Compared to the general population, BE patients have a 30-125-fold increased risk of developing EAC 
. Therefore, periodic endoscopic surveillance is generally practiced in the management of BE patients
. EAC detected during BE surveillance tends to occur at an earlier stage and have a better prognosis than EAC found in the non-surveillance setting
. However, in terms of cost-effectiveness, the impact of current BE surveillance recommendations is controversial 
, because the progression rate of BE to EAC is very low. Thus, stratification of BE patients to improve BE surveillance efficiency would be beneficial in terms of cost-effectiveness, as well as represent an improvement in quality of life due to diminished anxiety and inconvenience.
Current recommendations for the appropriate BE follow-up interval are as follows: two initial annual endoscopies, followed by a 3-year interval for BE cases without dysplasia, or less than 1 year for BE with LGD until dysplasia is no longer found
. To simplify calculations in the current study, we compared endoscopy savings between a uniform 2-year follow-up protocol and our three-tiered model. Using a simulation, we estimated that this 3-tiered risk stratification strategy would save approximately 5,300 endoscopies annually in the United States. If a 0.13% overall upper GI endoscopy complication rate is assumed, this endoscopy savings would prevent 6.9 unnecessary complications annually in the United States
These three risk tiers were defined using only progression status at 2 years and 4 years after analyzed specimens were obtained. Thus, theoretically, this stratification cannot guarantee differences in progression-free survival more than 4 years after sampling. However, Kaplan-Meier progression-free survival analysis () demonstrated that our prediction model could discriminate among the 3 risk groups well not only at 2 and 4 years post-sampling, but also over the entire follow-up period.
The Kaplan-Meier progression free survival curve showed that some LR patients progressed soon after the fourth year following their initial (index) BE EGD. However, the model recommends follow-up endoscopy within 4 years after any LR EGD. For example, patient #5 in had a LR specimen at 4.5 years prior to progression. This case does not represent a flaw in the model, since followup EGD was indeed performed as per the model's recommendation, and his risk level at 1.8 years before progression was upgraded to HR.
In this study, there were 6 sets of multiple specimens from the same timepoint in 5 patients (patients 4, 9, 15, 19, and 26; indicated by arrows
in ). The trained prediction model yielded conflicting risk grade outputs in 3 specimen sets (patients 4, 15, and 26). One possible explanation for this observed discrepancy was variation in biopsy sampling. Carcinogenic events, including histologic
, and epigenetic alterations
, do not occur uniformly throughout the BE epithelium. Therefore, as with histologic assessment, this discrepancy could have been caused by biopsy sampling variation. One potential solution to this issue is to perform sampling from multiple anatomic loci, as in histological assessment during current BE surveillance, and to apply the highest risk assessment obtained from these multiple loci to scheduling of the next BE surveilance endoscopy.
Our incremental analysis demonstrated that MI made a much greater contribution to prediction accuracy than did the other parameters. These findings are not surprising, since some researchers have reported that MI or CpG Island Methylator Phenotype (CIMP) status correlates with patient survival in esophageal cancer
and other malignancies, such as colorectal cancer
. However, mechanism(s) by which an “MI-high” epigenetic or methylator phenotype contributes to carcinogenesis remain(s) unclear. Possible explanations include: 1) methylator phenotype-positive tumors tend to be hypermethylated in promoter regions of other genes, including tumor suppressor genes (such as APC, CDH1, TIMP3, and others) 
; 2) methylator phenotype-positive tumors tend to undergo hMLH1 gene inactivation via promoter hypermethylation. Although hMLH1 hypermethylation is relatively uncommon in EAC compared to gastric, colorectal, or endometrial cancer
, hMLH1 hypermethylation in BE may cause microsatellite instability in the coding regions of the tumor suppressor genes
; 3) a methylator phenotype may be associated with chromatin remodeling
; and 4) methylated cytosines are hotspots for mutations, as with the p53 gene
Histopathologic assessment of dysplasia in BE is currently the most widely accepted parameter with which to predict BE progression. However, histopathologic assessment is plagued by inter-observer variation, which can lead to confusion during clinical BE surveillance. One aim in this study was to develop biomarkers that were more objective and quantifiable than histopathologic assessment, such as epigenetic parameters (including MI). However, MI data also risk being influenced by several factors. One such factor is the dichotomization of normalized methylation values (NMV) for each gene into positive vs.
negative classes. The significance and relevance to BE progression of methylation of each gene may vary. For this reason, we did not use uniform criteria to dichotomize NMV data, but rathere optimized criteria for each gene based on ROC curve analysis. Another factor potentially influencing MI data is endoscopic sampling bias. Methylation status in BE occurs heterogeneously 
, as does genomic clonality 
. Therefore, multiple biopsies during each endoscopic procedure are widely in BE surveillance.
In both the 2-year and 4-year prediction models, according to incremental value analysis, pathological assessment was the second-most influential parameter. The natural history of LGD is not well-described, with ultimate progression to HGD and EAC ranging from 5-12.5% 
. LGD also frequently regresses to BE, at rates ranging as high as 60-75% 
. In addition, the histological diagnosis of dysplasia in BE
, as well as in other premalignant lesions (esophageal squamous epithelium
, ulcerative colitis
, and others), is characterized by high inter-observer variability. Therefore, the value of LGD as a clinical cancer risk marker is controversial. However, some studies have demonstrated that LGD is a risk factor for the development of EAC in BE 
. Our findings corroborated this predictive value of LGD. In addition, our results emphasize the power of combining pathological assessment with methylation status to improve risk prediction accuracy.
In both the 2-year and 4-year models, segment length (SL) was also one of the parameters in the most optimal parameter set. Patients with long-segment (≥3cm) BE are widely believed to carry a greater risk of developing EAC than those with short-segment BE 
. However, other studies have demonstrated that the risk of developing EAC in patients with short-segment BE is not substantially lower than in patients with long-segment BE
. In the current study, SL was selected in the optimal parameter set for both the 2-year and the 4-year models (). This finding also suggests that SL is not strong as an independent clinical marker; however, it does contribute significantly as a member of a parameter set.
The NCI Early Detection Research Network (EDRN) defined five phases of biomarker development in the early detection of cancer
. Currently, flow cytometric (tetraploidy, aneuploidy)
and loss of heterozygosity (LOH) at the p53 locus
have advanced regarding biomarker validation in large-scale phase 4 studies as defined by EDRN classification. However, the AUROC for prediction of BE progression (to EAC) based on flow cytometry was 0.76
. Thus, our multi-tiered prediction method based on clinical and epigenetic parameters (AUROC
0.8387 and 0.7910 for the 2-year and 4-year models, respectively) exceeded published AUROCs based on single flow cytometric analysis alone
. Moreover, assessment of aberrant methylation in BE can be performed using formalin-fixed, paraffin-embedded specimens
. Finally, matching normal tissue is not necessary for methylation assays, in contrast to LOH. These advantageous features of methylation-based biomarkers may make specimen collection easier, thereby facilitating large-scale multi-institutional prospective or retrospective studies.
The work described in this report is now the subject of an EDRN Phase 3 validation study. In preparing to proceed to Phase 4 validation, we developed a prediction model to stratify BE patients, validated our model, and estimated its potential clinical impact (endoscopy savings) by applying a simulation. Because of the rarity of BE progressor specimens, the number of progressor patients and specimens was relatively small, despite collecting them from two institutions. Further studies may be needed to increase the number of BE progressor and non-progressor specimens by collecting specimens from multiple additional institutions prior to initiating a prospective Phase 4 study.
In conclusion, we developed a 3-tiered risk stratification strategy for neoplastic progression prediction in BE patients, based on epigenetic and clinical parameters. This strategy offers considerable promise to benefit the current BE surveillance health care system.