Development and validation of circulating protein signatures as diagnostic biomarkers for biliary tract cancer

Troels D Christensen; Emil Maag; Ole Larsen; Claus L Feltoft; Kaspar René Nielsen; Lars Henrik Jensen; Bonna Leerhøy; Carsten P Hansen; Inna M Chen; Dorte L Nielsen; Julia S Johansen

doi:10.1016/j.jhepr.2022.100648

Development and validation of circulating protein signatures as diagnostic biomarkers for biliary tract cancer

JHEP Rep. 2022 Dec 13;5(3):100648. doi: 10.1016/j.jhepr.2022.100648. eCollection 2023 Mar.

Authors

Troels D Christensen¹, Emil Maag², Ole Larsen¹, Claus L Feltoft³, Kaspar René Nielsen⁴, Lars Henrik Jensen⁵, Bonna Leerhøy⁶, Carsten P Hansen⁷, Inna M Chen¹, Dorte L Nielsen^{1

8}, Julia S Johansen^{1

3

8}

Affiliations

¹ Deparment of Oncology, Copenhagen University Hospital - Herlev and Gentofte Hospital, Herlev, Denmark.
² BioXpedia, Aarhus, Denmark.
³ Department of Medicine, Copenhagen University Hospital - Herlev and Gentofte Hospital, Herlev, Denmark.
⁴ Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark.
⁵ Department of Oncology, University Hospital of Southern Denmark, Vejle, Denmark.
⁶ Digestive Disease Center, Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark.
⁷ Department of Surgery, Copenhagen University Hospital - Rigshospitalet, Copenhagen, Denmark.
⁸ Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Abstract

Background & aims: Biliary tract cancer (BTC) is associated with a dismal prognosis, partly because it is typically diagnosed late, highlighting the need for diagnostic biomarkers. The purpose of this project was to identify and validate multiprotein signatures that could differentiate patients with BTC from non-cancer controls.

Methods: In this study, we included treatment-naïve patients with BTC, healthy controls, and patients with benign conditions including benign biliary tract disease. Participants were divided into three non-overlapping cohorts: a case-control-based discovery cohort (BTC = 186, controls = 249); a case-control-based validation cohort (validation cohort 1: BTC = 113, controls = 241); and a cohort study-based validation cohort including participants (BTC = 8, controls = 132) referred for diagnostic work-up for suspected cancer (validation cohort 2). Immuno-Oncology (I-O)-related proteins were measured in serum and plasma using a proximity extension assay (Olink Proteomics). Lasso and Ridge regressions were used to generate protein signatures of I-O-related proteins and carbohydrate antigen 19-9 (CA19-9) in the discovery cohort.

Results: Sixteen protein signatures, including 2 to 82 proteins, were generated. All signatures included CA19-9 and chemokine C-C motif ligand 20. Signatures discriminated between patients with BTC vs. controls, with AUCs ranging from 0.95 to 0.99 in the discovery cohort and 0.94 to 0.97 in validation cohort 1. In validation cohort 2, AUCs ranged from 0.84 to 0.94. Nine signatures achieved a specificity of 82% to 84% while keeping a sensitivity of 100% in validation cohort 2. All signatures performed better than CA19-9, and signatures including >15 proteins showed the best performance.

Conclusion: The study demonstrated that it is possible to generate protein signatures that can successfully differentiate patients with BTC from non-cancer controls.

Impact and implications: We attempted to find blood sample-based protein profiles that could differentiate patients with biliary tract cancer from those without cancer. Several profiles were found and tested in different groups of patients. The profiles were successful at identifying most patients with biliary tract cancer, pointing towards the utility of multiprotein signatures in this context.

Keywords: AUC, area under receiver-operating characteristic curve; BBTD, benign biliary tract disease; BP, best point; BTC, biliary tract cancer; CA19-9, carbohydrate antigen 19-9; CAIX, carbonic anhydrase IX; CASP8, caspase 8; CCA, cholangiocarcinoma; CCL, chemokine (C-C motif) ligand; CXCR, C-X-C motif chemokine; EDTA, ethylenediaminetetraacetic acid; GBC, gall bladder cancer; I-O, immuno-oncology; IL, interleukin; MMP-, matrix metalloproteinase-; NPX, normalized protein expression; TME, tumor microenvironment; biliary tract cancer; blood protein assay; cholangiocarcinoma; dCCA, distal cholangiocarcinoma; diagnosis; gall bladder cancer; iCCA, intrahepatic cholangiocarcinoma; multi-biomarker signature; pCCA, perihilar cholangiocarcinoma.