Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders

Podsumowanie

The present protocol describes how to perform ⁶⁴Cu PET/CT and PET/MRI imaging in humans to study copper-related disorders, such as Wilson disease, and the treatment effect on copper metabolism.

Streszczenie

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke's disease. A recent addition to this toolkit is 64-copper (⁶⁴Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the ⁶⁴Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, ⁶⁴Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, ⁶⁴Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform ⁶⁴Cu PET/CT or PET/MR in humans. Procedures for ⁶⁴Cu handling, patient preparation, and scanner setup are demonstrated here.

Wprowadzenie

Copper is a vital catalytic cofactor that drives multiple important biochemical processes essential for life, and defects in copper homeostasis are directly responsible for human diseases. Mutations in the ATP7A or ATP7B genes, encoding copper-transporting ATPases, cause Menke's and Wilson diseases, respectively. Menke's disease (ATP7A) is a rare lethal disorder of intestinal copper hyperaccumulation with severe copper deficiency in peripheral tissues and deficits in copper-dependent enzymes¹. Wilson disease (WD) (ATP7B) is a rare disease characterized by the inability to excrete excess copper to bile, resulting in copper overload and subsequent organ damage, most severely affecting the liver and brain².

Studies on copper metabolism have utilized radiolabeled copper (usually 64-copper [⁶⁴Cu] or 67-copper) for decades, and these studies have proven invaluable for our understanding of mammalian copper metabolism, including absorption site and excretion pathways^3,4,5,6. Previously, gamma counters were used to detect the radioactive signal with a limited anatomical resolution, but recently, ⁶⁴Cu positron emission tomography (PET) combined with computed tomography (CT) or magnetic resonance imaging (MRI) has been introduced in both human and animal studies. Today, PET scanners have such a high sensitivity that it is possible to track ⁶⁴Cu for up to 70 h after injection. The long half-life of 12.7 h for ⁶⁴Cu allows for the long-term assessment of copper fluxes. This improvement in resolution has just recently entered the field of copper studies, and studies on normal and pathological copper metabolism, as well as studies evaluating the impact of specific treatments, are starting to emerge. Additionally, the introduction of whole-body PET scanners with an extended field-of-view will further enhance the sensitivity of these examinations.

This methodological paper aims to enable clinicians and scientists to add ⁶⁴Cu PET CT/MRI to the existing repertoire of tools as a robust and easy-to-use method for assessing copper metabolism in a manner comparable between nuclear medicine departments. The production of ⁶⁴Cu copper can be carried out using different methods and is usually performed at special facilities. Among the nuclear reactions, the ⁶⁴Ni (p, n) ⁶⁴Cu method is widely used, since a high production yield of ⁶⁴Cu can be obtained with low energy protons in this route^7,8. A detailed description of the production methods is out of the scope of this work, and availability will differ by country and region.

In this article, we first describe the preparation of the necessary radiochemistry and the tracer. Then, the principles for preparing the PET/CT or PET/MRI scanners are demonstrated.

Protokół

A few clinical trials using this ⁶⁴Cu PET/CT or PET/MRI protocol have been approved by the Regional Ethics Committee of Region Midt, Denmark [1-10-72-196-16 (EudraCT 2016-001975-59), 1-10-72-41-19 (EudraCT 2019-000905-57), 1-10-72-343-20 (EudraCT 2020-005832-31), 1-10-72-25-21 (EudraCT 2021-000102-25), and 1-10-72-15-22 (EudraCT 2021-005464-21)]. Written informed consent was obtained from the participants at enrollment. The inclusion criteria for all participants were age >18, and for females the use of safe contraception. The exclusion criteria for Wilson disease patients were decompensated cirrhosis, a Model for End-stage Liver Disease (MELD) score >11, or a modified Nazer score >6. The exclusion criteria for all participants were a known hypersensitivity to ⁶⁴Cu or other ingredients in the tracer formula, pregnancy, breastfeeding, or a desire to become pregnant before the end of the trial.

1. Preparation of ⁶⁴CuCl₂

1. Dissolve solid ⁶⁴CuCl₂ in hydrochloric acid (0.1 M) and add sodium acetate buffer (0.5 M) to increase the pH to ~5. Formulate with saline and filter sterilize the solution by passing it through a 0.22 µm filter (see Table of Materials).
   NOTE: Sodium acetate buffer (0.5 M) is produced from sodium acetate trihydrate and sterile water that is passed through a 0.22 µm sterilizing filter.
2. For quality control of the produced ⁶⁴CuCl₂ solution, perform pH measurement, bacterial endotoxin testing, radiochemical purity determination, and radio nuclidic identification^7,8.
3. Store the product in a lead container at room temperature and keep it in quarantine until all quality control specifications have been satisfactorily met.
   NOTE: For the present study, ⁶⁴CuCl₂ was produced with a radio nuclidic purity ≥99% and a radiochemical purity ≥95%. Solid ⁶⁴CuCl₂, used as starting material, was obtained from a commercial source (see Table of Materials).

2. Preparation of PET scanner

1. Perform a quality check (QC)⁹ on the scanner, following the manufacturer's protocol (see Table of Materials).
   NOTE: QCs must be performed daily in the morning before patient scans.

3. Drawing of tracer for intravenous (IV) injection and per oral (PO) administration

1. Wear plastic gloves and remove the lid from the lead container.
2. Use long tweezers to disinfect the rubber membrane of the tracer-containing glass bottle inside the lead container with a disinfection swab.
3. Use tweezers to insert a short cannula (~0.5 mm x 16 mm) into the membrane to avoid spill-over from the vacuum inside the bottle.
4. Use tweezers to insert a longer cannula to draw from. This cannula should be long enough to reach the bottom of the bottle (usually 50 mm).
5. Ensure that the dose calibrator (see Table of Materials) is calibrated for ⁶⁴Cu. Calculate an approximate volume to draw for the first draw.
   NOTE: From the chemical quality control reports, the activity amount and volume of the liquid will be available, allowing for the calculation of an approximate volume to draw.
6. Wear plastic gloves, insert an appropriately sized plastic syringe into the long cannula, and draw the calculated volume. This volume will depend on the concentration of ⁶⁴Cu in the product and how much ⁶⁴Cu is decided for the protocol (see Dose calculations under the representative results).
7. Use tweezers to hold the cannula while moving the syringe to the dose calibrator to measure the radioactivity.
8. Keep drawing until the appropriate radioactivity amount is reached. Approximately 5% of the tracer will remain in the syringe and cannula after injection.
   NOTE: The ⁶⁴Cu should not be diluted in salt water, as the tracer may precipitate. Thus, the syringe cannot be rinsed with saline water after the injection (this is not relevant for PO administration).
9. With the tweezers, apply a cannula with a cap (~16 mm cannula) to close the syringe and store it in a lead container until application.

4. Application of the tracer

1. IV injection
   1. Insert an intravenous cannula (~22 G, 25 mm), preferably in a cubital vein, and rinse with saline water to ensure correct placement.
      NOTE: A worksheet with the participant's name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, injection, and leftover tracer should be available.
   2. Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.
   3. Transport the syringe in a lead container to the participant's bedside.
   4. If any spill-over occurs from the injection, place a napkin under the participant's elbow so the spilled radioactivity can be measured.
   5. With tweezers, remove the cap/cannula from the syringe, and with plastic gloves, connect the syringe to the IV access. Note the time on the worksheet and inject in one steady movement.
      NOTE: As mentioned earlier, the syringe should not be rinsed with saline as the tracer may precipitate.
   6. Remove the syringe from the IV access, put on the cap/cannula, and place it in the lead container with the napkin if necessary.
   7. Rinse the IV access through with saline water.
   8. Note the time and leftover radioactivity in the syringe on the worksheet.
      NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurements are entered into the PET scan protocol when the participant is scanned (see step 5).
   9. Dispose of the leftover material appropriately, according to institutional safety regulations.
   10. Remove the IV access. In case any allergic reactions appear, leave the IV access in for 30 min.
2. Oral administration
   NOTE: A worksheet with the participant's name, a stamp or signature for tracer quality control release, and time points and radioactivity for drawing, administration, and leftover tracer should be available.
   1. In a disposable and soft plastic cup, pour around 100 mL of water or cordial; the ⁶⁴Cu is tasteless. A disposable plastic straw and a small disposable plastic bag should be available.
   2. Measure the radioactivity in the syringe using the dose calibrator available and note the time and activity on the worksheet.
   3. Transport the syringe in a lead container to the participant's bedside. The participant should be seated in a bed or chair.
   4. Remove the cap/cannula from the syringe with tweezers and, wearing plastic gloves, inject the tracer into the cup, being careful not to spill any. Draw up a bit of the water/cordial and inject it into the cup again.
   5. Place a plastic straw in the cup (this is to minimize the risk of spill-over when the participant drinks).
   6. Note the time on the worksheet and let the participant drink. The cup should be as empty as possible.
   7. Put the empty cup and straw in the disposable plastic bag with the empty syringe and place them in the lead container.
   8. Note the time and measure the leftover radioactivity in the syringe. Note in the worksheet.
      NOTE: The injected activity is calculated as the difference between the syringe activity before and after injection, but using the PET scan protocol to correct for decay. Thus, all three time points (draw, injection, and leftover measurements) and the measured radioactivity at the draw and leftover measurement are entered into the PET scan protocol when the participant is scanned (see Scan).
   9. Dispose of the leftover material appropriately, according to institutional safety regulations.
      NOTE: Observing the participant for acute allergic reactions for 30 min after the intake may be appropriate.

5. PET scans

1. Place the participant in a supine position in the scanner.
2. Perform overview CT or MR scanning to plan the specific region to be examined during the PET scan.
3. Note the time of the draw, injection, and leftover measurement, and the radioactivity at the draw and leftover measurement in the PET protocol.
4. Perform PET scanning following the steps below.
   NOTE: The PET scan protocol must be standardized with regard to scan duration and image reconstruction parameters for all participants in the same study; published reports should be followed^10,11,12.
   1. Perform static PET scans with a scan time of 4.5 min/bed position for up to 24 h after tracer administration, and 10 min/bed position for up to 68 h after tracer administration (for further elaboration, see Scan under the representative results).
      NOTE: During dynamic PET scanning, decay is continuously recorded and subsequently segmented into a frame structure. This allows for selection of frames from short time intervals to emphasize the dynamics of ⁶⁴Cu distribution, and frames from longer time intervals to prioritize sensitivity. Typically, shorter intervals are selected right after injection and gradually increased thereafter¹⁰.

6. Image reconstruction

1. Reconstruct the images using the best available corrections for attenuation, scatter, time-of-flight, and point-spread function.
   NOTE: The image reconstruction parameters must be carefully selected to optimize the image properties, such as signal recovery and signal-to-noise. For multicenter studies, it is critical to standardize the image quality between centers.

7. Data analysis

NOTE: The present study describes a simple method to quantify ⁶⁴Cu content in the liver. The PET signal is measured as standard uptake value (SUV), the tissue radioactivity concentration adjusted for participant weight injected activity and/or kilobecquerel (kBq) per mL of tissue.

1. Download data to a suitable program, for example, Dicom files, to PMOD.
   NOTE: There are likely many different programs to analyze PET images, such as Hermes or PMOD (see Table of Materials).
2. Adjust the CT/MR scan tones to differentiate the anatomical structures.
3. Ensure that the anatomical scan and PET scan are overlapping.
4. Working in the horizontal plane with the best MRI or CT scan, localize the liver and the big structures.
5. Place an appropriate volume of interest (VOI) or multiple VOIs in the liver.
   NOTE: A VOI is a defined area of tissue where the SUV is measured. A VOI consists of multiple regions of interest (ROIs), which are tissue areas in one plane. Many programs have spherical VOIs as a pre-setting, meaning multiple ROIs (one in each plane) do not have to be drawn to constitute a VOI. The right liver lobe tends to be more homogenous, and thus a good position to place VOIs.
6. Place multiple VOIs in the right liver lobe in different horizontal planes to achieve the most precise measure of activity, as the SUV may vary somewhat (~5%) in the right liver lobe. Calculate the mean SUV of these VOIs.
7. To quantify the SUV, for example, in the entire liver, draw ROIs covering the whole liver volume in each plane for dosimetry studies.
   NOTE: Avoid big structures such as arteries and veins when using this method.

Wyniki

Dose calculation
Based on dosimetry calculations, the effective radioactivity dose for IV administration is 62 ± 5 µSv/MBq tracer¹⁰. Thus, a 50 MBq dose is recommended depending on the time frame. Up to 75-80 MBq is applicable for longer examinations and provides good-quality images without exceeding an ethically approved dose. The effective dose for oral administration is 113 ± 1 µSv/MBq tracer, due to intestinal accumulation of the tracer. Thus, a lower...

Dyskusje

The method is like any other PET method, but the long half-life of 12.7 h offers the opportunity to investigate long-term copper fluxes (we have good results from up to 68 h after IV tracer injection). All steps in the protocol must be handled by personnel familiar with PET, although they are no more critical than any other PET examination.

Troubleshooting
Because we often use ⁶⁴Cu for long-term investigations, the PET signal will be noisier than usual. This i...

Materiały

Name	Company	Catalog Number	Comments
0.22 micrometer sterilizing filter	Merck Life Science
Cannula 21 G 50 mm	BD Microlance	301155
Cannula 25 G 16 mm	BD Microlance	300600
Dose calibrator	Capintec CRC-PC calibrator
PET/CT scanner	Siemens: Biograph
PET/MR scanner	GE Signa
PMOD version 4.0	PMOD Technologies LLC
Saline solution 0.9% NaCl	Fresenius Kabi
Sodium acetate trihydrate BioUltra	Sigma Aldrich	71188
Solid 64CuCl2	Danish Technical University Risø
Sterile water	Fresenius Kabi
Venflon 22 G 25 mm	BD Venflon Pro Safety	393280