title: Serum Glycoprotein Biomarker Discovery and Qualification Pipeline Reveals Novel Diagnostic Biomarker Candidates for Esophageal Adenocarcinoma.
abstract: We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett's esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;
authors.forename: Alok K
authors.initials: AK
authors.lastname: Shah
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;
authors.forename: Kim-Anh Lê
authors.initials: KA
authors.lastname: Cao
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia; §School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia;
authors.forename: Eunju
authors.initials: E
authors.lastname: Choi
authors.affiliation: ¶School of Information and Communication Technology, Griffith University, Brisbane, Queensland, Australia;
authors.forename: David
authors.initials: D
authors.lastname: Chen
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;
authors.forename: Benoît
authors.initials: B
authors.lastname: Gautier
authors.affiliation: ‖QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia;
authors.forename: Derek
authors.initials: D
authors.lastname: Nancarrow
authors.affiliation: ‖QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia;
authors.forename: David C
authors.initials: DC
authors.lastname: Whiteman
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia;
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Saunders
authors.affiliation: **School of Medicine, The University of Queensland, Brisbane, Queensland, Australia;
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Barbour
authors.affiliation: ‡‡Ochsner Health System, Gastroenterology, New Orleans, Louisiana.
authors.forename: Virendra
authors.initials: V
authors.lastname: Joshi
authors.affiliation: From the ‡The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia; m.hill2@uq.edu.au.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Hill
mesh_terms: D000230:Adenocarcinoma / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D000368:Aged; D000818:Animals; D053299:Apolipoprotein B-100 / Q000097:blood / Q000235:genetics*; D001471:Barrett Esophagus / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D002138:Calibration; D016022:Case-Control Studies; D002645:Chickens; D003186:Complement C9 / Q000235:genetics* / Q000378:metabolism; D003937:Diagnosis, Differential; D004938:Esophageal Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D005260:Female; D018260:Gelsolin / Q000097:blood / Q000235:genetics*; D006023:Glycoproteins / Q000097:blood / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010047:Ovalbumin; D037121:Plant Lectins / Q000737:chemistry; D040081:Protein Array Analysis; D012015:Reference Standards; D053719:Tandem Mass Spectrometry
chemical_list: C569471:APOB protein, human; D053299:Apolipoprotein B-100; D014408:Biomarkers, Tumor; D003186:Complement C9; D018260:Gelsolin; D006023:Glycoproteins; D037121:Plant Lectins; D010047:Ovalbumin