Alok K. Shah

Early Diagnostic Biomarkers for Esophageal Adenocarcinoma—The Current State of Play

Esophageal adenocarcinoma (EAC) is one of the two most common types of esophageal cancer with alarming increase in incidence and very poor prognosis. Aiming to detect EAC early, currently high-risk patients are monitored using an endoscopic-biopsy approach. However, this approach is prone to sampling error and interobserver variability. Diagnostic tissue biomarkers related to genomic and cell-cycle abnormalities have shown promising results, although with current technology these tests are difficult to implement in the screening of high-risk patients for early neoplastic changes. Differential miRNA profiles and aberrant protein glycosylation in tissue samples have been reported to improve performance of existing tissue-based diagnostic biomarkers. In contrast to tissue biomarkers, circulating biomarkers are more amenable to population-screening strategies, due to the ease and low cost of testing. Studies have already shown altered circulating glycans and DNA methylation in BE/EAC, whereas disease-associated changes in circulating miRNA remain to be determined. Future research should focus on identification and validation of these circulating biomarkers in large-scale trials to develop in vitro diagnostic tools to screen population at risk for EAC development. Cancer Epidemiol Biomarkers Prev; 22(7); 1185–209. ©2013 AACR.

Glyco-centric lectin magnetic bead array (LeMBA) − proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals

This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA) – mass spectrometry techniques, “Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma” [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE) and esophageal adenocarcinoma (EAC) individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉) using search key “jn7qafftux”. The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS) data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Abstract 2492: Discovery and validation of novel serum glycoprotein biomarkers for Barrett's esophagus and esophageal adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Esophageal adenocarcinoma (EAC) is one of the most rapidly increasing cancers globally. The majority of EAC cases are diagnosed at very late stages during pathogenesis hence <15% of the patients survive 5 year post-diagnosis. There is an urgent need to improve diagnosis of EAC and its pre-cancer metaplastic condition, Barretts esophagus (BE). BE patients are monitored using upper gastro-esophageal endoscopy with biopsy for early neoplastic changes. However, being an asymptomatic condition, it is very difficult to identify BE patients for screening. Moreover, endoscopy is unsuitable for population screening due to high cost, requirement of technical expertise and patient non-compliance. The aim of this project is to identify serum biomarkers for diagnosis of BE and EAC, with the goal of translating to blood tests. APPROACH & METHODOLOGY: We focused on alterations in circulatory protein glycosylation, using a panel of 20 lectins to isolate different glycan structures on serum glycoproteins, as reported recently [1, 2]. Serum samples from healthy (n = 9), BE (n = 10) and EAC (n = 10) patient groups were analyzed by lectin magnetic bead array-coupled mass spectrometry (LeMBA-MS/MS) [1, 2]. Data analysis was performed using a customized database and analysis package “GlycoSelect” which incorporates outlier detection and sparse Partial Least Squares regression discriminant analysis (sPLS-DA) [3]. RESULTS & DISCUSSION: We identified a ranked list of candidate glycobiomarkers that distinguish a) EAC from BE b) BE from healthy and c) EAC from healthy group. In general, glycoproteins bound several lectins, reflecting heterogeneity and multiplicity of glycosylation. Specific glycan structure changes were observed as loss and gain of binding to a single lectin while maintaining binding to other lectins. Top two candidate biomarkers were validated using orthogonal validation technique LeMBA-immunoblotting in an independent patient cohort (n = 80). The biomarkers showed Area under Receiver Operating Characteristic curve (AUROC) of 0.74 to discriminate EAC from BE and 0.71 to discriminate BE from healthy patient group. Future work will validate all candidate protein-lectin pairs using lectin-affinity array coupled with triple quadrupole quantitative mass spectrometry measurements for the independent patient cohort. The specificity and sensitivity of panels of glycoprotein biomarkers will be determined for formulating a serum screening test for BE and EAC. [1] Choi et al., Electrophoresis 32, 3564-3575 (2011) [2] Loo et al., J Proteome Res 9, 5496-5500 (2010) [3] Le Cao et al., BMC Bioinformatics 12, 253-268 (2011) Citation Format: Alok K. Shah, David Chen, Kim-Anh Le Cao, Eunju Choi, Derek Nancarrow, David Whiteman, Nicholas A. Saunders, Andrew P. Barbour, Michelle M. Hill. Discovery and validation of novel serum glycoprotein biomarkers for Barretts esophagus and esophageal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2014-2492

Abstract 4942: Towards a screening blood test for esophageal adenocarcinoma

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has been rapidly increasing globally. The majority of EAC cases are diagnosed at late stages leading to poor 5-year survival of APPROACH & METHODOLOGY: Serum samples were collected from consenting patients undergoing endoscopy (Control - 16, BE - 13, and EAC - 10). The control group consisted of all patients not classified as BE with or without dysplasia, or EAC. LeMBA-MRM-MS was performed using 4 different lectins with different glycan specificities; AAL (Fucoseα1-2, -3, -6 linked glycan), EPHA (bisecting GlcNAc), JAC (Galα1-6GalNAc and Galβ1-3GalNAc), NPL (Manα1-6Man). Statistical analysis was performed using Shiny MixOmics as previously described [3]. RESULTS & DISCUSSION: Several lectin-protein candidate biomarkers cross-validated in the USA cohort. Most notably, differentially glycosylated complement component C9 is highly diagnostic in both cohorts, achieving area under the receiver operating curve (AUROC) of 0.74 (AAL-C9) to 0.90 (JAC-C9) as a single marker. Complement C9 binding to all four monitored lectins were increased 1.4 to 1.8 fold in EAC, compared to either BE or controls. Another consistent biomarker is gelsolin, with its binding to EPHA and NPL lectin significantly decreased in EAC, compared to either BE or controls. Hence, glycosylated C9 and gelsolin show promise as potential serum biomarkers for EAC screening, and will be further evaluated in prospective cohorts. REFERENCES: [1] Loo et al., J Proteome Res 9, 5496-5500 (2010); [2] Choi et al., Electrophoresis 32, 3564-3575 (2011); [3] Shah et al., Molecular & Cellular Proteomics 14:3023-39 (2015). Citation Format: Alok K. Shah, Virendra Joshi, Kim-Anh Le Cao, David C. Whiteman, Andrew P. Barbour, Michelle M. Hill. Towards a screening blood test for esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4942.