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Supplementary Data from Agrin Loss in Barrett's Esophagus-Related Neoplasia and Its Utility as a Diagnostic and Predictive Biomarker

Purpose:Sessile serrated lesions (SSL) are precursors to colon carcinoma, and their distinction from other polyps, in particular hyperplastic polyps (HP), presents significant diagnostic challenges. We evaluated expression patterns in colonic polyps of previously identified colon carcinoma–associated extracellular matrix (ECM) proteins to identify markers distinguishing SSLs from other polyps.Experimental Design:Gene-expression analyses of ECM proteins were performed using publicly available data on preneoplastic colonic polyps. In parallel, we evaluated by IHC the expression of agrin (AGRN) in over 400 colonic polyps, including HP, SSL with and without dysplasia, traditional serrated adenomas (TSA), and tubular adenomas (TA), and compared the consistency of standard histologic diagnosis of SSLs by experienced gastrointestinal pathologists with that of AGRN IHC.Results:Differential gene expression analysis and IHC identified AGRN, serine peptidase inhibitor (SERPINE2), and TIMP metallopeptidase inhibitor 1 (TIMP1) elevated in SSLs and HPs but decreased in TAs and absent in normal colon. AGRN-positive basal laminae were noted in all TA, TSA, HP, and SSL in distinguishable patterns, whereas other polyps and normal mucosa were negative. SSL with or without dysplasia consistently showed IHC staining for AGRN in the muscularis mucosae, which was absent in HP, TSA, TA, and other polyps. In contrast, histologic evaluation showed only weak interobserver agreement (kappa value = 0.493) in distinguishing SSLs.Conclusions:Muscularis mucosae–based AGRN immunostaining is a novel biomarker to distinguish SSL from HP, TSA, and TA, with a specificity of 97.1% and sensitivity of 98.9% and can assist in diagnosis of morphologically challenging colonic polyps.

Purpose:There is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus–related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus–related neoplasia and to predict neoplastic progression.Experimental Design:Gene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus–related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus–related neoplasia from 321 patients in three independent cohorts.Results:Differential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus–related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus–related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; P < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors (P < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus–related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%.Conclusions:We highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.

Serrated adenocarcinoma (SAC), a recognised WHO variant of colonic adenocarcinoma, is the purported end-product of serrated neoplasia. However, the diagnosis of SAC is infrequently rendered, and little is known about its prognosis, immune microenvironment and molecular alterations.We assessed 903 consecutive colon carcinomas and recognised tumours with ≥ 5% (n = 77) serrated and ≥ 50% serrated patterns (n = 13). We assessed precursor polyps and synchronous polyps. We recorded demographic/clinical parameters, histological features and mismatch repair (MMR) status. We performed immunohistochemistry and quantification on tissue microarray for HLA class I/II proteins, B2MG, CD8, CD163, LAG3, FoxP3, PD-L1 and BRAF V600E.We identified ≥ 5% epithelial serration prevalence in 8.5% of cases and ≥ 50% epithelial serration prevalence in 1.4% of cases. Precursor lesions were present in 21.4% of cases; these were mostly tubular adenomas with two traditional serrated adenomas identified. SAC with ≥ 5% serrations exhibited lower numbers of CD8-positive lymphocytes (P = 0.002) and lower B2MG expression (P = 0.048), although neither value was significant at ≥ 50% serration threshold. There was no difference in HLA class I/II, or PD-L1 expression on tumour cells and no difference in PD-L1, LAG3, FoxP3 and CD163 expression on immune cells. There was no association with MMR status, or BRAFV600E relative to conventional adenocarcinoma. There was improved disease-specific survival on univariate (but not multivariate) analysis between carcinomas with serrated pattern and non-mucinous conventional colonic carcinomas at ≥ 5% epithelial serrations (P = 0.04).SAC category shows a limited impact on survival, and this phenotype may harbour a unique immunological milieu.

AimsThe lack of accepted scoring criteria has precluded the use of p53 in routine practice. We evaluate the utility of automated quantitative p53 analysis in risk stratifying Barrett’s oesophagus (BE) patients using non-dysplastic BE (NDBE) biopsies in a multicentric cohort of BE progressor (P) and non-progressor (NP) patients.MethodsNDBE biopsies prior to the diagnosis of advanced neoplasia from 75 BE-P, and index and last surveillance biopsies from 148 BE-NP were stained for p53, and scored digitally as 1+, 2+ and 3+. A secondary cohort of 30 BE-P was evaluated.ResultsCompared with BE-NP, BE-P was predominantly men (p=0.001), ≥55 years of age (p=0.008), with longer BE segments (71% vs 33%; pConclusionsAutomated p53 analysis in NDBE biopsies serves as a promising tool for assessing BE neoplastic progression and risk stratification. Our study highlights the practical applicability of p53 assay to routine surveillance practice and its ability to detect prevalent dysplasia.