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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.

Mucinous adenocarcinoma (MAD), the most common subtype of colonic adenocarcinoma (CA), requires50% intratumoral mucin. There is limited data regarding the impact of MAD on key lymphocyte subsets and therapeutically critical immune elements. In this study we address: (1) the definition of MAD, (2) grading of MAD, and (3) the impact of MAD and extracellular mucin on intratumoral immune milieu. Estimation of the percentage of intratumoral mucin was performed by two pathologists. Tissue microarrays were stained for immune markers including CD8, CD163, PD-L1, FoxP3, β2 microglobulin, HLA class I, and HLA class II. Immunohistochemistry for BRAF V600E was performed. MMR status was determined on immunohistochemistry for MSH2, MSH6, MLH1, PMS2. Manual and automated HALO platforms were used for quantification. The 903 CAs included 62 (6.9%) MAD and 841 CA with ≤ 50% mucin. We identified 225 CAs with mucinous differentiation, defined by ≥10% mucin. On univariate analysis neither cut point, 50% (p = 0.08) and 10% (p = 0.08) mucin, correlated with disease-specific survival (DSS). There were no differences in key clinical, histological and molecular features between MAD and CA with mucinous differentiation. On univariate analysis of patients with MAD, tumor grade correlated with DSS (p = 0.0001) while MMR status did not (p = 0.86). There was no statistically significant difference in CD8 (P = 0.17) and CD163 (P = 0.05) positive immune cells between MAD and conventional CA. However, deficient (d) MMR MADs showed fewer CD8 (P = 0.0001), CD163 (P = 0.0001) and PD-L1 (P = 0.003) positive immune cells compared to proficient (p)MMR MADs, a finding also seen with at 10% mucin cut point. Although MAD does not impact DSS, this study raises the possibility that the immune milieu of dMMR MADs and tumors with =10% mucin may differ from pMMR MADs and tumors with10% mucin, a finding that may impact immune-oncology based therapeutics.

Genetically-engineered mouse models have greatly advanced our understanding of cancer, yet do not recapitulate the mutational complexity of human cancer, and likely lack neo-antigens capable of eliciting potent anti-tumor T cell responses. We developed a novel orthotopic organoid transplant model of colorectal cancer (CRC) harboring the strong T cell antigen SIINFEKL, and demonstrate the importance of antigen expression level in the anti-tumor T cell response. Although SIINFEKL low-expressing organoids (SIINLow) elicit an endogenous antigen-specific T cell response, the magnitude is substantially lower and kinetics delayed relative to SIINFEKL high-expressing organoids (SIINHi). Consistently, transplant of SIINHi results in rejection and T cell memory, while SIINLow results in tumor progression, terminally-exhausted T cells, and metastasis to the liver. We have shown that suboptimal T cell priming is the major factor underlying SIINLow tumor escape. Importantly, co-transplant of SIINHi and SIINLow organoids at distinct sites in the colon of the same animal results in complete rejection of both lines. In addition, co-transplant rescues the SIINLow tumor-infiltrating antigen-specific T cell response to a magnitude and quality comparable to that of SIINHi tumors. Single-cell RNA-sequencing of antigen-specific T cells from SIINHi and SIINLow tumors 8 days post-transplant revealed distinct clusters dominated by SIINLow-primed T cells, including a cluster enriched for immediate early response genes, Tox, and a number of immune checkpoints, indicative of early dysfunction. Collectively, our results establish the existence of a neo-antigen expression threshold at which T cell priming is limiting, resulting in attenuated magnitude and functionality of the T cell response, and tumor escape. To assess the therapeutic relevance of a poorly-expressed neo-antigen in CRC, we performed preclinical trials with immune checkpoint blockade and agonistic-CD40 (aCD40), which has been shown to potentiate T cell priming and response in poorly-immunogenic mouse and human pancreatic adenocarcinoma. While monotherapies showed only modest effects, the combination of checkpoint blockade and aCD40 resulted in an 80% response rate with a number of complete responses. In conclusion, antigen expression level is a critical determinant of T cell dysfunction, resulting from poor priming. Our results argue that targeting T cell priming may be a promising therapeutic strategy to invigorate anti-tumor immunity in human CRC, the majority of which remains refractory to immunotherapy. Citation Format: Peter Maxwell Kienitz Westcott, Nathan J. Sacks, Olivia Smith, Jason Schenkel, Zackery Ely, Daniel Zhang, Mary Clare Beytagh, William Hwang, George Eng, Jatin Roper, Omer Yilmaz, Tyler Jacks. T cell antigen expression levels govern progression and therapy response in a novel model of colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3447.

The small and large intestines are two of the fastest renewing epithelia in the body. The rapid turnover of intestinal epithelial cells requires continuous generation of new, functional cells fueled by stem cells that must balance between self-renewal and differentiation. The environment where stem cells reside, called the niche, provides key signals to dictate stem cell behavior. Because of its constant activity and well-defined cryptal structure, the intestinal stem cell niche is one of the most studied adult stem cell niches in the body. We will review current knowledge regarding the components that comprise the intestinal stem cell niche, including signaling molecules, nurse cells, extracellular matrix, and metabolites. We will discuss how to leverage current knowledge of the stem cell niche to develop a physiologically relevant intestinal model for tissue engineering and regenerative medicine.

Background To evaluate electrocardiographic parameters which are related with atrial and ventricular arrhythmias measured from 12-lead surface electrocardiogram (ECG) in workers occupationally exposed to lead. Methods Sixty lead-exposed workers and 60 healthy controls were enrolled. Twelve-lead surface ECG was recorded and measurements of P wave durations (Pmax, Pmin) and P wave dispersion (PWD), QT durations and dispersion (QTd), corrected QT (QTc), Tp-e interval, and Tp-e/QT ratio were analyzed. Results The lead-exposed and control groups were similar with respect to baseline demographic, laboratory, and transthoracic echocardiographic indices. PWD (26.3 ± 9.7 vs 22.0 ± 9.0 ms, P = 0.014), Pmin (89.9 ± 13.8 vs 79.2 ± 10.1 ms, P < 0.001), and Pmax (116.2 ± 15.0 vs 101.2 ± 14.2 ms, P < 0.001), QT maximum (377.0 ± 27.6 vs 364.9 ± 28.5 ms, P = 0.02), QTd (38.4 ± 16.5 vs 30.5 ± 12.4 ms, P = 0.004), Tp-e interval (78.9 ± 16.5 vs 70.3 ± 14.5 ms, P = 0.003), and Tp-e/QT ratio (0.22 ± 0.04 vs 0.20 ± 0.04, P = 0.013) were significantly higher in lead-exposed workers. QT minimum and QTc values did not differ significantly. QT maximum, QTd, and Tp-e/QT ratio were correlated with urine lead level and Tp-e interval was correlated with both blood and urine lead levels. Conclusions Lead-exposed workers have a higher risk for atrial and ventricular arrhythmias even without overt cardiac diseases compared with healthy subjects. These workers should be followed closely for adverse cardiovascular outcomes especially arrhythmias.