Determination of copper and other trace elements in serum samples from patients with biliary tract cancers: prospective noninterventional nonrandomized clinical study protocol

Essential trace elements are needed in minute amounts for normal physiology. Among them are iodine (I), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), selenium (Se), cobalt (Co) and molybdenum (Mo).¹¹ Alterations in levels and changes in the expression of proteins involved in metal metabolism have been demonstrated in a variety of cancers. First, the hyperproliferation of cancer cells renders them more reliant on iron than normal cells. Targeting iron metabolism in cancer cells is an emerging field of therapeutics.¹² When essential trace elements (Mn, Co, Zn, Cu, Se) in the serum, cell fraction, cerebrospinal fluid and tumor tissue samples of malignant brain cancer patients were analyzed, it was shown that elemental profiles in these samples were significantly altered in these cancer patients compared to the healthy individuals. Higher contents of trace elements (particularly Mn, Se, and lead (Pb)) could also be involved in the pathogenesis of brain tumors. Therefore, the urine-to-serum ratio of essential trace elements was proposed as an appropriate diagnostic biomarker in malignant brain tumors.¹³

Cu is an essential trace element with a precisely regulated amount in our bodies.¹⁴ Cu is present in all tissues. It is stored primarily in the liver and then in the muscles, heart, kidneys and brain. In the blood, it is transported bound to the protein Cp.^14,15,16 It is a coenzyme of many enzymes (e.g., Cu/Zn superoxide dismutase, ceruloplasmin, cytochrome oxidase, tyrosinase, dopamine hydroxylase, lysine oxidase, catalase, selenium-dependent peroxidase, etc.) that are important for cellular respiration and defense against free radicals. It also affects glutathione function. Consequently, Cu deficiency impairs cellular respiration and the regulation of reactive oxygen species. Deregulation of oxidative stress, due to excessive production of reactive oxygen species, impairs cellular DNA repair mechanisms and is an important mechanism in the development of cancer.¹⁴ In addition to malignant processes, an imbalance of Cu in the body affects the development and progression of chronic, inflammatory and neurodegenerative diseases. Cu deficiency leads to lower overall energy levels, abnormal glucose and cholesterol metabolism, increased oxidative damage, and changes in the function and structure of circulating blood and immune cells.¹⁴ Cu deficiency is associated with a higher incidence of infections and an increased risk of cardiovascular disease.¹⁴

Specifically, due to its role in inflammatory and antioxidant processes, Cu has an important role in the development of various cancers, such as gynecological cancers, lung cancer, colorectal cancer and other cancers of the digestive tract.¹⁴ Recent preclinical and clinical data confirmed that Cu concentrations are abnormal in malignant tissues of mice and in cancer patients. Namely, in humans, elevated Cu concentrations have been found in malignant tissues of the breast, ovary, lung and stomach. Cu is being investigated as a potential target for cancer treatment due to its elevated levels in malignant tissues and its ability to promote angiogenesis, cancer growth and metastasis.^14,15,16,17 In addition to malignant tissues, the concentration of Cu is also elevated in the serum of cancer patients. Elevated levels of Cu have been measured in the serum of patients with lung cancer, colorectal cancer, epithelial ovarian cancer and biliary tract cancers, and decreased levels in adrenocortical and hepatocellular carcinoma.^{14,15,16,17,18,19}

When Cu regulation is disrupted, the quantities and proportions of other essential trace elements may also be altered. Among these, the normal Cu/Zn ratio is known to be disturbed. An imbalance of Cu in the body affects the development and progression of chronic, inflammatory and neurodegenerative diseases and malignant processes. It has been found that high dietary intake of Zn can reduce intestinal absorption of Cu.²⁰ Altered intakes of only one of the two (similar observations are also made for the other essential trace elements) may cause an imbalance of the other. For example, relatively low levels of Zn and elevated levels of Cu can increase oxidative stress and impair the antioxidant activity of many enzymes.²⁰ Increased Cu/Zn ratios have been found in a wide variety of malignancies, including gastrointestinal cancers, gynecological cancers, breast cancer, and lung cancer, and have been correlated with the stage or condition of the disease at the time of treatment. ^{14,15,16,17,18,19,20} It has been suggested that the Cu/Zn ratio could be used for clinical diagnosis and as a prognostic biomarker to track response to treatment.

Trace element disorders (TEDs) are well established in diseases of genetic origin for which the levels of physiologically relevant metals in the blood are controlled by specific proteins. Inherited TED can result in protein malfunction and therefore, deficiency or toxic accumulation of metal in the body. Well-known examples are Wilson’s disease and hemochromatosis.^21–22 Diagnosis usually involves gene mutation testing, clinical observations and biochemical testing. Examples of such biochemical tests are determinations of non-Cp-bound Cu, exchangeable Cu, total blood Fe and serum ferritin (light chain). Despite growing evidence that TED are also associated with many types of cancer, knowledge in this field of research is still relatively scarce.^{11,12,14,15,16,17,18,19,20} It requires highly sophisticated interdisciplinary investigations, which promises to provide very useful information on the role of trace metals in cancers.

Several new findings have shown the potential to use TED identification as a biomarker for cancer.²³ It has been suggested that the imbalance in the Cu/Zn ratio could be used for clinical diagnosis and as a predictive biomarker to track the response to treatment. Cp correlates with immune infiltration and serves as a prognostic biomarker in breast cancer. Elevated serum Cu-Cp levels have been found in lung cancer, colon carcinoma, epithelial ovarian cancer and bile duct cancer, while the expression of Cu-Cp is significantly downregulated in adrenocortical carcinoma and hepatocellular carcinoma.²⁴ Serum Cu levels increase in several types of cancer. It was experimentally determined that in hepatocellular carcinoma patients, blood Cu and sulphur (S) are enriched in light isotopes compared with healthy individuals. Isotopic ratios of Cu (⁶⁵Cu/⁶³Cu) and S (³⁴S/³²S) were measured to elucidate their use as potential biomarkers of disease.²⁵ Changes in the isotopic compositions of Fe, Cu and Zn and their corresponding concentrations in plasma from hematological malignancy patients can be measured to assess their prognostic capability. Imbalances in trace metal concentrations, changes in their speciation and isotopic fractionation need to be further investigated to fully evaluate their emerging biomarker potential.²⁶

For improvements in cancer therapy efficacy, reliable and optimized analytical and imaging methods using contemporary instrumental techniques that allow investigations on the role of trace elements in cancer, quantitative determination of established or emerging biomarkers (exchangeable and Cp-bound Cu, stable isotope ratio of trace metals, such as Cu, Zn or Fe), as well as the monitoring of penetration, distribution and metabolism of a metallodrug within the target tissue/tumor are needed.^{27,28,29,30,31}

Cu toxicity is strongly related to its free (exchangeable) fraction, which is not bound to Cp.^32,33,34 Due to the important physiological functions and role of Cu in various diseases, it is necessary to quantify its exchangeable and bound to Cp fractions. In clinical practice, Cp in serum or plasma is commonly determined by nephelometry or turbidimetry. They unspecifically measure both holoCp (Cp with Cu) and apoCp (Cp without Cu). The latter Cp form (apoCp) is not relevant for medical diagnosis. To overcome this disadvantage, monolithic chromatography coupled to inductively coupled plasma‒mass spectrometry, which allows simultaneous quantification of exchangeable Cu, Cu bound to human albumin and holoCp, can be used. The chromatographic column used in this method comprises convective interaction media (CIM) affinity and weak anion-exchange disks (Protein G and diethylamine (DEAE) disks) assembled into a single housing forming a CLC monolithic column.^{35,36,37,38,39}

Isotopic fractionation of stable isotopes of essential metals was proposed as an emerging predictive biomarker for cancer diagnosis. In hepatocellular carcinoma patients, blood Cu and sulphur (S) are enriched in light isotopes compared with control subjects. Isotopic ratios of Cu (⁶⁵Cu/⁶³Cu) and S (³⁴S/³²S) were measured to elucidate their use as potential biomarkers of disease.^31,32 Changes in the isotopic compositions of Fe, Cu and Zn and their corresponding concentrations in plasma from hematological malignancy patients can be measured to assess their prognostic capability.^31,32

Trace elements as biomarkers in oncology are promising fields for detecting, diagnosing and predicting responses to treatment. To date, there has been no published clinical trial investigating copper as a predictive biomarker of response to systemic therapy. In this context, we selected patients with locoregional advanced, inoperable or metastatic BTCs for this noninterventional non-randomized prospective clinical trial, treated with first-line systemic chemotherapy or immunochemotherapy, 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. The proportion of free Cu and Cu bound to Cp and the isotopic ratio of ⁶⁵Cu/⁶³Cu in serum samples will be determined in enrolled healthy volunteers to establish reference values for the general population and to provide the foundation for the development of BTC cancer screening methodology. Determination of the total concentration of trace elements and speciation analysis will be carried out on a quadrupole inductively coupled plasma mass spectrometer (ICP-MS), while isotopic ratios will be precisely determined by multicollector ICP-MS.

In a prospective, noninterventional, nonrandomized clinical study started in 2023 at the Institute of Oncology Ljubljana, 20 patients with BTC and 20 healthy volunteers are planning to enroll to provide the development of a BTC cancer screening methodology. The proportion of free Cu and Cu bound to Cp and the isotopic ratio of ⁶⁵Cu/⁶³Cu in serum samples will be determined in enrolled healthy volunteers to establish reference values. The inclusion of 20 BTC patients will enable the identification of serum Cu levels as a potential predictive biomarker of response to systemic therapy in correlation with radiological CT evaluation for response to systemic therapy. The clinical study was approved by the Ethics Committee ERIDEK-0095/2022 and the Institutional Review Board ERID-KSOPKR-0091/2022 at the Institute of Oncology Ljubljana, approved 13.12.2022 by the Commission of the Republic of Slovenia for Medical Ethics (0120-472/2022/3). It was registered with ClinicalTrials.gov under the registration number NCT06060990. All patients entering the study signed informed consent forms. Monitoring will be carried out throughout the study. The flow diagram of the study is presented in Figure 1.

FIGURE 1.

The main purpose of the research is to determine the total concentration of selected trace elements (Cu, Zn, Fe), the proportion of free Cu and Cu bound to Cp, and the isotopic ratio between ⁶⁵Cu/⁶³Cu in blood serum samples of healthy volunteers and BTC cancer patients using methods based on ICP-MS. We will statistically evaluate the results and evaluate the possibilities of using the used analytical methods and the results of these clinical trials in cancer diagnostics and therapy.

Our hypothesis is that serum Cu levels and the ⁶⁵Cu/⁶³Cu isotope ratio in cancer patients differ significantly from those in healthy volunteers and that these levels vary according to the response to systemic chemotherapy or chemoimmunotherapy.

The first primary objective is to establish reference levels of Cu, Zn and Fe in the serum of healthy volunteers and their levels in locally advanced inoperabile and metastatic BTC patients to establish a framework for reference Cu, Zn and Fe values.

The second primary objective of the study is 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.

The secondary objective of the study is to apply newly developed analytical methods based on ICP-MS to metal-based biomarker research in oncology.

In this prospective, noninterventional, nonrandomized clinical study, we aim to include 20 patients with locoregionally advanced, inoperable or metastatic BTC who will start with first-line systemic chemotherapy or immunochemotherapy at the Institute of Oncology Ljubljana. Potential study participants will be identified at the institutional multidisciplinary tumor board, comprised of diagnostic radiologists, interventional radiologists, hepatobiliary surgeons, medical oncologists, and radiation oncologists. The study protocol will be explained to all eligible patients in detail. Only those who sign the consent form will enter the clinical study.

Patients’ blood levels of Cu and other trace metals will be determined before starting systemic therapy and at least during one cycle of systemic therapy will be included in the analysis.

Twenty healthy volunteers will also be enrolled, after prior signed written consent to participate in the clinical study, for a single 7 ml blood draw for analysis.

Patients will be followed for up to 12 consecutive months. All patients for whom the medical oncologist decides to be treated with first-line systemic therapy with combination chemotherapy with cisplatin and gemcitabine or combination chemotherapy combined with immunotherapy with the anti-PD-L1 inhibitor durvalumab during the course of treatment will be invited to participate in the clinical trial. Systemic chemotherapy and immunotherapy treatment, duration of treatment and other medical procedures will be performed independently of the study at the discretion of the medical oncologist and according to the recommendations of good clinical practice.

Systemic chemotherapy with cisplatin and gemcitabine will be administered in cycles every 3 weeks, with cisplatin and gemcitabine administered on days 1 and 8 of each cycle, for a total of 8 cycles, or systemic chemotherapy with cisplatin and gemcitabine administered in cycles every 3 weeks, with cisplatin and gemcitabine administered on days 1 and 8 of each cycle, in combination with immunotherapy with durvalumab administered on day 1 of each cycle for a total of 8 cycles, followed by maintenance treatment with durvalumab immunotherapy every 4 weeks until disease progression, unacceptable toxicity or a decision to discontinue treatment by the patient or treating medical oncologist. Standard chest and abdomen CT imaging with contrast will be performed before the start of systemic treatment as baseline imaging to delineate the extent of disease, then 3 months after the start of systemic treatment to assess the efficacy of treatment, and finally 6 months after the start of treatment. All further diagnostic and therapeutic procedures will be part of the standard management of patients undergoing systemic chemotherapy and immunotherapy. All decisions on additional treatment, either surgery or radiotherapy during systemic therapy, will be made by the multidisciplinary gastrointestinal cancer consortium. In the case of adverse events of systemic chemotherapy and immunotherapy, actions will follow standard recommendations for the treatment of complications and discontinuation of systemic treatment. Despite discontinuation of systemic treatment, patients will continue with the planned investigations in accordance with good clinical practice and according to the protocol of the clinical trial, in line with the primary and secondary objectives of the trial.

All patients will give blood for laboratory tests, blood counts and biochemical tests, including a blood draw to determine the serum Cu level, its speciation and isotopic fractionation before starting treatment and then before each application of systemic therapy. A blood sample will be obtained before the start of systemic therapy and then on days 1 and 8 of each cycle at a regular outpatient check-up at the Institute of Oncology Ljubljana. Patients will additionally have 7 ml of blood drawn into a standard serum tube. The blood sample will be send to the Department of Experimental Oncology of the Institute of Oncology Ljubljana, where the sample will be centrifuged (1300 × g, 10 min, 4°C) and the serum needed for the analysis will be stored at −20°C until the analysis is performed by ICP-MS-based techniques at the Jožef Stefan Institute.

Once patient enrollment in the clinical study has been completed and 20 patients have been enrolled, this will be followed by the enrollment of 20 age- and sex-matched healthy volunteers. The blood sample will be prepared, stored, and analyzed as for the patients.

All patients will undergo diagnostic imaging (chest and abdominal CT with contrast) up to 4 weeks prior to enrollment in the clinical trial and then at 12 (± 7 days) and 24 weeks (± 7 days) after initiation of treatment and if disease progression or adverse events of systemic therapy are suspected. CT scans will be evaluated according to RECIST (response evaluation criteria in solid tumors) and irRECIST criteria (immune-related response evaluation criteria in solid tumors).^40,41 The IrRECIST criteria divide the response to treatment into different groups: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Pseudoprogression is defined as transient radiological disease progression in the absence of clinical progression and a progressive reduction in the burden of the underlying disease according to irRECIST criteria in patients who will receive durvalumab immunotherapy in addition to systemic chemotherapy.

In case of adverse events of treatment with systemic chemotherapy and immunotherapy, measures will follow standard recommendations for treatment of complications and interruption of systemic treatment. Adverse events of systemic therapy will be treated in accordance with the recommendations of the NCI Common Terminology Criteria for Adverse Events (CTCAE) v 5.0 and in accordance with good clinical practice.⁴² Despite discontinuation of systemic therapy, patients will continue with planned investigations in accordance with good clinical practice and according to the protocol in the clinical trial according to the primary and secondary objectives of the trial.

Before each cycle of systemic therapy, a laboratory blood sample will be taken as a standard before the decision to continue treatment. Standard peripheral blood sampling will follow hygiene protocols. At the same time, we will add an additional peripheral blood sample to determine Cu in the serum. The collection of additional samples does not pose a major health risk, and the possible complications of blood collection are mainly local: the appearance of a hematoma or infection.⁴² Imaging evaluation poses potential hazards due to contrast agent administration, anaphylactic reaction, and ionizing radiation.⁴²

CT imaging diagnostics will take place at the same time intervals as planned for the evaluation of the effectiveness of systemic treatment. In this way, the subjects will not be exposed to additional imaging tests. In the case of a known allergy to the contrast agent, the procedure will be performed with appropriate premedication or with other methods. Hydration and other measures will be taken before the planned imaging diagnostics in case of deterioration of renal function.

Total concentrations of trace metals and quantitative determination of relevant species (Cu-Cp, exchangeable Cu) will be determined by ICP-MS or high-performance liquid chromatography coupled to inductively coupled plasmamass spectrometry (HPLC-ICPMS), respectively.^36,37,39 Emerging metal-based biomarkers (stable isotope fractionation of Cu, and, if relevant, Zn and S) will be followed by multicollector ICP-MS.

Data from the determination of relevant trace elements (with special attention to Cu) before systemic therapy and at least during one cycle of systemic therapy will be included in the analysis. The association between the change in, for example, Cu concentration, speciation, and/or isotopic fractionation between healthy individuals and those suffering from biliary tract cancer will be statistically analyzed to evaluate the applicability of such an approach as a diagnostic biomarker for disease. The same data will be used to assess response to treatment by a logistic regression model and a multivariate model including different variables. Paired t test or an appropriate nonparametric alternative will be used to compare values at different time points, and analysis of variance (ANOVA) or Kruskal – Wallis’s test will be used when comparing several groups simultaneously. Statistical analysis will be performed using GraphPad Prism (GraphPad, San Diego, CA, USA), and differences will be considered statistically significant if p<0.05.

Response to treatment will be determined radiologically using RECIST or irRECIST criteria.^40,41 The objective response to treatment will be calculated as the percentage of patients who have a partial or complete response according to the RECIST or irRECIST criteria among all patients who will receive at least one cycle of systemic therapy and have at least one radiological assessment during systemic treatment. For patients who will not progress or die, the end date for analysis will be the date of last follow-up. Time to disease progression will be defined as the time interval from the date of first therapy administration to the date of disease progression or death, using the Kaplan‒Meier method. Comparison of survival of several groups will be calculated using the log-rank test. The association between the change in Cu concentration, speciation and/or fractionation and response to treatment will be assessed by a logistic regression model and a multivariate model including different variables. Statistical analysis will be performed as previously described in the Data analysis chapter.

The clinical protocol was approved by the Ethics Committee ERIDEK-0095/2022 and the Clinical Trials Protocol Review Committee ERIDK-SOPKR-0091/2022 at the Institute of Oncology Ljubljana, and 13.12.2022 was approved by the Commission of the Republic of Slovenia for Medical Ethics (0120-472/2022/3). The clinical study will be performed in accordance with the ethical principles of the Declaration of Helsinki. The clinical trial number: NCT06060990. Informed consent will be obtained from each participating patient and healthy volunteer in written form.