LIQUID BIOPSY PROTEINS AS PSC-SPECIFIC AND PAN-CCA BIOMARKERS OF CANCER RISK, EARLY DIAGNOSIS AND SURVIVAL MIRRORING TUMOR CELLS
1Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute-Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian. 2Proteomics Platform, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), ProteoRed-ISCIII, Bizkaia Science and Technology Park, Derio. 3National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), ISCIII, Madrid. 4Liver and Internal Medicine Unit, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Poland. 5Translational Medicine Group, Pomeranian Medical University, Szczecin, Poland. 6Biotech Research and Innovation Centre, Department of Health and Medical Sciences, University of Copenhagen, Denmark. 7Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway. 8European Reference Network Hepatological Diseases (ERN RARE-LIVER), Hamburg, Germany. 91st Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 10Martin Zeitz Centre for Rare Diseases, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. 11Department of Clinical Science, University of Bergen, Bergen, Norway. 12Department of Medical Biology, Pomeranian Medical University in Szczecin, Poland. 13Department of Gastroenterology, Infectious Diseases and Intoxication, Heidelberg University Hospital, Heidelberg, Germany. 14Experimental Hepatology, Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany. 15Department of Medical Oncology, The Christie NHS Foundation Trust/Division of Cancer Sciences, University of Manchester, Manchester, UK. 16Experimental Hepatology and Drug Targeting (HEVEPHARM), University of Salamanca, Biomedical Research Institute of Salamanca (IBSAL), Salamanca. 17Osakidetza Basque Health Service, Bidasoa IHO, Bidasoa Hospital, Department of Digestive System, Irun. 18Department of Medicine II, Saarland University Medical Centre, Saarland University, Homburg, Germany. 19Laboratory of Metabolic Liver Diseases, Centre for Preclinical Research, Department of General, Transplant and Liver Surgery, Warsaw, Poland. 20Osakidetza Basque Health Service, Ezkerraldea-Enkarterri-Cruces IHO, Cruces University Hospital, Barakaldo. 21Osakidetza Basque Health Service, Donostialdea IHO, Donostia University Hospital, Department of Pathology, San Sebastian. 22Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Exosomes Laboratory, Derio. 23Ikerbasque, Basque Foundation for Science, Bilbao. 24Department of Medicine, Faculty of Medicine and Nursing, University of the Basque Country, UPV/EHU, Leioa. 25Section of Gastroenterology, Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway. 26Department of Biochemistry and Genetics, School of Sciences, University of Navarra, Pamplona.
Introduction and objectives: Cholangiocarcinomas (CCAs), heterogeneous biliary tumors with dismal prognosis, lack accurate early-diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs).
Methods: EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 42), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC etiology (n = 56), hepatocellular carcinoma (n = 34) and healthy individuals (n = 55) were characterized by mass-spectrometry. Diagnostic biomarkers of PSC-CCA, non-PSC CCA or CCAs regardless etiology (pan-CCAs) were defined, and their expression was evaluated in human organs/tissues and within CCA tumors at single-cell level. Prognostic EV-biomarkers for CCA were investigated.
Results: High-throughput proteomics identified candidate diagnostic biomarkers for PSC-CCA, non-PSC CCA or pan-CCA, as well as and for differential diagnosis of intrahepatic CCA and HCC, that were cross-validated by ELISA using total serum. Machine learning logit modelling disclosed CRP/FRIL/Fibrinogen algorithm with diagnostic value for early-stage PSC-CCA v s isolated PSC (AUC = 0.944; OR = 82.0), overpowering CA19-9 (AUC = 0.735; OR = 9.3). An algorithm combining CRP/VWF/PIGR//Fibrinogen allowed the diagnosis of early-stage non-PSC CCAs compared to healthy individuals (AUC = 0.999; OR = 1,115). Noteworthy, levels of Fibrinogen/CRP/PIGR/FRIL showed predictive capacity for CCA development in patients with PSC before clinical evidences of malignancy. Multi-organ transcriptomic analysis revealed that serum EVbiomarkers were mostly expressed in hepatobiliary tissues, and scRNA-seq and immunofluorescence analysis of CCA tumors showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV-prognostic biomarkers independent to clinical features, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively or positively to patients’ survival, respectively.
Conclusions: Serum EVs contain protein biomarkers for the prediction, early diagnosis and prognosis estimation of CCA, representing a novel tumor cell-derived liquid biopsy for personalized medicine.
