ (Gedragscode Goed Gebruik - 2011) applies. Both Codes of Conduct are issued and published by the Dutch Federation of Medical Research Associations (Federatie van Medisch Wetenschappelijke Verenigingen). These Codes of Conduct were adhered to, to the extent applicable to this project. Residual human tissue for this project was gathered from referring hospitals throughout the Netherlands. In order to transfer ownership of the material and to ensure that third parties can use the tissue material for research purposes, material transfer agreements (MTAs) have been signed with all 58 referring hospitals. Ethical approval was obtained from the ethics committee and the biobank review committee of the AMC, as well as the ethics committees of all 10 hospitals participating in the retrospective regional Barrett's registry. Only patients who gave written informed consent for this project are included in the cohort. Designing the legal and ethical framework for the ReBus-project was performed in 2010. Acquiring the MTAs from all 58 hospitals were the biopsy specimens resided was performed between 2010 and 2012. Identification of eligible patients, obtaining informed consent, data collection, and sample retrieval was performed between 2011 and 2013. Statistical analysis Categorical variables are described using percentages. Continuous variables with a normal distribution are presented as mean and standard deviation, median, and interquartile range are used for continuous variables with a skewed distribution. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 20.0, IBM Corp., Armonk, NY, USA). RESULTS Progressor patients The selection of progressor patients in the study is summarized in Figure 2. Demographics and surveillance characteristics of this cohort are summarized in Table 1. Between 2000 and 2013, 629 patients were referred to the AMC, 138 were referred to the St Antonius Hospital, and 120 were referred to the Catharina Hospital for endoscopic work-up of esophageal neoplasia and were screened for eligibility. Of these 887 patients, 286 patients were diagnosed with HGD or early EAC (T1N0M0) and had a surveillance history before the diagnosis of neoplastic progression, irrespective of the duration of the surveillance interval and the modality that was used to diagnose progression. Fig. 2 View largeDownload slide Flowchart of patients into the ReBus progressor cohort and the regional Barrett's registration cohorts in the Amsterdam region. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; ER, endoscopic resection; HGD, high-grade dysplasia; * We did not ask patients for informed consent due to advanced age, or severe comorbidities. ** Includes patients with confirmed LGD as well as patients for whom the original histology slides demonstrating LGD could not be retrieved. Fig. 2 View largeDownload slide Flowchart of patients into the ReBus progressor cohort and the regional Barrett's registration cohorts in the Amsterdam region. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; ER, endoscopic resection; HGD, high-grade dysplasia; * We did not ask patients for informed consent due to advanced age, or severe comorbidities. ** Includes patients with confirmed LGD as well as patients for whom the original histology slides demonstrating LGD could not be retrieved. Table 1 Baseline and surveillance characteristics for progressor and non-progressor patients and information on progression diagnosis for progressor patients   NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA    NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA  †Calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed >2 years before the endpoint. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; HGD, high-grade dysplasia; NA, not applicable; NDBE, nondysplastic BE. . View Large Table 1 Baseline and surveillance characteristics for progressor and non-progressor patients and information on progression diagnosis for progressor patients   NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA    NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA  †Calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed >2 years before the endpoint. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; HGD, high-grade dysplasia; NA, not applicable; NDBE, nondysplastic BE. . View Large Diagnosis of progression to HGD/EAC One hundred patients were diagnosed with HGD, 186 patients with early adenocarcinoma (134 patients with intramucosal carcinoma (T1m1/m2/m3), 35 patients with submucosal cancer (T1sm1/sm2/sm3) and 17 with unknown infiltration depth). Patients with advanced carcinoma (≥T2) were not included (n = 81). The worst histological diagnosis was based on an ER specimen in 216 patients, on an esophagectomy specimen in seven patients and on biopsies in 63 patients. Of this last patient group, 50 patients were diagnosed with HGD/EAC based on biopsies from two or more endoscopies. The remaining 13 patients with a single biopsy diagnosis of HGD/EAC were excluded. Surveillance history of progressor patients Of the patients with incident HGD/EAC and an unequivocal progression diagnosis (n = 273), 59 patients had a BE surveillance history less than 2 years and were excluded from the study. Of the remaining 214 patients, we excluded 13 patients with insufficient quality of baseline biopsy sampling and eight patients who did not consent to the study. In 41/55 patients with baseline LGD the original histology slides were retrieved and LGD was confirmed in 14 of those patients who were subsequently excluded. We also excluded the 14 patients in whom we could not retrieve the original histology slides, resulting in a progressor cohort of 165 patients. Treatment of progressor patients and follow-up after treatment Progressor patients diagnosed with EAC who had ≥1 risk factors for lymph node metastasis (i.e. submucosal invasion, lymphovascular invasion, poor tumor differentiation) or in whom the depth of infiltration was unknown were considered high risk (n = 39). Patients with HGD or intramucosal EAC and none of the above mentioned risk factors were classified as low risk (n = 126). Treatment strategy and outcome for patients at low- and high-risk of lymph node metastasis are shown in Figure 3. Fig. 3 View largeDownload slide Treatment modality and outcome of treatment for progressor patients at high and low risk of lymph node metastasis. APC, argon plasma coagulation; CR-IM, complete remission of intestinal metaplasia; CR-N, complete remission of neoplasia; PDT photodynamic therapy; RFA, radiofrequency ablation. Fig. 3 View largeDownload slide Treatment modality and outcome of treatment for progressor patients at high and low risk of lymph node metastasis. APC, argon plasma coagulation; CR-IM, complete remission of intestinal metaplasia; CR-N, complete remission of neoplasia; PDT photodynamic therapy; RFA, radiofrequency ablation. Selection of nonprogressors Selection of non-progressors is depicted in Figure 2 and baseline characteristics are summarized in Table 1. A total of 2350 Barrett's patients were entered in a retrospective regional registration database. Of these, 2206 were never diagnosed with HGD/EAC and 1280 of these nonprogressor patients had an endoscopic surveillance history (i.e. underwent at least 2 surveillance endoscopies). In 1040 patients, the duration of the endoscopic surveillance history was 2 years or more. We obtained informed consent for the study in 780 of these patients. In 56/105 patients with baseline LGD, the original histology slides were retrieved and LGD was confirmed in 8 of those patients who were subsequently excluded. We further excluded all 49 patients with baseline LGD for whom we could not retrieve the original histology slides, resulting in a nonprogressor cohort of 723 patients. DISCUSSION In this publication, we have outlined the selection and characteristics of a community-based nested case-control cohort of BE patients available for biomarker research. Using stringent selection criteria, 165 Barrett's surveillance patients were identified who progressed from NDBE to HGD or EAC. Controls were identified from a large population-based cohort of BE surveillance patients who never demonstrated neoplastic progression (n = 723). Depending on the set-up and sample size of intended biomarker studies, progressors can be matched with nonprogressors in different ratios by baseline variables (i.e. age, gender, length of the BE segment and duration of surveillance). Several research groups have investigated biomarkers in case-control cohorts and published promising results suggesting that some of these markers may be of value in risk stratifying BE patients (Table 2). There are three main clusters of relevant publications we would like to discuss. The Seattle group has performed several studies in a prospective cohort of BE patients enrolled in the Seattle Barrett's Esophagus Study from 1992 onwards. They published studies on LOH for 17p (RR 16; 95% CI 6–39) and measures of clonal diversity (RR 11; 95% CI 6–21).10,11 Subsequently, this group has published a panel of markers consisting of abnormal DNA ploidy and LOH for 9p and 17p, which was highly associated with neoplastic progression (RR 39; 95% CI 11–139).12 There are, however, major limitations in the selection of patients for the Seattle cohort that need to be addressed. The main limitation in this cohort is the lack of a reliable baseline diagnosis before progression. These studies have assessed progression to EAC alone, but have also included BE patients with HGD at baseline in the progressor cohort. It is likely that a proportion of these patients had synchronous EAC at baseline since a diagnosis of HGD is often indicative of a visible lesion elsewhere in the BE segment. These lesions are often hard to detect during standard endoscopy, especially with low-resolution endoscopes that were used in the 90s. Additionally, the agreement among expert GI pathologists for the distinction between HGD and EAC is limited, especially when based on histological interpretation of biopsy specimens.22 In conjunction with the fact that patients were not excluded when progression to EAC was diagnosed shortly after the baseline endoscopy, this may have led to the inclusion of patients with prevalent neoplasia at baseline. Therefore, the reported odds of progression for the Reid biomarker panel are likely inflated. Furthermore, most international guidelines currently recommend endoscopic eradication therapy for BE patients with HGD, which renders the prediction of progression from HGD to EAC irrelevant. In addition, external validity of the findings may be limited since the Seattle cohort originates in a tertiary referral setting and the biomarker research was performed on snap-frozen specimens. DNA extraction is easier from snap-frozen biopsy specimens compared with formalin fixed paraffin embedded specimens, however, fresh frozen samples are not a practical and readily available source of material outside the academic setting. Table 2 Summary of main clusters of phase 3 biomarker studies in Barrett's esophagus patients           Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes            Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes  †Diagnosis of HGD/EAC based on endoscopic resection specimen, esophagectomy specimen or biopsies obtained at two separate endoscopies; ‡Studies assessed progression to EAC alone and included patients with HGD at baseline. AUC, area under receiver-operating characteristic curve; EAC, esophageal adenocarcinoma; LGD, low-grade dysplasia; LOH, loss of heterozygosity; NDBE, nondysplastic Barrett's esophagus; OR, odds ratio; RR, relative risk. View Large Table 2 Summary of main clusters of phase 3 biomarker studies in Barrett's esophagus patients           Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes            Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes  †Diagnosis of HGD/EAC based on endoscopic resection specimen, esophagectomy specimen or biopsies obtained at two separate endoscopies; ‡Studies assessed progression to EAC alone and included patients with HGD at baseline. AUC, area under receiver-operating characteristic curve; EAC, esophageal adenocarcinoma; LGD, low-grade dysplasia; LOH, loss of heterozygosity; NDBE, nondysplastic Barrett's esophagus; OR, odds ratio; RR, relative risk. View Large The Baltimore group has studied epigenetic markers in the Baltimore VA and University of Maryland Barrett's cohort. Focusing on promoter methylation in a panel of 10 genes hypermethylation of p16, RUNX3 and HPP1 were statistically significant predictors of neoplastic progression in a multivariable analysis.13 Subsequently, this 3-marker panel was combined with a set of clinical features to stratify patients into low-risk, intermediate-risk, and high-risk groups of progression (4-year prediction model, area under the receiver-operating characteristic curve (AUC) = 0.791).14 Subsequent studies identified 5 genes that were methylated early and often in neoplastic progression (i.e. nel-like 1 (NELL1), tachykinin-1 (TAC1), somatostatin (SST), AKAP12, and CDH13). These 5 genes were combined with p16, RUNX3 and HPP1, to form an 8-gene panel that was validated in a subsequent study.15 The AUC for the 2- and 4-year prediction models combined was 0.840. These results from the Baltimore group are promising, but again the selection of patients has several limitations. First, for all 3 studies it is uncertain if the baseline endoscopy of patients who progressed did not contain HGD or EAC. The study by Schulman and colleagues had no minimum surveillance interval required to exclude prevalent HGD/EAC. In fact, most of the samples that generated high hazard ratios in this study were obtained less than 2 years before progression. The other two studies stratified progressor patients according to the duration of their progression free surveillance interval. However, they did not exclude progressor patients with advanced lesions, which may still have led to inclusion of patients with prevalent disease at baseline. Second, for the latter 2 studies we are not informed about the modality that was used to establish the progression diagnosis, which may have diluted the progressor cohort with patients who lacked genuine neoplastic progression (‘single biopsy HGD-cases’). Finally, the external validity may be limited since patients in all three studies were enrolled at tertiary referral centers and biomarker research was performed on snap-frozen biopsies. The Cambridge group has recently published a promising panel of markers consisting of a consensus diagnosis of LGD, abnormal DNA ploidy, and AOL.17 In patients with consensus LGD, the adjusted OR for progression was 3.74 (95% CI 2.43–5.79) for each additional biomarker and the risk increased by 2.99 (95% CI 1.72–5.20) for each additional marker in patients without LGD. This study was performed on a population-based matched case-control cohort from the Northern Ireland Barrett's Esophagus Registry (NIBR). Also in this cohort, the main limitation is the lack of a reliable baseline diagnosis without HGD/EAC. Minimum required progression-free surveillance interval for progressor patients was only 6 months. Additionally, patients with advanced EAC were not excluded, increasing the likelihood of prevalent neoplasia at baseline. Finally, patients with a single endoscopy, single biopsy diagnosis of HGD qualified as progressor patients, which might have diluted the progressor cohort as described previously. A further limitation of all BE case-control cohorts described above is the limited size of the progressor cohorts. Apart from the study by Fitzgerald et al. in the NIBR cohort (89 progressors), the number of progressor patients does not exceed 50 (Table 2). This is likely to be an insufficient number of patients to validate findings from biomarker identification studies. In conclusion, recent studies reported promising results in the field of biomarkers for progression in BE. However, patient selection in these studies was flawed in several ways, as we have outlined above and summarized in Table 2. Major limitations are the lack of a reliable baseline diagnosis without HGD/EAC, the absence of an unequivocally diagnosed progression endpoint and the limited size of the progressor cohorts. Additionally, almost all studies were performed in a tertiary referral setting and have used biopsy material that is not widely available in the community setting. In order to overcome the previously described limitations in the selection of patients for biomarker research in BE, we have described a stringently selected population-based cohort. We only included patients with an unequivocal diagnosis of HGD/EAC, based on either an endoscopic resection specimen, an esophagectomy specimen or biopsy specimens obtained at two separate endoscopies. Progressor patients diagnosed with advanced cancer were excluded. We selected progressor patients with a minimum of two years surveillance before neoplastic progression and only included those who had sufficient quality of baseline biopsy sampling. We excluded all patients in whom baseline LGD was confirmed after central expert histology review as well as those for whom we could not retrieve the original histology slides demonstrating baseline LGD. In our opinion, these patients should be excluded, since previous studies have demonstrated that patients with a confirmed diagnosis of LGD have a highly increased risk of progression.20,23–25 Outcomes of biomarker studies that include patients with LGD are, therefore, likely affected by the outcome of LGD patients. To allow for matching of progressors with Barrett's patients without neoplastic progression, we selected nonprogressor patients from a population-based Barrett's registry, avoiding the tertiary referral bias as present in other biomarker studies. For each biomarker research project to be performed in this cohort, progressors and controls can be matched depending on the size and ratio of the intended cohort. The matched case-control cohort that ensues is an optimal selection of patients for phase 3 biomarker studies in the field of BE. Notes Conflicts of interest: JJGHMB receives research support from Olympus Endoscopy, Cook Medical, Boston Scientific, GI Solutions Covidien, Erbe and Ninepoint Medical; receives financial support for training programs from GI Solutions Covidien; and receives honorarium-consultancy-speakers fee from Cook Medical, Boston Scientific and GI Solutions Covidien. BLAMW receives research support from Covidien GI solutions, Erbe Medical and C2Therapeutic and has received consulting fees from Boston Scientific and C2Therapeutic. All other authors have no conflicts of interest to declare. Specific author contributions: Study concept and design: Lucas C. Duits, David F. Boerwinkel, Roos E. Pouw, Jacques J.G.H.M. Bergman; Acquisition of data: Lucas C. Duits, Esther Klaver, Angela Bureo Gonzalez, David F. Boerwinkel, Fiebo J.W. ten Kate, G. Johan A. Offerhaus, Sybren L. Meijer, Mike Visser, Cees A. Seldenrijk, Kausilia K. Krishnadath, Erik J. Schoon, Bas L.A.M. Weusten; Analysis and interpretation of data: Lucas C. Duits, Esther Klaver, Angela Bureo Gonzalez, David F. Boerwinkel, Rosalie C. Mallant-Hent, Roos E. Pouw, Jacques J.G.H.M. Bergman; Drafting of the manuscript: Lucas C. Duits, David F. Boerwinkel; Critical revision of the manuscript for important intellectual content and final approval of the manuscript: All authors; study supervision: Roos E. Pouw, Jacques J.G.H.M. Bergman. Grant support: None. References 1 Shaheen N J, Richter J E. Barrett's oesophagus. Lancet  2009; 373: 850– 61. Google Scholar CrossRef Search ADS PubMed  2 Spechler S J, Sharma P, Souza R F et al.   American gastroenterological association technical review on the management of Barrett's esophagus. Gastroenterology  2011; 140: e18– 52. Google Scholar CrossRef Search ADS PubMed  3 Fitzgerald R C, Pietro M di, Ragunath K et al.   British Society of Gastroenterology guidelines on the diagnosis and management of Barrett's oesophagus. Gut  2014; 63: 7– 42. Google Scholar CrossRef Search ADS PubMed  4 Gordon L G, Mayne G C, Hirst N G et al.   Cost-effectiveness of endoscopic surveillance of non-dysplastic Barrett's esophagus. Gastrointest Endosc  2014; 79: 242– 56. e6. Google Scholar CrossRef Search ADS PubMed  5 Curvers W L, Bansal A, Sharma P et al.   Endoscopic work-up of early Barrett's neoplasia. Endoscopy  2008; 40: 1000– 7. Google Scholar CrossRef Search ADS PubMed  6 Tschanz E R. Do 40% of patients resected for Barrett esophagus with high-grade dysplasia have unsuspected adenocarcinoma? Arch Pathol Lab Med Online  2005; 129: 177– 80. 7 Montgomery E, Bronner M P, Goldblum J R et al.   Reproducibility of the diagnosis of dysplasia in Barrett esophagus: a reaffirmation. Hum Pathol  2001; 32: 368– 78. Google Scholar CrossRef Search ADS PubMed  8 Phoa K N, Vilsteren F G I van, Weusten B L A M et al.   Radiofrequency ablation vs endoscopic surveillance for patients with Barrett esophagus and low-grade dysplasia. JAMA  2014; 311: 1209– 17. Google Scholar CrossRef Search ADS PubMed  9 Rubenstein J H, Kwon R S. Radiofrequency ablation for Barrett's esophagus with low-grade dysplasia: a hammer looking for a nail. Gastroenterology  2014; 147: 706– 7. Google Scholar CrossRef Search ADS PubMed  10 Reid B, Prevo L, Galipeau P. Predictors of progression in Barrett's esophagus II: baseline 17p (p53) loss of heterozygosity identifies a patient subset at increased risk for neoplastic progression. Am J Gastroenterol  2001; 96: 2839– 48. Google Scholar CrossRef Search ADS PubMed  11 Maley C C, Galipeau P C, Finley J C et al.   Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat Genet  2006; 38: 468– 73. Google Scholar CrossRef Search ADS PubMed  12 Galipeau P C, Li X, Blount P L et al.   NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma. PLoS Med  2007; 4: e67. Google Scholar CrossRef Search ADS PubMed  13 Schulmann K, Sterian A, Berki A et al.   Inactivation of p16, RUNX3 and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk. Oncogene  2005; 24: 4138– 48. Google Scholar CrossRef Search ADS PubMed  14 Sato F, Jin Z, Schulmann K et al.   Three-tiered risk stratification model to predict progression in Barrett's esophagus using epigenetic and clinical features. PLoS One  2008; 3: e1890. Google Scholar CrossRef Search ADS PubMed  15 Jin Z, Cheng Y, Gu W et al.   A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett's esophagus. Cancer Res  2009; 69: 4112– 5. Google Scholar CrossRef Search ADS PubMed  16 Lao-Sirieix P, Lovat L, Fitzgerald R C. Cyclin a immunocytology as a risk stratification tool for Barrett's esophagus surveillance. Clin Cancer Res  2007; 13: 659– 65. Google Scholar CrossRef Search ADS PubMed  17 Bird-Lieberman E L, Dunn J M, Coleman H G et al.   Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus. Gastroenterology  2012; 143: 927– 935.e3. Google Scholar CrossRef Search ADS PubMed  18 Murray L, Sedo A, Scott M et al.   TP53 and progression from Barrett's metaplasia to oesophageal adenocarcinoma in a UK population cohort. Gut  2006; 55: 1390– 7. Google Scholar CrossRef Search ADS PubMed  19 Peters F P, Curvers W L, Rosmolen W D et al.   Surveillance history of endoscopically treated patients with early Barrett's neoplasia: nonadherence to the Seattle biopsy protocol leads to sampling error. Dis Esophagus  2008; 21: 475– 9. Google Scholar CrossRef Search ADS PubMed  20 Curvers W L, Kate F J ten, Krishnadath K K et al.   Low-grade dysplasia in Barrett's esophagus: overdiagnosed and underestimated. Am J Gastroenterol  2010; 105: 1523– 30. Google Scholar CrossRef Search ADS PubMed  21 Casparie M, Tiebosch A T M G, Burger G et al.   Pathology databanking and biobanking in The Netherlands, a central role for PALGA, the nationwide histopathology and cytopathology data network and archive. Cell Oncol  2007; 29: 19– 24. Google Scholar PubMed  22 Ormsby A H, Petras R E, Henricks W H et al.   Observer variation in the diagnosis of superficial oesophageal adenocarcinoma. Gut  2002; 51: 671– 6. Google Scholar CrossRef Search ADS PubMed  23 Duits L C, Phoa K N, Curvers W L et al.   Barrett's oesophagus patients with low-grade dysplasia can be accurately risk-stratified after histological review by an expert pathology panel. Gut  2015; 64: 700– 6. Google Scholar CrossRef Search ADS PubMed  24 Lim C, Treanor D, Dixon M et al.   Low-grade dysplasia in Barrett's esophagus has a high risk of progression. Endoscopy  2007; 39: 581– 7. Google Scholar CrossRef Search ADS PubMed  25 Vieth M, Schubert B, Lang-Schwarz K et al.   Frequency of Barrett's neoplasia after initial negative endoscopy with biopsy: a long-term histopathological follow-up study. Endoscopy  2006; 38: 1201– 5. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diseases of the Esophagus Oxford University Press

The Amsterdam ReBus progressor cohort: identification of 165 Barrett's surveillance patients who progressed to early neoplasia and 723 nonprogressor patients

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The International Society for Diseases of the Esophagus
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© The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus.
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1120-8694
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1442-2050
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10.1093/dote/doy037
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Abstract

SUMMARY Patient selection is suboptimal in most studies focused on identifying biological markers for neoplastic progression in Barrett's esophagus (BE). This study aims to describe a stringently selected community-based case-control cohort of non-dysplastic BE (NDBE) patients who progressed to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) and BE patients who never progressed to be used for future biomarker studies. We identified all patients referred for endoscopic work-up of BE neoplasia at three tertiary referral centers for treatment of BE neoplasia between 2000 and 2013. We performed a detailed registration of any endoscopic surveillance history before neoplastic progression. Controls were selected from a retrospective BE surveillance registration in 10 community hospitals. A total of 887 patients were referred for endoscopic work-up of BE neoplasia. Based on predefined selection criteria, we identified 165 progressor patients (82% men; mean age 55 years ± 10.4) with a baseline endoscopy demonstrating NDBE > 2 years before neoplastic progression. Using the same predefined selection criteria, 723 nonprogressor patients (67% men; mean age 57 years ± 11.3) with >2 years of endoscopic surveillance were identified. Median length of the BE segment was 5 cm (IQR 4–7) in progressors and 4 cm (IQR 2–6) in controls. Median duration of surveillance was 89 months (IQR 54–139) in progressors and 76 months (IQR 47–116) in nonprogressors. Paraffin embedded biopsies are available for biomarker research in all patients. Ethical approval was obtained and material transfer agreements were signed with all 58 contributing pathology labs. This is the largest community-based case-control cohort of BE patients with and without progression to early neoplasia. The stringent selection criteria and the availability of paraffin embedded biopsy specimens make this a unique cohort for biomarker studies. ABBREVIATIONS ABBREVIATIONS BE Barrett's esophagus NDBE non-dysplastic BE LGD low-grade dysplasia HGD high-grade dysplasia EAC esophageal adenocarcinoma LOH loss of heterozygosity AOL Aspergillus oryzae lectin AUC area under the receiver-operating characteristic curve INTRODUCTION In Barrett's esophagus (BE), the squamous epithelial lining of the distal esophagus has been replaced by columnar epithelium containing intestinal metaplasia upon histological investigation of biopsies.1,2 International guidelines recommend endoscopic surveillance with biopsies in patients with BE to detect early neoplasia at a curable stage.2,3 However, endoscopic surveillance has several limitations, such as questionable cost-effectiveness, difficulties with endoscopic identification of early neoplasia, biopsy sampling error, and considerable interobserver variation among pathologists for diagnosing dysplasia.4–7 Despite these known shortcomings, management of BE patients is still based on the subjective histological evaluation of biopsy specimens obtained during surveillance. Patients with non-dysplastic BE (NDBE) are offered lifelong endoscopic surveillance with a 3–5 year interval, whereas patients with low-grade dysplasia (LGD) undergo intensified endoscopic surveillance or may be considered for endoscopic therapy.8,9 A finding of high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) warrants therapy.2,3 To optimize management of BE patients, much research is focused on identifying biological markers. Such markers may enable objective evaluation of the presence of neoplasia during BE surveillance and may be used for risk-stratification to identify patients with an increased risk to develop HGD/EAC over time. High-risk patients can then undergo intensified endoscopic surveillance or prophylactic ablation of their BE segment. On the other hand, surveillance may be discontinued or the interval may be prolonged in patients with a low risk profile. Several promising biological markers have been identified such as loss of heterozygosity (LOH) at 17p (p53 gene) or 9p (CDKN2A or p16 gene), chromosomal aneuploidy, hypermethylation of promoter sites for genes such as p16, RUNX3 and HPP1, markers of cell proliferation such as cyclin A and MCM2, and decreased binding of a specific glycan-binding protein Aspergillus oryzae lectin (AOL).10–18 These biological markers have generally been identified in retrospectively identified cohorts of patients who progressed to HGD/EAC (‘progressors’) and in whom biopsy material of their prior surveillance history was available. These progressors were matched for age and sex with a comparable group of BE patients without progression to HGD/EAC during follow-up (‘non-progressors’). All published biomarker studies, however, have significant limitations relating to the selection of patients and the selection of samples. First, many studies have included progressors in whom the diagnosis of HGD/EAC may be questioned: in these patients the diagnosis was based only on a single biopsy, obtained at a single endoscopy, and evaluated by a single pathologist.10–12 Given the interobserver variability in histological diagnosis of HGD/EAC, this may imply that the group of progressors may have been diluted by inclusion of patients without real progression.7 Second, most studies have included progressors with only a short interval between the index BE diagnosis and the diagnosis of HGD/EAC: many studies only excluded cases that progressed within 6 months and in the majority of progressors the interval between the baseline endoscopy used for biomarker testing and the date of progression was less than 2 years.10–15,17,18 With such a relatively short interval between the baseline diagnosis of NDBE and the detection of HGD/EAC, the possibility of prevalent HGD/EAC at baseline cannot be excluded. In order to exclude the presence of HGD/EAC at baseline, the quality of the index endoscopy and the extent of random biopsy sampling at baseline is also important. None of the published biomarker studies have used the quality of baseline sampling as a selection criterion. Apart from the time interval between baseline and HGD/EAC diagnosis and the quality of random biopsy sampling at the baseline endoscopy, the stage of disease at the time of progression is also relevant to exclude prevalent HGD/EAC. Most published cohorts have included all patients with HGD/EAC irrespective of the stage of disease at the time of diagnosis.10–12,14,15,17,18 However, in patients with advanced cancer, even a long interval (e.g. 5 years) between the baseline BE diagnosis and cancer diagnosis may not suffice to exclude the possibility of prevalent neoplasia at baseline. Finally, another limitation of many published biomarker studies is that these have used fresh-frozen biopsy specimens, which is not a practical and readily available source of material outside the academic setting.10–15 In our opinion, for a biomarker panel to be applicable in the setting of community BE surveillance, the assays should perform well on paraffin embedded biopsy specimens. The main objective of this project (the Predict neoplastic progREssion in Barrett's esophagUS, or Amsterdam ReBus project) was to construct a case-control cohort that can be used to identify and validate biological markers for neoplastic progression in BE. We first aimed to identify a cohort of BE patients who progressed to HGD/EAC using strict criteria for the diagnosis and stage of neoplastic progression, the interval between baseline sampling and progression diagnosis, and the quality of the baseline endoscopy. Second, we aimed to identify a cohort of community-based BE patients who did not manifest HGD/EAC using the same strict selection criteria to allow for matching with progressors for age, gender, length of BE, and duration of follow-up. Third, we aimed to collect all relevant patient data and tissue blocks with informed consent of patients and legal and ethical transfer of all rights to our unit. The identification of this cohort of progressors and nonprogressors is anticipated to be the starting point for multiple biomarker studies in this field. This publication describes the selection process and characteristics of this cohort. METHODS Setting and source population BE surveillance patients who progressed to HGD/EAC were identified at three centers in the Netherlands: the Academic Medical Center (AMC) Amsterdam, the St Antonius Hospital in Nieuwegein, and the Catharina Hospital in Eindhoven. These three centers have a tertiary referral function for endoscopic work-up and treatment of early Barrett's neoplasia, with approximately 120 BE patients undergoing endoscopic work-up for HGD/EAC per year. In approximately 50% of these patients, the early neoplasia is a coincidental finding at the initial endoscopy. The other 50% of patients, however, have been diagnosed with BE at an earlier stage and have a BE surveillance history prior to the diagnosis of HGD/EAC.19 Controls without progression were identified from a retrospective Barrett's surveillance registration cohort that contains approximately 2500 BE patients from 10 community hospitals in the Amsterdam region.20 Identification, data collection, and inclusion criteria for progressors Two researchers independently performed a chart evaluation of all patients who underwent endoscopic work-up for BE neoplasia at the three participating centers between January 2000 and December 2013. Endoscopic imaging and treatment of early neoplasia were performed at the three participating sites, but the prior surveillance history in community hospitals was only superficially summarized on referral. For the purpose of this project, all surveillance endoscopies with biopsies were identified in the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA database). The PALGA database has nationwide coverage since 1991, archiving the reports of all pathology laboratories in the Netherlands.21 Subsequently, all original surveillance endoscopy and pathology reports were retrieved from the referring hospitals. Irrespective of the final histological diagnosis or presence of a surveillance history, standardized case report forms were completed for all patients. This included information on baseline characteristics (e.g. relevant medical history, esophageal landmarks), surveillance history (e.g. baseline BE diagnosis, quality of baseline biopsy sampling, number and outcome of surveillance endoscopies, interval between baseline BE diagnosis and first HGD/EAC diagnosis), the endpoint of progression (e.g. worst histological diagnosis, number of endoscopies with HGD/EAC, and specimen type in which HGD/EAC was diagnosed) and treatment modality and outcome of treatment. We only included patients with maximum T1-disease at time of progression based on either an endoscopic resection specimen, esophagectomy specimen, or biopsy specimens obtained at two separate endoscopies. All histological diagnoses of progression were made by at least two pathologists. The minimum required surveillance interval before neoplastic progression was 2 years. The baseline endoscopy demonstrating NDBE was required to be of sufficient quality (i.e. > 50% of required number of biopsies according to Seattle protocol were obtained). We only included patients with a baseline diagnosis of NDBE. For this a histological diagnosis by a single pathologist sufficed. In case of a baseline diagnosis of LGD, slides were reviewed by two expert pathologists. Such patients were only included if this baseline diagnosis was unanimously downstaged to NDBE. Identification, data collection and inclusion criteria for nonprogressors In 2003, the Amsterdam Gastroenterological Association (also known as the ‘Amsterdam Gutclub’) initiated a retrospective registration of all Barrett's patients in 16 hospitals in the Amsterdam region. At that time, the PALGA database of each participating hospital was searched with the search strings ‘Barrett’ and ‘intestinal metaplasia and esophagus.’ Two research nurses, each with at least 3 years of experience with Barrett's surveillance, reviewed all endoscopy and pathology reports in the participating hospitals for verification of the diagnosis of BE, defined as columnar lined epithelium on endoscopy and intestinal metaplasia on histology. After verification of the diagnosis, patient demographics, history, and information from endoscopy and pathology reports were entered in a dedicated registration database. Following this retrospective analysis, a prospective BE surveillance registry was initiated in 6 of these 16 hospitals. In the remaining 10 hospitals, the retrospective analysis was updated in 2012, which enabled us to identify newly diagnosed BE patients and retrieve long-term follow-up information for the patients that were identified in the first retrospective analysis. All patients who did not eventually develop HGD or EAC and who underwent at least 2 endoscopies with a minimum surveillance interval of 2 years were eligible for inclusion as nonprogressor. We performed central expert pathology review of all baseline biopsies with an original diagnosis of LGD and only included patients in whom this diagnosis was downstaged to NDBE. The prospective surveillance cohort in the remaining 6 hospitals includes progressor and nonprogressor patients and remains available as an independent validation cohort for phase 4 biomarker studies. The relation between the respective patient cohorts is depicted in Figure 1. Fig. 1 View largeDownload slide The Amsterdam ReBus project: a graphical representation of the source population for the different patient cohorts. Fig. 1 View largeDownload slide The Amsterdam ReBus project: a graphical representation of the source population for the different patient cohorts. Endpoints and duration of surveillance The endpoint of the study was the first diagnosis of HGD/EAC (progressors) or the most recent endoscopic procedure with a histological diagnosis (nonprogressors). Duration of surveillance was calculated as the interval between the endoscopy date on which intestinal metaplasia was first detected and the endpoint of the study. The minimum duration of surveillance was calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed at least 2 years before the endpoint. Patient material, ethical and legal considerations All selected patients were contacted to obtain written informed consent for transferring medical data and paraffin embedded tissue, obtained during surveillance endoscopies in referring hospitals, to the AMC for biomarker research. The Dutch Medical Research Involving Human Subjects Act (Wet Medisch-wetenschappelijk Onderzoek met mensen, WMO) does not apply to this project and the ethics committee of the AMC has, therefore, exempted this study from formal ethical review. With respect to the processing of personal (medical) data, the Dutch Data Protection Act (Wet Bescherming Persoonsgegevens) and the Dutch Civil Code on Agreements with Regard to Medical Treatment (de Wet Geneeskundige BehandelingsOvereenkomst) apply. The principals of these laws regarding the use of data for research purposes are structured in the Code of Conduct ‘Use of Data in Health Research’ (Gedragscode Gezondheidsonderzoek - 2004). All human tissue samples that were collected under this protocol, are samples obtained for diagnosis and medical treatment purposes. These samples are considered residual human tissue. To this type of tissue, the Dutch Code of Conduct ‘Proper Secondary Use of Human Tissue’ (Gedragscode Goed Gebruik - 2011) applies. Both Codes of Conduct are issued and published by the Dutch Federation of Medical Research Associations (Federatie van Medisch Wetenschappelijke Verenigingen). These Codes of Conduct were adhered to, to the extent applicable to this project. Residual human tissue for this project was gathered from referring hospitals throughout the Netherlands. In order to transfer ownership of the material and to ensure that third parties can use the tissue material for research purposes, material transfer agreements (MTAs) have been signed with all 58 referring hospitals. Ethical approval was obtained from the ethics committee and the biobank review committee of the AMC, as well as the ethics committees of all 10 hospitals participating in the retrospective regional Barrett's registry. Only patients who gave written informed consent for this project are included in the cohort. Designing the legal and ethical framework for the ReBus-project was performed in 2010. Acquiring the MTAs from all 58 hospitals were the biopsy specimens resided was performed between 2010 and 2012. Identification of eligible patients, obtaining informed consent, data collection, and sample retrieval was performed between 2011 and 2013. Statistical analysis Categorical variables are described using percentages. Continuous variables with a normal distribution are presented as mean and standard deviation, median, and interquartile range are used for continuous variables with a skewed distribution. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS 20.0, IBM Corp., Armonk, NY, USA). RESULTS Progressor patients The selection of progressor patients in the study is summarized in Figure 2. Demographics and surveillance characteristics of this cohort are summarized in Table 1. Between 2000 and 2013, 629 patients were referred to the AMC, 138 were referred to the St Antonius Hospital, and 120 were referred to the Catharina Hospital for endoscopic work-up of esophageal neoplasia and were screened for eligibility. Of these 887 patients, 286 patients were diagnosed with HGD or early EAC (T1N0M0) and had a surveillance history before the diagnosis of neoplastic progression, irrespective of the duration of the surveillance interval and the modality that was used to diagnose progression. Fig. 2 View largeDownload slide Flowchart of patients into the ReBus progressor cohort and the regional Barrett's registration cohorts in the Amsterdam region. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; ER, endoscopic resection; HGD, high-grade dysplasia; * We did not ask patients for informed consent due to advanced age, or severe comorbidities. ** Includes patients with confirmed LGD as well as patients for whom the original histology slides demonstrating LGD could not be retrieved. Fig. 2 View largeDownload slide Flowchart of patients into the ReBus progressor cohort and the regional Barrett's registration cohorts in the Amsterdam region. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; ER, endoscopic resection; HGD, high-grade dysplasia; * We did not ask patients for informed consent due to advanced age, or severe comorbidities. ** Includes patients with confirmed LGD as well as patients for whom the original histology slides demonstrating LGD could not be retrieved. Table 1 Baseline and surveillance characteristics for progressor and non-progressor patients and information on progression diagnosis for progressor patients   NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA    NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA  †Calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed >2 years before the endpoint. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; HGD, high-grade dysplasia; NA, not applicable; NDBE, nondysplastic BE. . View Large Table 1 Baseline and surveillance characteristics for progressor and non-progressor patients and information on progression diagnosis for progressor patients   NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA    NDBE patients with neoplastic progression during surveillance (n = 165)  NDBE patients without neoplastic progression during surveillance (n = 723)  Male, n (%)  135 (82)  483 (67)  Age at BE diagnosis, years ± SD  55 ± 10.4  57 ± 11.3  Age at HGD/EAC diagnosis, years ± SD  64 ± 9.5  NA  Length of BE segment, cm (IQR)  5 (4–7)  4 (2–6)  Circumferential BE extent, cm (IQR)  2 (3–5)  2 (1–4)  Duration of surveillance, months (IQR)  89 (54–139)  76 (47–116)  Minimum duration of surveillance, months (IQR)†  34 (27–45)  37 (28–49)  Number of surveillance endoscopies (IQR)  5 (3–7)  4 (2–6)  Progression diagnosis, n (%)     High-grade dysplasia  49 (30)  NA   Intramucosal carcinoma  81 (49)  NA   Submucosal carcinoma  26 (16)  NA   EAC with unknown infiltration depth  9 (5)  NA  Diagnosis based on, n (%)     Endoscopic resection specimen  129 (78)  NA   Esophagectomy specimen  5 (3)  NA   Biopsies from ≥ 2 separate endoscopies  31 (19)  NA  †Calculated as the interval between the endpoint of the study and the date of the last surveillance endoscopy that was performed >2 years before the endpoint. BE, Barrett's esophagus; EAC, esophageal adenocarcinoma; HGD, high-grade dysplasia; NA, not applicable; NDBE, nondysplastic BE. . View Large Diagnosis of progression to HGD/EAC One hundred patients were diagnosed with HGD, 186 patients with early adenocarcinoma (134 patients with intramucosal carcinoma (T1m1/m2/m3), 35 patients with submucosal cancer (T1sm1/sm2/sm3) and 17 with unknown infiltration depth). Patients with advanced carcinoma (≥T2) were not included (n = 81). The worst histological diagnosis was based on an ER specimen in 216 patients, on an esophagectomy specimen in seven patients and on biopsies in 63 patients. Of this last patient group, 50 patients were diagnosed with HGD/EAC based on biopsies from two or more endoscopies. The remaining 13 patients with a single biopsy diagnosis of HGD/EAC were excluded. Surveillance history of progressor patients Of the patients with incident HGD/EAC and an unequivocal progression diagnosis (n = 273), 59 patients had a BE surveillance history less than 2 years and were excluded from the study. Of the remaining 214 patients, we excluded 13 patients with insufficient quality of baseline biopsy sampling and eight patients who did not consent to the study. In 41/55 patients with baseline LGD the original histology slides were retrieved and LGD was confirmed in 14 of those patients who were subsequently excluded. We also excluded the 14 patients in whom we could not retrieve the original histology slides, resulting in a progressor cohort of 165 patients. Treatment of progressor patients and follow-up after treatment Progressor patients diagnosed with EAC who had ≥1 risk factors for lymph node metastasis (i.e. submucosal invasion, lymphovascular invasion, poor tumor differentiation) or in whom the depth of infiltration was unknown were considered high risk (n = 39). Patients with HGD or intramucosal EAC and none of the above mentioned risk factors were classified as low risk (n = 126). Treatment strategy and outcome for patients at low- and high-risk of lymph node metastasis are shown in Figure 3. Fig. 3 View largeDownload slide Treatment modality and outcome of treatment for progressor patients at high and low risk of lymph node metastasis. APC, argon plasma coagulation; CR-IM, complete remission of intestinal metaplasia; CR-N, complete remission of neoplasia; PDT photodynamic therapy; RFA, radiofrequency ablation. Fig. 3 View largeDownload slide Treatment modality and outcome of treatment for progressor patients at high and low risk of lymph node metastasis. APC, argon plasma coagulation; CR-IM, complete remission of intestinal metaplasia; CR-N, complete remission of neoplasia; PDT photodynamic therapy; RFA, radiofrequency ablation. Selection of nonprogressors Selection of non-progressors is depicted in Figure 2 and baseline characteristics are summarized in Table 1. A total of 2350 Barrett's patients were entered in a retrospective regional registration database. Of these, 2206 were never diagnosed with HGD/EAC and 1280 of these nonprogressor patients had an endoscopic surveillance history (i.e. underwent at least 2 surveillance endoscopies). In 1040 patients, the duration of the endoscopic surveillance history was 2 years or more. We obtained informed consent for the study in 780 of these patients. In 56/105 patients with baseline LGD, the original histology slides were retrieved and LGD was confirmed in 8 of those patients who were subsequently excluded. We further excluded all 49 patients with baseline LGD for whom we could not retrieve the original histology slides, resulting in a nonprogressor cohort of 723 patients. DISCUSSION In this publication, we have outlined the selection and characteristics of a community-based nested case-control cohort of BE patients available for biomarker research. Using stringent selection criteria, 165 Barrett's surveillance patients were identified who progressed from NDBE to HGD or EAC. Controls were identified from a large population-based cohort of BE surveillance patients who never demonstrated neoplastic progression (n = 723). Depending on the set-up and sample size of intended biomarker studies, progressors can be matched with nonprogressors in different ratios by baseline variables (i.e. age, gender, length of the BE segment and duration of surveillance). Several research groups have investigated biomarkers in case-control cohorts and published promising results suggesting that some of these markers may be of value in risk stratifying BE patients (Table 2). There are three main clusters of relevant publications we would like to discuss. The Seattle group has performed several studies in a prospective cohort of BE patients enrolled in the Seattle Barrett's Esophagus Study from 1992 onwards. They published studies on LOH for 17p (RR 16; 95% CI 6–39) and measures of clonal diversity (RR 11; 95% CI 6–21).10,11 Subsequently, this group has published a panel of markers consisting of abnormal DNA ploidy and LOH for 9p and 17p, which was highly associated with neoplastic progression (RR 39; 95% CI 11–139).12 There are, however, major limitations in the selection of patients for the Seattle cohort that need to be addressed. The main limitation in this cohort is the lack of a reliable baseline diagnosis before progression. These studies have assessed progression to EAC alone, but have also included BE patients with HGD at baseline in the progressor cohort. It is likely that a proportion of these patients had synchronous EAC at baseline since a diagnosis of HGD is often indicative of a visible lesion elsewhere in the BE segment. These lesions are often hard to detect during standard endoscopy, especially with low-resolution endoscopes that were used in the 90s. Additionally, the agreement among expert GI pathologists for the distinction between HGD and EAC is limited, especially when based on histological interpretation of biopsy specimens.22 In conjunction with the fact that patients were not excluded when progression to EAC was diagnosed shortly after the baseline endoscopy, this may have led to the inclusion of patients with prevalent neoplasia at baseline. Therefore, the reported odds of progression for the Reid biomarker panel are likely inflated. Furthermore, most international guidelines currently recommend endoscopic eradication therapy for BE patients with HGD, which renders the prediction of progression from HGD to EAC irrelevant. In addition, external validity of the findings may be limited since the Seattle cohort originates in a tertiary referral setting and the biomarker research was performed on snap-frozen specimens. DNA extraction is easier from snap-frozen biopsy specimens compared with formalin fixed paraffin embedded specimens, however, fresh frozen samples are not a practical and readily available source of material outside the academic setting. Table 2 Summary of main clusters of phase 3 biomarker studies in Barrett's esophagus patients           Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes            Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes  †Diagnosis of HGD/EAC based on endoscopic resection specimen, esophagectomy specimen or biopsies obtained at two separate endoscopies; ‡Studies assessed progression to EAC alone and included patients with HGD at baseline. AUC, area under receiver-operating characteristic curve; EAC, esophageal adenocarcinoma; LGD, low-grade dysplasia; LOH, loss of heterozygosity; NDBE, nondysplastic Barrett's esophagus; OR, odds ratio; RR, relative risk. View Large Table 2 Summary of main clusters of phase 3 biomarker studies in Barrett's esophagus patients           Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes            Reliable baseline NDBE/LGD diagnosis    Patient cohort  Study setting and biopsy material  Reference  Investigated biomarkers and results  Sample size (cases:controls)  Quality of baseline sampling assessed?  Minimum surveillance interval  Advanced EAC excluded?  Unequivocal progression diagnosis†  Seattle Barrett's Esophagus Study  Tertiary referral, prospective cohort Fresh frozen biopsies  Reid et al.10  17p LOH (p53); RR = 16 (95% CI, 6.2–39)  269 (26:243)  No  NA‡  No  No      Maley et al.11  Measures of clonal diversity; Shannon LOH diversity index: RR = 11.0 (95% CI, 5.8–21.0)  268 (37:231)  No  NA‡  No  No      Galipeau et al.12  Abnormal DNA ploidy, 9p LOH (p16), 17p LOH (p53); RR = 38.7 (95% CI 10.8–138.5)  243 (34:209)  No  NA‡  No  No  Baltimore VA and University of Maryland Barrett's cohort  Tertiary referral, retrospective cohort fresh frozen biopsies  Schulmann et al.13  Promoter methylation of P16, RUNX3 and HPP1; p16: OR = 1.7 (95% CI, 1.3–2.2), RUNX3: OR = 1.8 (95% CI, 1.1–2.8), HPP1: OR = 1.8 (95% CI, 1.1–2.8)  53 (8:45)  No  No  Yes  Yes      Sato et al.14  P16, HPP1, RUNX3 (combined with clinical markers); AUC 0.84 at 2 years, AUC 0.79 at 4 years  62 (35:27)  No  No (stratified analysis)  Unknown  Unknown      Jin et al.15  Promotor methylation of 8-gene panel; AUC 0.84 at 2 years; AUC 0.83 at 4 years; AUC 0.84 for combined model  195 (50:145)  No  No (stratified analysis)  Unknown  Unknown  Northern Ireland Barrett's Registry  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Murray et al.18  P53; OR 8.42 (95% CI, 2.37–30.0)  210 (35:175)  No  6 months  No  No      Bird-Lieberman et al.17  Consensus LGD, abnormal DNA ploidy, Aspergillus oryzae lectin; OR for each additional positive biomarker 3.74 (95% CI, 2.43–5.79) and 2.99 (95% CI, 1.72–5.20) for pts with and without LGD respectively  380 (89:291)  No  6 months  No  No  Amsterdam ReBus cohort  Population-based nested case-control Formalin fixed paraffin embedded biopsies  Current study  –  888 (165:723)  Yes  2 years  Yes  Yes  †Diagnosis of HGD/EAC based on endoscopic resection specimen, esophagectomy specimen or biopsies obtained at two separate endoscopies; ‡Studies assessed progression to EAC alone and included patients with HGD at baseline. AUC, area under receiver-operating characteristic curve; EAC, esophageal adenocarcinoma; LGD, low-grade dysplasia; LOH, loss of heterozygosity; NDBE, nondysplastic Barrett's esophagus; OR, odds ratio; RR, relative risk. View Large The Baltimore group has studied epigenetic markers in the Baltimore VA and University of Maryland Barrett's cohort. Focusing on promoter methylation in a panel of 10 genes hypermethylation of p16, RUNX3 and HPP1 were statistically significant predictors of neoplastic progression in a multivariable analysis.13 Subsequently, this 3-marker panel was combined with a set of clinical features to stratify patients into low-risk, intermediate-risk, and high-risk groups of progression (4-year prediction model, area under the receiver-operating characteristic curve (AUC) = 0.791).14 Subsequent studies identified 5 genes that were methylated early and often in neoplastic progression (i.e. nel-like 1 (NELL1), tachykinin-1 (TAC1), somatostatin (SST), AKAP12, and CDH13). These 5 genes were combined with p16, RUNX3 and HPP1, to form an 8-gene panel that was validated in a subsequent study.15 The AUC for the 2- and 4-year prediction models combined was 0.840. These results from the Baltimore group are promising, but again the selection of patients has several limitations. First, for all 3 studies it is uncertain if the baseline endoscopy of patients who progressed did not contain HGD or EAC. The study by Schulman and colleagues had no minimum surveillance interval required to exclude prevalent HGD/EAC. In fact, most of the samples that generated high hazard ratios in this study were obtained less than 2 years before progression. The other two studies stratified progressor patients according to the duration of their progression free surveillance interval. However, they did not exclude progressor patients with advanced lesions, which may still have led to inclusion of patients with prevalent disease at baseline. Second, for the latter 2 studies we are not informed about the modality that was used to establish the progression diagnosis, which may have diluted the progressor cohort with patients who lacked genuine neoplastic progression (‘single biopsy HGD-cases’). Finally, the external validity may be limited since patients in all three studies were enrolled at tertiary referral centers and biomarker research was performed on snap-frozen biopsies. The Cambridge group has recently published a promising panel of markers consisting of a consensus diagnosis of LGD, abnormal DNA ploidy, and AOL.17 In patients with consensus LGD, the adjusted OR for progression was 3.74 (95% CI 2.43–5.79) for each additional biomarker and the risk increased by 2.99 (95% CI 1.72–5.20) for each additional marker in patients without LGD. This study was performed on a population-based matched case-control cohort from the Northern Ireland Barrett's Esophagus Registry (NIBR). Also in this cohort, the main limitation is the lack of a reliable baseline diagnosis without HGD/EAC. Minimum required progression-free surveillance interval for progressor patients was only 6 months. Additionally, patients with advanced EAC were not excluded, increasing the likelihood of prevalent neoplasia at baseline. Finally, patients with a single endoscopy, single biopsy diagnosis of HGD qualified as progressor patients, which might have diluted the progressor cohort as described previously. A further limitation of all BE case-control cohorts described above is the limited size of the progressor cohorts. Apart from the study by Fitzgerald et al. in the NIBR cohort (89 progressors), the number of progressor patients does not exceed 50 (Table 2). This is likely to be an insufficient number of patients to validate findings from biomarker identification studies. In conclusion, recent studies reported promising results in the field of biomarkers for progression in BE. However, patient selection in these studies was flawed in several ways, as we have outlined above and summarized in Table 2. Major limitations are the lack of a reliable baseline diagnosis without HGD/EAC, the absence of an unequivocally diagnosed progression endpoint and the limited size of the progressor cohorts. Additionally, almost all studies were performed in a tertiary referral setting and have used biopsy material that is not widely available in the community setting. In order to overcome the previously described limitations in the selection of patients for biomarker research in BE, we have described a stringently selected population-based cohort. We only included patients with an unequivocal diagnosis of HGD/EAC, based on either an endoscopic resection specimen, an esophagectomy specimen or biopsy specimens obtained at two separate endoscopies. Progressor patients diagnosed with advanced cancer were excluded. We selected progressor patients with a minimum of two years surveillance before neoplastic progression and only included those who had sufficient quality of baseline biopsy sampling. We excluded all patients in whom baseline LGD was confirmed after central expert histology review as well as those for whom we could not retrieve the original histology slides demonstrating baseline LGD. In our opinion, these patients should be excluded, since previous studies have demonstrated that patients with a confirmed diagnosis of LGD have a highly increased risk of progression.20,23–25 Outcomes of biomarker studies that include patients with LGD are, therefore, likely affected by the outcome of LGD patients. To allow for matching of progressors with Barrett's patients without neoplastic progression, we selected nonprogressor patients from a population-based Barrett's registry, avoiding the tertiary referral bias as present in other biomarker studies. For each biomarker research project to be performed in this cohort, progressors and controls can be matched depending on the size and ratio of the intended cohort. The matched case-control cohort that ensues is an optimal selection of patients for phase 3 biomarker studies in the field of BE. Notes Conflicts of interest: JJGHMB receives research support from Olympus Endoscopy, Cook Medical, Boston Scientific, GI Solutions Covidien, Erbe and Ninepoint Medical; receives financial support for training programs from GI Solutions Covidien; and receives honorarium-consultancy-speakers fee from Cook Medical, Boston Scientific and GI Solutions Covidien. BLAMW receives research support from Covidien GI solutions, Erbe Medical and C2Therapeutic and has received consulting fees from Boston Scientific and C2Therapeutic. All other authors have no conflicts of interest to declare. Specific author contributions: Study concept and design: Lucas C. Duits, David F. Boerwinkel, Roos E. Pouw, Jacques J.G.H.M. Bergman; Acquisition of data: Lucas C. Duits, Esther Klaver, Angela Bureo Gonzalez, David F. Boerwinkel, Fiebo J.W. ten Kate, G. Johan A. Offerhaus, Sybren L. Meijer, Mike Visser, Cees A. Seldenrijk, Kausilia K. Krishnadath, Erik J. Schoon, Bas L.A.M. Weusten; Analysis and interpretation of data: Lucas C. Duits, Esther Klaver, Angela Bureo Gonzalez, David F. Boerwinkel, Rosalie C. Mallant-Hent, Roos E. Pouw, Jacques J.G.H.M. Bergman; Drafting of the manuscript: Lucas C. Duits, David F. Boerwinkel; Critical revision of the manuscript for important intellectual content and final approval of the manuscript: All authors; study supervision: Roos E. Pouw, Jacques J.G.H.M. Bergman. Grant support: None. References 1 Shaheen N J, Richter J E. Barrett's oesophagus. Lancet  2009; 373: 850– 61. Google Scholar CrossRef Search ADS PubMed  2 Spechler S J, Sharma P, Souza R F et al.   American gastroenterological association technical review on the management of Barrett's esophagus. Gastroenterology  2011; 140: e18– 52. Google Scholar CrossRef Search ADS PubMed  3 Fitzgerald R C, Pietro M di, Ragunath K et al.   British Society of Gastroenterology guidelines on the diagnosis and management of Barrett's oesophagus. Gut  2014; 63: 7– 42. Google Scholar CrossRef Search ADS PubMed  4 Gordon L G, Mayne G C, Hirst N G et al.   Cost-effectiveness of endoscopic surveillance of non-dysplastic Barrett's esophagus. Gastrointest Endosc  2014; 79: 242– 56. e6. Google Scholar CrossRef Search ADS PubMed  5 Curvers W L, Bansal A, Sharma P et al.   Endoscopic work-up of early Barrett's neoplasia. Endoscopy  2008; 40: 1000– 7. Google Scholar CrossRef Search ADS PubMed  6 Tschanz E R. Do 40% of patients resected for Barrett esophagus with high-grade dysplasia have unsuspected adenocarcinoma? Arch Pathol Lab Med Online  2005; 129: 177– 80. 7 Montgomery E, Bronner M P, Goldblum J R et al.   Reproducibility of the diagnosis of dysplasia in Barrett esophagus: a reaffirmation. Hum Pathol  2001; 32: 368– 78. Google Scholar CrossRef Search ADS PubMed  8 Phoa K N, Vilsteren F G I van, Weusten B L A M et al.   Radiofrequency ablation vs endoscopic surveillance for patients with Barrett esophagus and low-grade dysplasia. JAMA  2014; 311: 1209– 17. Google Scholar CrossRef Search ADS PubMed  9 Rubenstein J H, Kwon R S. Radiofrequency ablation for Barrett's esophagus with low-grade dysplasia: a hammer looking for a nail. Gastroenterology  2014; 147: 706– 7. Google Scholar CrossRef Search ADS PubMed  10 Reid B, Prevo L, Galipeau P. Predictors of progression in Barrett's esophagus II: baseline 17p (p53) loss of heterozygosity identifies a patient subset at increased risk for neoplastic progression. Am J Gastroenterol  2001; 96: 2839– 48. Google Scholar CrossRef Search ADS PubMed  11 Maley C C, Galipeau P C, Finley J C et al.   Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat Genet  2006; 38: 468– 73. Google Scholar CrossRef Search ADS PubMed  12 Galipeau P C, Li X, Blount P L et al.   NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma. PLoS Med  2007; 4: e67. Google Scholar CrossRef Search ADS PubMed  13 Schulmann K, Sterian A, Berki A et al.   Inactivation of p16, RUNX3 and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk. Oncogene  2005; 24: 4138– 48. Google Scholar CrossRef Search ADS PubMed  14 Sato F, Jin Z, Schulmann K et al.   Three-tiered risk stratification model to predict progression in Barrett's esophagus using epigenetic and clinical features. PLoS One  2008; 3: e1890. Google Scholar CrossRef Search ADS PubMed  15 Jin Z, Cheng Y, Gu W et al.   A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett's esophagus. Cancer Res  2009; 69: 4112– 5. Google Scholar CrossRef Search ADS PubMed  16 Lao-Sirieix P, Lovat L, Fitzgerald R C. Cyclin a immunocytology as a risk stratification tool for Barrett's esophagus surveillance. Clin Cancer Res  2007; 13: 659– 65. Google Scholar CrossRef Search ADS PubMed  17 Bird-Lieberman E L, Dunn J M, Coleman H G et al.   Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus. Gastroenterology  2012; 143: 927– 935.e3. Google Scholar CrossRef Search ADS PubMed  18 Murray L, Sedo A, Scott M et al.   TP53 and progression from Barrett's metaplasia to oesophageal adenocarcinoma in a UK population cohort. Gut  2006; 55: 1390– 7. Google Scholar CrossRef Search ADS PubMed  19 Peters F P, Curvers W L, Rosmolen W D et al.   Surveillance history of endoscopically treated patients with early Barrett's neoplasia: nonadherence to the Seattle biopsy protocol leads to sampling error. Dis Esophagus  2008; 21: 475– 9. Google Scholar CrossRef Search ADS PubMed  20 Curvers W L, Kate F J ten, Krishnadath K K et al.   Low-grade dysplasia in Barrett's esophagus: overdiagnosed and underestimated. Am J Gastroenterol  2010; 105: 1523– 30. Google Scholar CrossRef Search ADS PubMed  21 Casparie M, Tiebosch A T M G, Burger G et al.   Pathology databanking and biobanking in The Netherlands, a central role for PALGA, the nationwide histopathology and cytopathology data network and archive. Cell Oncol  2007; 29: 19– 24. Google Scholar PubMed  22 Ormsby A H, Petras R E, Henricks W H et al.   Observer variation in the diagnosis of superficial oesophageal adenocarcinoma. Gut  2002; 51: 671– 6. Google Scholar CrossRef Search ADS PubMed  23 Duits L C, Phoa K N, Curvers W L et al.   Barrett's oesophagus patients with low-grade dysplasia can be accurately risk-stratified after histological review by an expert pathology panel. Gut  2015; 64: 700– 6. Google Scholar CrossRef Search ADS PubMed  24 Lim C, Treanor D, Dixon M et al.   Low-grade dysplasia in Barrett's esophagus has a high risk of progression. Endoscopy  2007; 39: 581– 7. Google Scholar CrossRef Search ADS PubMed  25 Vieth M, Schubert B, Lang-Schwarz K et al.   Frequency of Barrett's neoplasia after initial negative endoscopy with biopsy: a long-term histopathological follow-up study. Endoscopy  2006; 38: 1201– 5. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Diseases of the EsophagusOxford University Press

Published: Jun 4, 2018

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