TY - JOUR

T1 - Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis and prognostication of cholangiocarcinoma

AU - Lapitz, Ainhoa

AU - Azkargorta, Mikel

AU - Milkiewicz, Piotr

AU - Olaizola, Paula

AU - Zhuravleva, Ekaterina

AU - Grimsrud, Marit M

AU - Schramm, Christoph

AU - Arbelaiz, Ander

AU - O'Rourke, Colm J

AU - La Casta, Adelaida

AU - Milkiewicz, Malgorzata

AU - Pastor, Tania

AU - Vesterhus, Mette

AU - Jimenez-Agüero, Raul

AU - Dill, Michael T

AU - Lamarca, Angela

AU - Valle, Juan W

AU - Macias, Rocio I R

AU - Izquierdo-Sanchez, Laura

AU - Castaño, Ylenia Pérez

AU - Caballero-Camino, Francisco Javier

AU - Riaño, Ioana

AU - Krawczyk, Marcin

AU - Ibarra, Cesar

AU - Bustamante, Javier

AU - Nova-Camacho, Luiz M

AU - Falcon-Perez, Juan M

AU - Elortza, Felix

AU - Perugorria, Maria J

AU - Andersen, Jesper B

AU - Bujanda, Luis

AU - Karlsen, Tom H

AU - Folseraas, Trine

AU - Rodrigues, Pedro M

AU - Banales, Jesus M

N1 - Copyright © 2023. Published by Elsevier B.V.

PY - 2023

Y1 - 2023

N2 - BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks 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=44), PSC who developed CCA during follow-up (PSC to CCA; n=25), CCAs from non-PSC etiology (n=56), hepatocellular carcinoma (HCC; n=34) and healthy individuals (n=56) were characterized by mass-spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA or CCAs regardless etiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumors at single-cell level. Prognostic EV-biomarkers for CCA were investigated.RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and HCC, that were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease (LD)) vs isolated PSC (AUC=0.947;OR=36.9), and combined with CA19-9, overpowers CA19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs healthy individuals (AUC=0.992;OR=387.5). Noteworthy, CRP/FRIL accurately diagnosed LD Pan-CCA (AUC=0.941;OR=89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidences of malignancy. Multi-organ transcriptomic analysis revealed that serum EV-biomarkers 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, 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 detectable using total serum, representing a tumor cell-derived liquid biopsy tool for personalized medicine.IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumor biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and etiology-related logistic models with predictive, diagnostic or prognostic capacities by combining 2-4 circulating protein biomarkers, moving a step forward into personalized medicine. These novel liquid biopsy tools may allow the: i) easy and non-invasive diagnosis of sporadic CCAs, ii) identification of patients with PSC with higher risk for CCA development, iii) establishment of cost-effective surveillance programs for the early detection of CCA in high-risk populations (e.g., PSC), and iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

AB - BACKGROUND & AIMS: Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks 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=44), PSC who developed CCA during follow-up (PSC to CCA; n=25), CCAs from non-PSC etiology (n=56), hepatocellular carcinoma (HCC; n=34) and healthy individuals (n=56) were characterized by mass-spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA or CCAs regardless etiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumors at single-cell level. Prognostic EV-biomarkers for CCA were investigated.RESULTS: High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and HCC, that were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease (LD)) vs isolated PSC (AUC=0.947;OR=36.9), and combined with CA19-9, overpowers CA19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs healthy individuals (AUC=0.992;OR=387.5). Noteworthy, CRP/FRIL accurately diagnosed LD Pan-CCA (AUC=0.941;OR=89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidences of malignancy. Multi-organ transcriptomic analysis revealed that serum EV-biomarkers 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, 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 detectable using total serum, representing a tumor cell-derived liquid biopsy tool for personalized medicine.IMPACT AND IMPLICATIONS: The accuracy of current imaging tests and circulating tumor biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and etiology-related logistic models with predictive, diagnostic or prognostic capacities by combining 2-4 circulating protein biomarkers, moving a step forward into personalized medicine. These novel liquid biopsy tools may allow the: i) easy and non-invasive diagnosis of sporadic CCAs, ii) identification of patients with PSC with higher risk for CCA development, iii) establishment of cost-effective surveillance programs for the early detection of CCA in high-risk populations (e.g., PSC), and iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

U2 - 10.1016/j.jhep.2023.02.027

DO - 10.1016/j.jhep.2023.02.027

M3 - Journal article

C2 - 36868481

VL - 79

SP - 93

EP - 108

JO - Journal of Hepatology, Supplement

JF - Journal of Hepatology, Supplement

SN - 0169-5185

IS - 1

ER -