Real-time diagnosis of sampled lesions in targeted biopsy of prostate Cancer using a novel tracer [⁶⁴Cu] Cu-DOTA-PSMA-3Q: a pilot preclinical study

Abstract

Purpose

Prostate-specific membrane antigen positron emission tomography (PSMA-PET) guided targeted biopsy has shown potential for detecting more prostate cancer (PCa) cases. However, relying exclusively on imaging may risk missing lesions, an issue that could be addressed by incorporating radiation-guided technology. Accordingly, we aimed to develop a novel PSMA radiotracer with a long half-life, [⁶⁴Cu]Cu-DOTA-PSMA-3Q, and evaluate its targeting accuracy. Additionally, this research explored the practicability of real-time lesion verification during surgical interventions.

Methods

In this study, we synthesized the radiotracer [⁶⁴Cu]Cu-DOTA-PSMA-3Q and verified its PSMA specificity and safety. We prospectively enrolled 18 patients with suspected PCa for PET/CT imaging to assess the efficacy of PCa detection; 10 patients underwent targeted biopsy combined with standard template biopsy at an interval of 24 h after injection. The radioactivity of the biopsy tissue was quantified in counts per minute (CPM) using a gamma spectrometer intraoperatively.

Results

The efficacy and specificity of [⁶⁴Cu]Cu-DOTA-PSMA-3Q were confirmed through preclinical cell and animal studies. In clinical settings, PET/CT imaging performed 2 h after injection visualized all of the PSMA-positive lesions both within and outside the prostate. The assessment of human organ radiation exposure indicated that the kidneys received the highest absorbed dose, followed by the bladder wall, salivary glands, and liver. In 10 patients, a total of 132 biopsy cores were extracted, with 53 cores histologically confirmed as PCa. The median CPM for PCa tissues was significantly greater at 134,148 CPM compared to 18,39 CPM for normal prostate tissues (P < 0.001). Receiver operating characteristic (ROC) curve analysis yielded an AUC of 0.8837, suggesting high diagnostic accuracy. The optimal diagnostic cut-off was established at 66 CPM, achieving a sensitivity of 77.36%.

Conclusions

Radiation-guided technology using [⁶⁴Cu]Cu-DOTA-PSMA-3Q improves the reliability of PSMA-PET image-guided biopsy, enables instant confirmation of sample lesions during surgical procedures, and holds potential for achieving optimized puncture outcomes.

Trial registration

Chinese Clinical Trial Registry ChiCTR2300072655, Registered 20 June 2023.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00259-024-07000-5.

Introduction

Prostate cancer (PCa) is the second most common cancer among men and a major cause of cancer-related deaths worldwide [1]. Prostate-specific membrane antigen (PSMA) has been identified as a pivotal molecular target for the initial diagnosis, staging, and follow-up of PCa and has increasingly been used in radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC) over the last decade [2, 3]. Histopathological biopsy remains the definitive method for diagnosing PCa. However, transrectal ultrasound-guided needle biopsy (TRUS-GB) is plagued by significant trauma and low accuracy [4]. A single-centre randomized study demonstrated that, compared with TRUS-GB, [¹⁸F]/[⁶⁸Ga]Ga-PSMA PET/CT targeted biopsy (TB) can increase the detection rate of clinically significant prostate cancer (csPCa) to an accuracy of 80.6% [5, 6], with per-patient accuracy reaching 93% [7]. Moreover, compared with multiparametric magnetic resonance imaging targeted biopsy (mpMRI TB), the diagnostic accuracy of [⁶⁸Ga]Ga-PSMA PET TB for csPCa was found to be noninferior, ranging from 78 to 85%, compared with 74-77% for mpMRI TB [8, 9]. Consequently, PSMA-TB is emerging as a novel and more effective method, enabling the precise differentiation of PCa tumours from normal prostate (NP) tissues with greater intuition, speed, and reliability [10].

While PSMA PET effectively localizes PCa, these imaging techniques primarily provide support through improved visualization, with the actual confirmation of cancerous regions still reliant on histopathology, which is often challenging to verify instantly during a biopsy. The lack of operator experience, the complexities of image-guided biopsies, and the potential misplacement of targets may necessitate repeat biopsies, increasing the risk of postoperative complications [11]. The high accumulation of [¹⁸F]/[⁶⁸Ga]Ga-PSMA in csPCa allows for the rapid identification of target lesions by measuring the radioactivity of the biopsy needle shortly after injection. A previous study demonstrated that the use of [¹⁸F]PSMA-1007 uptake in PCa could reliably confirm accurate intraoperative tumour sampling, thus reducing the necessity for saturation biopsies [11]. However, the short half-life of [¹⁸F]/[⁶⁸Ga]Ga-PSMA limits the time available for conducting biopsies post-PET/CT imaging, complicating interdisciplinary collaboration. Alternatively, ^99mTc or ¹²³I-labelled PSMA offers a cost-effective solution, although the lower resolution of SPECT imaging and the potential to miss small lesions hinder its effectiveness for targeted biopsies [12]. In this research, we utilized the longer half-life radionuclide ⁶⁴Cu to label PSMA and conducted both preclinical and preliminary clinical studies to assess its targeting specificity and the feasibility of immediate intraoperative confirmation of target lesions.

Materials and methods

General materials, cell culture and animal models

All of the chemicals, reagents, and solvents used for synthesis and analysis were of analytical grade (all procured from Maclin Biochemical Technology Co., Ltd. (Shanghai, China)). The ⁶⁴Cu radionuclide was obtained from Guangdong Sinotau Molecular Imaging Technology Co., Ltd (Guangdong, China). The cell lines were generously provided by GuYan Biotech Co., Ltd. (Shanghai, China). 22RV1 (moderately PSMA-positive) and PC-3 (PSMA-negative) cells were cultured in RPMI 1640 medium or F12K medium supplemented with 1% penicillin-streptomycin and 10% foetal bovine serum (FBS) (Gibco Life Technologies, Grand Island, NY, USA). The cells were cultured in a humidified incubator with 5%CO₂ at 37 °C and passaged when they reached approximately 80–90% confluence.

Male BALB/c nude mice (4–5 weeks) were purchased from Charles River laboratories (Beijing, China), and 22RV1 or PC-3 cells (1 × 10⁷ cells/mL, 0.2 mL) were inoculated into the right axilla region. The mice were utilized for biodistribution studies and micro-PET imaging once the tumour volume reached 100 to 200mm³. All animal experiments were conducted in accordance with the protocol approved by the Animal Care and Use Committee of the PLA General Hospital (approval number: S2023-295-01).

Chemical synthesis, Radiolabelling and Quality Control

The radiosynthesis pathways for [⁶⁴Cu]Cu-DOTA-PSMA-3Q are illustrated in Scheme 1. Additional information regarding the radioactive synthesis and quality control procedures is provided in the Supplementary Material.

Scheme 1.

Radiosynthesis of [⁶⁴Cu]Cu-DOTA-PSMA-3Q

In vitro studies

The partition coefficient was determined by adding [⁶⁴Cu]Cu-DOTA-PSMA-3Q to a mixture of n-octanol and phosphate-buffered saline (PBS) at a volume ratio of 1:1, and the in vitro stability was assessed in PBS and 5% bovine serum albumin (BSA). The 22Rv1 and PC-3 cell lines were utilized for cell uptake and blocking experiments and for establishing a xenograft model.

Radiotoxicity, pharmacokinetics, biodistribution, and micro‑PET imaging

Two groups of male ICR mice (n = 5/group) were injected with [⁶⁴Cu]Cu-DOTA-PSMA-3Q (37 MBq, 150 µL) and an equal volume of normal saline via the tail vein to assess the radiotoxicity of the tracer. [⁶⁴Cu]Cu-DOTA-PSMA-3Q (0. 74 MBq, 150 µL) was injected into the tail vein of male ICR mice (n = 5), and blood samples were collected at various time points (1, 2, 5, 10, 15, 30, 60, 90,120 and 180 min) after injection for pharmacokinetic studies. The biodistribution of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in 22RV1 tumour-bearing mice (n = 4/time point) was studied, and the radioactive uptake value (%ID/g) in each tissue and organ was determined. Micro-PET imaging was conducted on 22Rv1 and PC-3 tumour-bearing mice (n = 4/group) at different time points (1, 2, 6, and 24 h) after injection. ZJ-43 ((S)-2-(3-((S)-1-carboxy-3-methylbutyl)ureido), pentanedioic acid) was utilized as a blocking agent for PSMA (for all of the details see the supplementary materials).

Preliminary clinical PET/CT imaging in humans

The preliminary clinical study was authorized by the Ethics Committee of the Chinese PLA General Hospital (Approval No. S2023-208-02). The recruitment phase ran from July 2023 to May 2024, during which volunteers were enlisted and subsequently underwent PET/CT imaging. All participants provided written informed consent prior to their involvement. The cohort consisted of two healthy volunteers and 18 individuals suspected of having prostate cancer, as detailed in Table 1. PET/CT scans were performed using a uEXPLORER system (United Imaging Healthcare, China) at 2 h post-injection of [⁶⁴Cu]Cu-DOTA-PSMA-3Q (148 Mbq per person), spanning from the skull base to the upper thigh. The PET images were reconstructed using various algorithms including ordered subset expectation maximization (OSEM), time-of-flight (TOF), and the point spread function (PSF), with settings of 3 iterations, 20 subsets, a 256 × 256 matrix, a 600 mm field of view (FOV), a 1.443 mm slice thickness, and a Gaussian postfilter with a 2 mm full width at half maximum (FWHM). The PET/CT images were assessed by two experienced physicians who recorded the maximum standard uptake value (SUVmax) of each identified lesion. For the two volunteers, whole-body dynamic imaging was performed within the first 60 min following injection, and static images were captured at 2, 4, and 24 h post-injection. The dynamic images were segmented and reconstructed at intervals of 5, 15, 35, and 60 min to analyse the radioactivity concentration (Bq/ml) and the volume of each organ at these time points. These data facilitated the assessment of the biodistribution across human organs and the estimation of radiation dosimetry. The time-integrated activity curve for each organ was measured, and the dosimetric parameters were calculated using OLINDA/EXM software (version 2.0).

Table 1.

Individual patient characteristics and the CPM of prostate cancer tissue

Pat	Age	PSA (ng/ml)	Primary tumour (SUVmax)	Lymph node lesions(SUVmax)	Bone lesions (SUVmax)	miPSMA	Gleason score	ISUPmax on Biopsy	Maxinum cpm of tissue	Total cores (12 + X)
1	66	57.65	29.34	7.32, 7.65, 13.15	3.01 ~ 56.57	3	4 + 4	4	309	6 + 1
2	70	30.78	48.22	5.12 ~ 15.38	5.13, 15.60	3	4 + 3	3	802	12 + 1
3	72	5.36	5.06	None	None	1	0	0	60	12 + 1
4	84	4.58	8.68	None	None	2	3 + 4	2	189	12 + 1
5	70	23.10	54.65	None	None	3	4 + 3	3	853	12 + 2
6	65	15.43	12.30	None	None	2	4 + 3	3	90	12 + 3
7	67	9.71	22.47	None	None	3	3 + 4	2	265	12 + 1
8	76	23.14	24.58	None	None	3	3 + 5	4	204	12 + 3
9	80	18.04	9.90	None	None	2	3 + 4	2	310	12 + 1
10	68	45.12	26.46	None	None	3	4 + 3	3	295	12 + 2
11	72	4.06	NE	None	None	0	0	0	——	——
12	61	21.78	NE	None	None	0	0	0	——	——
13	80	11.28	14.92	None	None	3	4 + 5	5	——	——
14	69	19.21	40.28	None	None	3	4 + 3	3	——	——
15	73	19.03	22.43	None	None	3	4 + 4	4	——	——
16	65	202.7	13.14	3.54, 6.34	5.82 ~ 21.94	2	4 + 5	5	——	——
17	68	16.35	8.90	None	None	2	4 + 3	3	——	——
18	61	12.46	NE	None	None	0	0	0	——	——

NE: Negative expression

Study design of PSMA PET-guided targeted biopsy for prostate cancer

Ten patients (patients 1–10) with positive or equivocal positive PET/CT images were enrolled from among the subjects who underwent [⁶⁴Cu]Cu-DOTA-PSMA-3Q PET/CT imaging. The miPSMA score of the lesions was determined based on the Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE) criteria [13], and a miPSMA score of 1 was defined as suspicious lesions. The inclusion criteria included persistent elevation of prostate-specific antigen (PSA) levels > 4 ng/mL or multiparameter magnetic resonance imaging (mpMRI) findings suggesting suspected disease, and final biopsy pathological results could be obtained. The exclusion criteria were as follows: patients with other serious diseases, patients who had previously received treatment, and patients with contraindications to prostate biopsy or active urinary tract infection. All enrolled patients underwent biopsy at 24 -hour intervals after the injection of [⁶⁴Cu]Cu-DOTA-PSMA-3Q. Before commencing the biopsy, exposure rates were assessed at distances of 10 cm and 1 m from the patient to ensure safe radiation exposure for the team in the operating room. Following local anaesthesia administration in the pelvic region, the patient underwent standard transperineal biopsy combined with targeted biopsy (12 + X core). One of the patients had only seven punctures because anticoagulants were not discontinued before surgery as required. The target area was determined by the doctor in advance, which was usually the positive lesion suggested by PSMA PET/CT images. The PSMA PET/CT scans revealed one PSMA-positive lesion in the prostate for all patients, with 4 requiring 2–3 targeted biopsies due to large size (over half of one lobe or involving both lobes). The remaining patients had a single core-targeted biopsy.

The CPM of the biopsy tissue was quantified using a well-type gamma counter (Wiper PHILIPS-05-500), with a measurement duration of one minute and an energy window set from 0 to 1000 KeV. To ensure accuracy, environmental background counts were recorded before the formal measurement of each patient’s biopsy sample. Post-biopsy, the samples were immediately placed into 5 ml microcentrifuge tubes to measure CPM. Following the radiometric assessment, the tissues were fixed in formalin for histopathological analysis, which included measurements of tissue length, tumour proportion, and WHO/ISUP grade determination.

Pathological evaluations served as the reference standard for confirming the presence of tumour tissue. A comparative analysis was conducted between the cancer detection results from targeted biopsy tissues and those from standard biopsy procedures. Additionally, this study analysed the correlation between CPM values and WHO/ISUP grades to assess the efficacy of radiotracer uptake as an indicator of tumour grade and density. This correlation analysis aimed to increase the precision of PCa diagnostics and potentially refine the biopsy-targeting strategies based on quantitative radioactive measurements.

Statistical analysis

SPSS 24.0 (New York, America) and GraphPad Prism 9.0 (San Diego, America) were used for statistical analysis. The significance level of all of the statistical tests was set at P = 0.05. Quantitative data are reported as the means ± SDs (standard deviations) or medians and quartiles for continuous variables, and statistical comparisons were made using either Student’s t test or the Mann‒Whitney U test. However, qualitative data are presented as frequencies and percentages [n (%)] of categorical variables, and statistical comparisons were performed using the chi-square test. The Spearman rank test was used to analyse the correlation between radioactive counts and the ISUP grade. The receiver operating characteristic (ROC) curve and the area under the curve (AUC) were used to evaluate the diagnostic efficacy of the optimal cut-off value for predicting positive lesions. The Youden index (maximum sensitivity + specificity − 1) was used to determine the optimal cut-off value for the ROC curve.

Results

Radiochemistry and quality control

[⁶⁴Cu]Cu-DOTA-PSMA-3Q was prepared, and a radiochemical purity over 98% was confirmed by radio-HPLC. The mobile phase consisted of a mixture of 12% acetonitrile (v/v) containing 0.4% phosphoric acid, which flowed at a rate of 1 mL/min (Fig. S1). The log P value of [⁶⁴Cu]Cu-DOTA-PSMA-3Q was − 2.48 ± 0.06, indicating its hydrophilic nature. After incubation for 24 h in PBS or 5% BSA at 37 °C, the radiochemical purity of [⁶⁴Cu]Cu-DOTA-PSMA-3Q was greater than 95%, indicating the stability of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in both systems in vitro (Fig. S2a).

In vitro cellular studies

The cell-specific binding affinity of the radiotracer [⁶⁴Cu]Cu-DOTA-PSMA-3Q was assessed using the 22Rv1 and PC-3 cell lines. The uptake of [⁶⁴Cu]Cu-DOTA-PSMA-3Q by 22Rv1 cells, which are mildly PSMA- positive, was consistently and significantly greater than that observed in PSMA-negative PC-3 cells at all of the assessed time points, as shown in Fig. 1a (p < 0.01). Specifically, the uptake in 22Rv1 cells increased in a time-dependent manner, increasing from 4.83 ± 0.87 IA%/10⁶ cells at 10 min to 21.56 ± 0.72 IA%/10⁶ cells at 4 h. Co-incubation with excess ZJ-43, a PSMA inhibitor, for 4 h significantly reduced the uptake to 4.63 ± 0.56 IA%/10⁶ cells (p < 0.001). In contrast, the uptake in PC-3 cells remained significantly lower throughout and was unaffected by ZJ-43, with values of 4.51 ± 0.91 IA%/10⁶ cells at 4 h and 4.15 ± 0.75 IA%/10⁶ cells at 4 h when blocked (p > 0.05).

Fig. 1.

a Cell uptake of [⁶⁴Cu]Cu-DOTA-PSMA-3Q (0.074 MBq/well) in 22Rv1 and PC-3 cells at different time points. 10, 30, 60, 120 min and 4 h for [⁶⁴Cu]Cu-DOTA-PSMA-3Q; b The distribution of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in 22Rv1 tumor-bearing mice; c I ~ IV: Micro-PET maximum intensity projection (MIP) images of 22Rv1 tumor-bearing mice after administration of 5.55 MBq of [⁶⁴Cu]Cu-DOTA-PSMA-3Q at 1, 2, 6, 24 h p.i.; V, VI: Micro-PET MIP images of 22Rv1 tumor-bearing mice co-injected with ZJ-43 and PC-3 tumor-bearing mice

Radiotoxicity, pharmacokinetics, biodistribution, and micro-PET imaging

The radiotoxicity assessment of [⁶⁴Cu]Cu-DOTA-PSMA-3Q revealed no significant differences in body weight or histopathological changes in major organs (H&E staining) between the two groups of ICR mice during the observation period (Figs. S2a, b). As shown in Figure S3, pharmacokinetic analysis revealed that the distribution and elimination half-life of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in ICR mice were 1.20 min and 82.72 min, respectively. As depicted in Fig. 1b, the uptake of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in the tumour peaked at 6 h, increasing from 13.05 ± 4.12 ID%/g at 1 h to 17.81 ± 3.60 ID%/g at 6 h. Additionally, the tumour-to-blood and tumour-to-muscle ratios were 7.08 and 11.41, respectively.

Notably, the kidneys presented the highest initial uptake of the tracer (146.99 ± 32.96 ID%/g at 1 h), which then sharply decreased to 9.86 ± 1.12 IDv/g at 24 h post-injection (p.i.). The liver uptake decreased modestly from 19.23 ± 2.95 ID%/g to 16.76 ± 3.02 ID%/g over 24 h. The 22Rv1 tumours in tumour-bearing mice showed significant uptake and retention of radioactivity, in contrast with the non-visualization of PC-3 tumours. The SUVmax for the 22Rv1 tumour was 2.84 ± 0.46 at 24 h p.i., with a corresponding tumour-to-muscle ratio of 18.06 ± 1.59, as shown in Fig. 1c. These results underscore the potential of [⁶⁴Cu]Cu-DOTA-PSMA-3Q for specific targeting and visualization of PSMA-expressing tumours with minimal radiotoxic effects.

Preliminary PET/CT imaging in humans

The subject recruitment for the clinical study spanned from July 2023 to May 2024 and included 2 healthy volunteers and 18 patients with suspected PCa. The average age of the participants was 70.4 ± 6.4 years, and their PSA levels averaged 30.0 ± 45.2 ng/ml. Within one hour post-injection of [⁶⁴Cu]Cu-DOTA-PSMA-3Q, pronounced radioactive accumulation was observed in the kidneys, bladder, parotid glands, submandibular glands, liver, and spleen of healthy subjects, as illustrated in Fig. 2. The marked accumulation in the salivary glands is due to the physiological expression of PSMA in these tissues.

Fig. 2.

The MIP image of [⁶⁴Cu]Cu-DOTA-PSMA-3Q PET/CT of a 52-year-old healthy volunteer at 5, 15, 35, 60 min and 2, 4, 24 h p.i. Physiological uptake was observed in the salivary glands, kidneys, bladder, liver, spleen, and small intestine

The notable accumulation in the kidneys and bladder is attributed to the physiological expression of PSMA in these organs and the renal excretion pathway of [⁶⁴Cu]Cu-DOTA-PSMA-3Q. Additionally, the accumulation in the liver is likely due to the interaction of endogenous proteins involved in copper metabolism with the DOTA chelator, which may lead to the gradual dissociation of ⁶⁴Cu from DOTA-PSMA-3Q and subsequent hepatic uptake over time. These biodistribution characteristics are consistent with those observed in previous studies with similar tracers, such as [⁶⁴Cu]Cu-PSMA-617 and [⁶⁴Cu]Cu-DOTHA2-PSMA [14, 15], highlighting the potential of these tracers for effective imaging in clinical settings.

In the final pathological assessment, 4 out of the 18 patients with suspected PCa tested negative, with 3 of these showing no discernible lesions on PSMA PET/CT imaging, which is consistent with the histopathological findings. Conversely, at least one lesion was identified in each of the remaining 14 patients. Notably, PSMA-positive lesions in the lymph nodes and bones were clearly visualized in the images acquired 2 h p.i. in 3 patients, as depicted in Fig. 3. Although it is difficult to obtain the pathological results of these lesions, in combination with clinical symptoms, PSA values, PSMA PET/CT and subsequent follow-up reviews, these patients were still considered to have prostate cancer with multiple metastases by physicians. For example, after 8 months of endocrine drug treatment, Patient 16 underwent reexamination with PSMA PET/CT. The results indicated that the original uptake in the lymph nodes and bones had largely vanished, which was considered a change post-treatment. This could also imply that the patient indeed experienced metastasis.

Fig. 3.

[⁶⁴Cu]Cu-DOTA-PSMA-3Q PET/CT images of a 66-year-old prostate cancer patient (no. 1) with a PSA of 57.65 ng/mL and a Gleason score of 4 + 4 = 8. a The MIP image at 2 h p.i. clearly showed extensive lesions throughout the body; b-e Fused PET/CT images, which clearly depicted focal uptakes in the prostate primary ( SUVmax: 29.34 ), as well as in lymph node ( SUVmax: 13.1 ) and bone metastases ( SUVmax: 56.5 )

Detailed data on the tumour burden and uptake values for the tumour lesions are presented in Table 1. The mean maximum standardized uptake value (SUVmax) was as follows: 22.89 ± 14.87 for primary tumors, 7.93 ± 3.83 for lymph node-positive lesions, 21.46 ± 16.97 for bone-positive lesions, and 46.70 ± 33.11 for the tumour-muscle ratio. These findings indicate significant radioactive uptake in lesions both inside and outside the prostate, demonstrating the efficacy of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in detecting and evaluating the extent of disease in patients with PCa.

The absorbed and effective doses for each tissue and organ are detailed in Table 2. The kidneys received the highest absorbed dose at 0.165 mGy/MBq, followed by the urinary bladder wall at 0.148 mGy/MBq, the salivary glands at 0.142 mGy/MBq, and the liver at 0.0987 mGy/MBq. The overall effective dose averaged 0.0244 mSv/MBq. This dosage implies that the administration of 4 mCi (megacuries) of [⁶⁴Cu]Cu-DOTA-PSMA-3Q would result in an effective dose of 3.6 mSv to a patient. This dose level is comparable to or lower than those observed with previous formulations of [⁶⁴Cu]Cu-PSMA drugs, demonstrating the radiotracer’s suitability for clinical use with a favourable radiation safety profile.

Table 2.

Human organ radiation dosimetry estimation of [⁶⁴Cu]Cu-DOTA-PSMA-3Q

Organs	Obsorbed dose(mGy/MBq)
Adrenals	3.91E-02
Brain	2.68E-03
Esophagus	1.30E-02
Eyes	8.74E-03
Gallbladder Wall	5.20E-02
Left colon	2.90E-02
Small Intestine	3.10E-02
Stomach Wall	1.30E-02
Right colon	3.07E-02
Rectum	1.61E-02
Heart Wall	1.98E-02
Kidneys	1.65E-01
Liver	9.87E-02
Lungs	1.10E-02
Pancreas	3.25E-02
Salivary Glands	1.42E-01
Red Marrow	1.15E-02
Osteogenic Cells	1.08E-02
Spleen	4.48E-02
Testes	1.72E-02
Thymus	1.11E-02
Thyroid	1.43E-02
Urinary Bladder Wall	1.48E-01
Effective Dose(mSv/MBq)	0.0244

Analysis of intraoperative real-time targeted biopsy results using [⁶⁴Cu]Cu-DOTA-PSMA-3Q

This prospective study enrolled 10 patients with an average age of 71.8 ± 6.3 years and a PSA level of 21.6 ± 16.5 ng/ml. The baseline characteristics of these patients are outlined in Table 1. Nine out of the ten patients (90%) were diagnosed with sigPCa, with a maximum ISUP grade range of 2 to 4.

A total of 132 biopsy samples were collected. After the exclusion of one patient who did not discontinue anticoagulant therapy prior to surgery, ultrasound-guided standard plus targeted biopsy was performed on the remaining nine patients. Pathology revealed that 53 out of 132 cores (40.15%) presented positive findings. According to the ISUP GG, the distribution of ISUP grades 1, 2, 3, and 4 was 4 (7.55%), 20 (37.74%), 24 (45.28%), and 5 (9.43%) respectively. These results are visually represented in Fig. 4a, illustrating the predominance of higher-grade cancer findings among the patients studied.

Fig. 4.

a Bar graph for the number of needles for different biopsy ISUP grade; b Box plot for the distribution of CPM according to the absence (0) or presence (1) of PCa; c Box plot illustrations for CPM distribution according to biopsy ISUP grade; d The ROC curve of 140 MBq [⁶⁴Cu]Cu-DOTA-PSMA-3Q-injected patients

The analysis of biopsy cores revealed a significantly greater median CPM for PCa tissues (134, 148) than for NP tissues (8, 39) (p < 0.0001) (Fig. 4b). This marked difference underscores the utility of CPM in differentiating between cancerous and noncancerous tissue during intraoperative procedures. Additionally, a moderate positive correlation was found between the ISUP grade and CPM (rs = 0.691, p < 0.0001), indicating that higher CPM values are associated with higher-grade tumours, as depicted in Fig. 4c. The optimal threshold of 66 CPM for detecting PCa was determined based on its diagnostic performance, achieving an area under the curve (AUC) of 0.88, with a sensitivity of 77.4% and a specificity of 93.7% (Fig. 4d). The utility of PSMA PET/CT in accurately identifying PCa lesions is further exemplified by patient 7’s imaging results (Fig. 5). The primary lesion in this patient displayed a maximum CPM of 265, which was sevenfold greater than that observed in NP tissue, clearly delineating the malignant area. This example highlights the efficacy of CPM and PSMA PET/CT imaging in enhancing the accuracy of PCa detection and grading during surgical interventions.

Fig. 5.

The MIP image (a), low-dose axial CT image (b), and fused PET/CT image (c) of patient no. 7 at 2 h post-injection in a 67-year-old primary PCa patient with a rising PSA level of 9.71 ng/mL and a Gleason score of 3 + 4 = 7. Histopathological images of the apical right target were captured at 40x (d) and 400x (e) magnification levels. The primary tumor was clearly visualized in PET/CT images (SUVmax = 22.47), and the corresponding CPM count in the target region measured 265

In this study, histopathological analysis revealed that 86 out of 132 needle biopsies registered counts below the established threshold of 66 CPM. Among these cores, 12 (14.0%) were classified as false-negative, with 3 measuring less than 5 mm in length, and all having a tumour area proportion of less than 40%. Conversely, of the 132 total needle biopsies, 46 (34.8%) exceeded the 66 CPM threshold. Only 5 of these (10.9%) were identified as false-positives, indicating a relatively high specificity of the threshold value. Excluding one patient who tested negative, targeted biopsies were performed on the remaining nine patients, with each undergoing at least one biopsy in the area targeted by PSMA PET/CT imaging. Among these 18 targeted biopsies, 16 yielded positive results for PCa. In comparison, the standard biopsy group utilized 104 cores, with cancer detected in 39. Although there was no statistically significant difference in the median CPM values between the two groups (123, 214 vs. 133, 172; p = 0.436), the detection rate of PCa was significantly greater in the targeted biopsy group than in the standard group (88.9% vs. 37.5%; p = 0.001). This enhanced detection rate in targeted biopsies underscores the efficacy of using PSMA PET/CT-guided biopsies to improve the accuracy of PCa diagnostics, particularly in identifying and confirming cancerous lesions within the prostate.

After a mean dose of 140 MBq of [⁶⁴Cu]Cu-DOTA-PSMA-3Q was administered, the radiation exposure rates measured 24 h post-injection were relatively low. At a distance of 10 cm from the patient, the exposure rate was less than 2.4 µSv/h, and at a distance of 1 m, it was less than 0.4 µSv/h. These low exposure rates indicate that [⁶⁴Cu]Cu-DOTA-PSMA-3Q has a favourable radiation safety profile, facilitating safer handling and proximity during clinical procedures and monitoring postadministration. This property is particularly advantageous in clinical settings, ensuring minimal radiation exposure to health care personnel and other patients.

Discussion

The remarkably elevated expression of PSMA in csPCa renders it an ideal target for targeted molecular imaging [16, 17]. PSMA-TB not only facilitates the diagnosis and staging of prostate cancer but also enhances its detection rate through image guidance, as supported by increasing evidence. Its utility is found to be on par with mpMRI TB and may even reduce the frequency of unnecessary biopsies. A multicentre study revealed that combining PSMA with MRI improved the negative predictive value (NPV) and sensitivity for detecting csPCa, especially by increasing the detection rate of csPCa in patients with PI-RADS 2/3 lesions, suggesting potential targeted screening to possibly forego biopsy in certain subgroups [18, 19].

However, relying solely on imaging for real-time identification of PCa lesions can be inadequate. To overcome the limitations associated with surgeon expertise and the accuracy of preoperative imaging, a study demonstrated the utility of quantifying [¹⁸F]-PSMA-1007 uptake to confirm lesion sampling intraoperatively, a promising advancement though challenging to implement owing to the short half-lives of ¹⁸F and ⁶⁸Ga [11]. Additionally, the acquisition of high-resolution images via single-photon radionuclides such as ^99mTc and ¹²³I is problematic because of their inherent limitations.

To address these challenges, [⁶⁴Cu]Cu-PSMA-BCH was used in another study for PET-ultrasound fusion-guided biopsy, confirming its potential for targeted approaches through autoradiography [20]. However, real-time verification of biopsy samples as target lesions is still unachievable. To overcome these limitations, we developed [⁶⁴Cu]Cu-DOTA-PSMA-3Q, which features an extended half-life, allowing for a longer biopsy time window and facilitating multidisciplinary collaboration while ensuring the capture of high-quality images. The excellent specificity and safety profile of this novel tracer have been validated in preliminary preclinical studies, setting the stage for its future application in real-time PSMA-TB assessments.

The use of [⁶⁴Cu]Cu-DOTA-PSMA-3Q has significant potential for real-time confirmation of PCa lesions during surgical interventions. By establishing 66 CPM as the optimal cutoff value, this study identified discrepancies in 17 out of 132 needles (12.9%) compared with pathology findings. The primary causes of false-negatives were insufficient specimen length and a low tumour area ratio. Compared with the previous study of [¹²³I]-PSMA-7, this study yielded a lower cutoff (1312 CPM vs. 66 CPM). The predominant factor lies in inherent disparities between single-photon and positron radionuclides. ⁶⁴Cu exhibits distinctive decay characteristics, including β+, β-, and electron capture processes, with the β + decay pathway accounting for a mere 14.7% of its total decay events. Additionally, a portion of the high-energy γ-rays generated by the remaining decay modes will directly pass through the instrument and go undetected. However, this study’s initial findings showed that the CPM value of PCa tissue remained significantly greater than that of normal prostate tissue, up to 7 times greater, which aids in accurate lesion identification.

In one particular case (patient 3), clinical suspicion of PCa was present, yet PSMA PET/CT imaging produced equivocal results. However, both intraoperative radioactivity measurements (mean CPM, 33 ± 15) and subsequent pathology confirmed the absence of PCa. These findings not only reinforce the link between PSMA tracer accumulation and tumour aggressiveness but also illustrate a moderate positive correlation between the ISUP grade and CPM [21, 22].

In this study, 18 targeted biopsies were performed on 9 patients who tested positive for PCa. The detection rate of PCa using targeted biopsy was significantly higher than that with standard template biopsy, with the potential to reach a patient-based detection rate of up to 100%. This outcome reveals the efficacy of targeted biopsy in potentially reducing the number of biopsy needles needed, thereby minimizing patient discomfort and postoperative complications [23]. This approach supports the feasibility of replacing standard template biopsy with targeted biopsy.

Additionally, the application of radioactive guidance technology helps address potential spatial deviations between preoperative imaging positioning and actual biopsy sites, enabling real-time adjustments and more precise targeting. Such enhancements bolster operator confidence and reduce the risk of missed detections. Daniela et al. pioneered the application of this technique to the localization of lesions during PCa biopsy, in which all five patients underwent PET/CT guided saturated template biopsies (113 cores in total) within 3 h after [¹⁸F]-PSMA-1007 injection. The ROC analysis revealed an AUC of 0.81, and identified the optimal threshold for determining PCa as 75 CPM (sens/spec of 65.1%/87%) [11]. This finding is similar to the results of our study, but [⁶⁴Cu]Cu-DOTA-PSMA-3Q exhibited marginally superior sensitivity and specificity. Luan et al. performed a similar study using [¹²³I]-PSMA-7 with an AUC of 0.97 and a cutoff of 1312 (sens/spec of 94.40%/91.90%) [12]. In addition, previous studies have explored the use of radiolabelled PSMA tracers such as ¹¹¹In and ^99mTc for guiding surgeries, particularly in detecting lymph node or local recurrences that are PSMA- avid [24–26]. Furthermore, [⁶⁸Ga]Ga-PSMA Cerenkov luminescence imaging (CLI) has been effectively used to detect positive surgical margins intraoperatively, adding another layer of precision to surgical interventions in prostate cancer management [27].

The radiotracer [¹⁸F]-PSMA-1007, which is primarily eliminated via the hepatobiliary route, minimizes urinary interference with tumour lesion imaging, providing a clearer diagnostic picture [28]. In contrast, [⁶⁴Cu]Cu-DOTA-PSMA-3Q is excreted through the urinary system. Despite this, its accumulation in the bladder significantly decreases 24 h p.i., thus minimizing interference with radioactive count measurements during biopsy procedures. A study conducted by Cui et al. demonstrated that in the mouse liver, [⁶⁴Cu]-DOTA undergoes transchelation with superoxide dismutase (SOD), resulting in a decrease in the stability of the [⁶⁴Cu]-DOTA complex in vivo [29]. However, considering the significant variations in protein type and abundance within the liver across different species, the metabolic processes of the [⁶⁴Cu]-DOTA complex may differ between animals and humans [30]. The distribution and excretion pathways of the same Glu-Ureido-Lys-based PSMA tracer were found to vary across different species, as reported by Zhang and Liu et al. [31, 32]. Admittedly, the accumulation of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in the liver is inevitable; however, our findings suggest that this limitation does not compromise its diagnostic efficacy for PCa. The utilization of triaza macrocyclic compounds or cross-bridged tetraazamacrocyclic complexes has been demonstrated to enhance tracer stability in vivo and diminish hepatic uptake [20, 33–35]. In the future, modifications will be made to the structure of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in order to further optimize its performance.

The preliminary findings of this study, which were based on a sample size of 132 cores, suggest a potential benefit of radiation-guided biopsy in patients with positive PSMA PET/CT results, especially those who are medically fragile and may not tolerate more invasive procedures well. This technique offers real-time guidance for accurately sampling target lesions during surgery, thereby minimizing tissue damage. The safety and effectiveness of robot-assisted biopsy procedures are well- documented [36, 37]. The future could benefit from a greater emphasis on technological advancements, such as the integration of gamma counters with robotic arms. Considering the limitations of the small sample size in this study, we intend to conduct a subsequent investigation in collaboration with the urology department, employing a larger sample size. Additionally, we aim to implement robot-assisted biopsy technology to minimize surgeons’ radiation exposure and further increase biopsy accuracy. Such innovations could significantly improve the utility and efficacy of radioactive-guided biopsies in clinical practice.

Conclusions

The integration of radiation-guided technology using [⁶⁴Cu]Cu-DOTA-PSMA-3Q enhances the reliability of PSMA-PET image-guided biopsies, facilitating real-time verification of lesion sampling during surgical procedures. This approach not only improves the accuracy of biopsies but also holds promise for optimizing puncture outcomes in clinical settings. This study underscores the potential of [⁶⁴Cu]Cu-DOTA-PSMA-3Q in advancing prostate cancer diagnostics and treatment.

Electronic supplementary material

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Abbreviations

AUC

Area under the ROC curve

BSA

Bovine serum albumin

CLI

Cerenkov luminescence imaging

CPM

Counts per minute

FBS

Fetal bovine serum

GG

Grade group

HPLC

High performance liquid chromatography

LN

Lymph node

mpMRI TB

multiparametric magnetic resonance targeted biopsy

Mcrpc

metastatic castration-resistant prostate cancer

MIP

Maximum intensity projection

NP

normal prostate cancer

PCa

Prostate cancer

PET/CT

Positron emission tomography/Computed tomography

PSA

Prostate-specific antigen

PSMA

Prostate-specific membrane antigen

PROMISE

Prostate Cancer Molecular Imaging Standardized Evaluation

PBS

Phosphate-buffered saline

RLT

Radioligand therapy

ROC

Receiver operating characteristics

SigPCa

Significant prostate cancer

SUVmax

the Maximum Standard Uptake Value

TRUS-GB

Transrectal ultrasound-guided needle biopsy

TB

Targeted biopsy

ZJ-43

(S)-2-(3-((S)-1-carboxy-3-methylbutyl)ureido), pentanedioic acid

Authors contributions

Jingfeng Zhang, Shaoxi Niu and Yachao Liu contributed equally to this work. Corresponding authors: Baixuan Xu, Jinming Zhang and Ruimin Wang. Data curation, formal analysis, writing original draft: Jingfeng Zhang, Shaoxi Niu and Yachao Liu. Acquisition of data: Xiaohui Luan, Yue Pan, Xiaodan Xu, Shina Wu, Yuan Wang, Baojun Wang and Zhiqiang Chen. Pharmaceutical synthesis and control: Xiaojun Zhang, Huanhuan Liu and Jinming Zhang. Study concept and design: Xu Zhang, Ruimin Wang, Jinming Zhang, Baixuan Xu. Surpervision: Ruimin Wang, Jinming Zhang, Baixuan Xu.

Funding

The authors state that this work has not received any funding.

Data availability

Data are available for legitimate researchers who request it from the authors.

Declarations

Ethics approval

This study was approved by the Ethics Committee of Chinese PLA General Hospital (approval of No. S2023-208-02, Registration number of ChiCTR2300072655).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

All authors have read this manuscript and would like to have it considered exclusively for publication.

Competing interests

The authors declare that they have no competing interests.