Trace metals disorder as a biomarker in biliary tract cancers, 1.10.2023-30.9.2026, Janez Ščančar



Trace metals disorders (MD) in cancer patients are associated with health problems for which treatments are poorly effective. In this project, we set out to identify and investigate the MD in biliary tract cancers at pre-clinical and clinical levels by integrating ICP-MS-based methods into complex analytical methodology in order to test the hypothesis that MD can be used as biomarkers for disease. Objectives will be met by the application of newly developed analytical methods to metal-based biomarker research in oncology at the pre-clinical and clinical levels. The clinical study is designed with the aim of identifying serum Cu, Zn, and Fe levels and their ratios. Cu speciation and its isotopic fractionation will be investigated as a predictive biomarker of response to systemic therapy. Radiological CT evaluation and other clinical tests will be used to confirm the feasibility of the approach proposed. Pre-clinical investigations are carried out at the Jozef Stefan Institute, Ljubljana Slovenia, and clinical at the Institute of Oncology Ljubljana, Ljubljana, Slovenia where a prospective, non-interventional, non-randomized clinical trial is designed with the goal to identify MD specific for biliary tract cancers. Clinical trial is led by Assist. Prof. Martina Reberšek.

Background: Biliary tract cancers (BTCs) are a heterogeneous group of uncommon and rare epithelial tumors. In the early stages, patients are asymptomatic without specific clinical presentation and diagnosed at an advanced stage, when the disease is incurable. Currently used tumor biomarkers have limited diagnostic value for BTCs, so there is an urgent need for sensitive and specific biomarkers for their earlier diagnosis. Deregulation of homeostasis of trace elements is involved in the carcinogenesis of different cancers including BTCs.

Objective:  The objective is to determine the total concentration of copper, zinc, and iron and, if important, other trace elements, the proportion of free copper and copper bound to ceruloplasmin and the isotopic ratio of 65-copper/63-copper in blood serum samples from healthy volunteers and cancer patients using inductively coupled plasma mass spectrometry-based methods. The results will be statistically evaluated for usability of MD identified as biomarker of BTCs and the potential of the analytical methodology used in cancer diagnosis and therapy will be assessed.


Posnetek zaslona 2024 02 26 111644

Methods and analysis:  A prospective, non-interventional, non-randomized study, in which 20 patients and 20 healthy volunteers enroll, will be carried out. Objectives are to identify serum copper, zinc, iron, and other trace elements levels, Cu speciation and/or isotopic fractionation as a predictive biomarker of response to systemic therapy of BTCs, which will be evaluated by radiological computer tomography. In the investigations, newly developed analytical methods based on inductively coupled plasma mass spectrometry will be applied to metal-based biomarker research in oncology.

Ethics and dissemination:  The trial will be conducted according to the ethical principles of the Declaration of Helsinki and has been approved by the ethics committee of all participating centers. The results of this study will be published in peer-reviewed scientific journals and presented at relevant academic conferences.

Objectives will be met by the development and the application of newly developed analytical methods to metal-based biomarker research in oncology at pre-clinical and clinical level. Clinical study is designed to identify serum Cu, Zn, and Fe levels and ratios, and Cu speciation and isotopic fractionation as a predictive biomarker of response to systemic therapy in correlation with radiological CT evaluation for response to systemic therapy and thereby confirm the feasibility of the approach proposed.


The project consists of 4 work packages. Within WP1, protocols for sample collection, selection, storage, and preparation (3D models, tissue, cells, and plasma/serum) will be selected. Special attention will be paid to preserving the chemical species' analyzed integrity. These include also preparation and characterization of calibrants, isotopic standards, model samples, and reference test materials. WP 2 is devoted to the developments and applications of ICP-MS-based analytical methods (total metal concentration, speciation and isotopic fractionation analysis) for the identification of MD and altered speciation and isotopic ratios of essential trace elements (mainly Cu, then Zn and Fe) in the investigation on the possibility to use MD as bio-marker of biliary tract cancers. Additionally, methods for the determination of the quantitative distribution of the selected cancer-related analytes (including possible biomarkers)relevant for disease diagnosis and treatment in tumor spheroid (3D model) at the low ng/g concentration level by laser ablation – ICP-MS and supporting imaging techniques for possible future application at the clinical level (cancer biopsy samples) will be developed and validate and single cell ICP-MS technique introduced to pre-clinical investigation for the correlation of individual cell metal content with other important biochemical parameters to predict the response of tumor in cancer therapy. WP3 is a prospective, non-interventional, non-randomized clinical trial with 20 patients and 20 healthy volunteers carefully prepared to identify serum Cu levels, its speciation and/or isotopic fractionation as a predictive biomarker of response to systemic therapy in correlation with radiological CT evaluation for response to systemic therapy. With this, newly developed analytical methods based on ICP-MS will be directly integrated to metal-based biomarker research in oncology.



Statistical data handling is part of all the experimental WPs (1 to 3), while WP4 is devoted to management (Creating impact, management, and coordination). Such organization of the project is optimal for the number of partners participating, experimental work planned, and amount of finance.

The topic of this project is oriented to the needs of end-users. The beneficiaries from the results of this project will include academics, health protection bodies and clinics, clinical laboratories, oncological patients and wider human society.

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Jožef Stefan Institute (JSI), Ljubljana, Slovenia:

Janez Ščančar / 18359: leader

Radmila Milačič / 8314: researcher

Tea Zuliani / 25667: researcher

Janja Vidmar / 36350: researcher

Katarina Marković / 52052: researcher

Stefan Marković / 57296: researcher

Pia Leban / 55787: young researcher

Tjaša Žerdoner / 54691: young researcher

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Institute of Oncology (OIL), Ljubljana, Slovenia:

Martina Reberšek / 20056: leader on OIL

Nežka Hribernik / 52649: technician

Tanja Mesti / 32614: researcher

Janja Ocvirk / 13541; researcher

Maja Čemažar / 14575: researcher

Katja Uršič Valentinuzzi / 38223: researcher

Teja Valant / 55607: researcher

Katarina Žnidar / 37534: researcher

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