Abstract
Background/Aim: To test the correlation of 68Ga-PSMA-11 uptake and the expression of PSMA (prostatic specific membrane antigen) with the Gleason score, apparent diffusion coefficient (ADC) and pharmacokinetic parameters obtained from dynamic contrast agent-enhanced MRI/PET. Patients and Methods: Forty newly diagnosed, therapy naïve patients with prostatic carcinoma (PC) (mean age of 56.7, range=34-79), who were referred for 68Ga-PSMA-11-PET/MRI for primary staging and had undergone radical prostatectomy (RAPE) were included in this prospective study. Their blood samples were tested for serum levels of prostate-specific antigen (PSA) and proPSA. The patients’ prostates were evaluated using whole-mount sections, which helped determine the extent and grade of the tumor; tests were performed to determine immunohistochemical PSMA expression. Results: A correlation between PSMA expression and the accumulation of 68Ga-PSMA-11 was found using the Spearman correlation coefficient (p=0.0011). A stronger correlation was found between Gleason patterns 3 or 4 and PSMA expression (p=0.06). Furthermore, the correlation of Gleason score with the overall 68Ga-PSMA-11 accumulation within the tumor or non-tumor tissue was found to be significant (p=0.0157). A significant relation was found only with the Kep elimination rate constant, which was stronger in Gleason pattern 4 than in Gleason pattern 3. A weaker correlation was found between the accumulation of 68Ga-PSMA-11 and Ktrans in Gleason pattern 4: the most significant relation being between ADCmin and Gleason pattern 3 and 4 (p=0.0074). The total size of the tumor correlated with levels of proPSA (p<0.0001), and its extra prostatic extension correlated with levels of proPSA (p<0.0001). Conclusion: 68Ga-PSMA-11 correlates well with the expression of PSMA. Gleason pattern 3 and 4 had a higher correlation with 68Ga-PSMA-11 levels than did Gleason pattern 5. Either no correlation, or a weak correlation, was established with pharmacokinetics.
In patients with newly detected prostate carcinoma (PC), the assessment of the exact tumor aggressiveness is most reliably determined by establishing the extent of the tumor along with its metastasis. This is the most reliable parameter for planning treatment.
The Gleason score is the gold standard measure of tumor aggressiveness in tissue samples collected in transrectal biopsies. Tumor behavior can furthermore be predicted based on the level of prostatic specific antigen (PSA) and, as of recent years, the level of the proPSA and its reflection in the prostate health index (PHI).
It is usually assumed that there is a pre-treatment risk of metastatic disease if the level of PSA is over 20 ng/ml and the Gleason score confirmed from the biopsy sample is 4+4 or higher. Over the past few years, the prostate specific membrane antigen (PSMA) ligand 68Ga-PSMA-11 has become a promising PET radiopharmaceutical in the staging of PC. The PSMA-11 molecule shows high specificity in binding to the PSMA molecule (1-3). PSMA is a transmembrane protein; it is a metalloproteinase expressed not only in prostate glandular tissue, but also in salivary, lacrimal, or other glands of the aerodigestive system. In PC, PSMA is thought to be overexpressed in most of the prostate cancer cells but is thought to be more pronounced in metastatic disease (2, 3) than in the primary tumor (4, 5). Its expression is well-documented in patients with disseminated disease, but it remains unclear to what extent it is typically expressed in treatment-naïve tumor tissue.
Since the aggressiveness of PC corresponds to the grading of the tumor using the Gleason score, determining PSMA expression using 68Ga-PSMA-11 could be helpful in establishing tumor aggressiveness. Some doubts, however, remain: If there is a correlation between radiopharmaceutical uptake and the Gleason score, perhaps some low-grade tumors could go unnoticed, or some low-grade parts of tumorous tissue could be left undetected; or perhaps the accumulation of 68Ga-PSMA-11 is a universal feature of all PC.
Even though some studies have confirmed that metastases in the lymph node could be detected using 68Ga-PSMA-11-PET/CT, its accumulation within tumor tissue remains low. This raises the question of whether 68Ga-PSMA-11 can be used in the primary detection of prostate carcinoma inside the prostate and in the assessment of its aggressiveness. The aggressiveness and development of prostatic carcinoma depends on a variety of mechanisms.
In the early years of PC therapy, the combination of measuring 68Ga-PSMA and the performance of PET/CT improved the detection of PC (4, 5). With the advent of PET/MRI as methods of clinical imaging, it became clear that the combination of MRI and PET could improve the diagnosis of the in-prostate spread of PC and that this kind of imaging could be used to navigate biopsy (6-9). This study has three aims: the primary aim of the study is to use 68Ga-PSMA-11-PET/MRI to test the correlation between the intensity of 68Ga-PSMA-11 uptake and the level of staining obtained with PSMA by immunochemistry. The second goal is to gauge whether the intensity of PSMA expression is related to the Gleason score and the third aim is to discover how the expression of PSMA correlates with the apparent diffusion coefficient (ADC), the pharmacokinetic parameters of the transfer constant (Ktrans), rate constant (kep), extracellular volume (ve), the initial area under the curve (iAUC), as well as the extracapsular spread of the tumor.
Furthermore, having obtained PSA and proPSA serum levels from all patients, making it possible to calculate their health prostate index (PHI), an additional aim of the study is to compare blood tests with the 68Ga-PSMA-11 uptake and the Gleason score.
Patients and Methods
Patient population and general study concept. Forty therapy naïve patients (mean age 65.77, range=50-74) with newly diagnosed PC who were referred for 68Ga-PSMA-11-PET/MRI for primary staging were included in this prospective study. They had all undergone radical prostatectomy. PC was confirmed histologically in all patients following a transrectal ultrasound-guided biopsy. The serum of all patients was tested for PSA levels and the proPSA fraction of free PSA. All forty patients underwent 68Ga-PSMA-11 PET/MRI before RAPE and all prostate specimens were evaluated using whole-mount sections according to the extent and grade of the tumor. An additional histopathological evaluation was based on the immunohistochemical expression of PSMA within several components of a tissue. As it was impossible to stain the whole area of the whole-mount section, a pathologist collected additional samples from benign tissue, as well as histological components of Gleason score 3, 4 and 5, if present.
Written informed consent was obtained from each patient, in compliance with institutional guidelines and an ethical committee. The study was conducted according to the principles of the Declaration of Helsinki and approved by a local ethics committee. It was a part of the Clinical Investigation approved by the National Institute of Drug Control.
Radiopharmaceutical preparation and administration. A cold kit (ANMI SA a Telix company, Herstal, Belgium) of PSMA-11 (Glu-NH-CO-NH-Lys(Ahx)-HBED-CC) was used to prepare the 68Ga-PSMA-11. The labeling was done in a local radiopharmaceutical facility by a radiopharmacist, using the eluate of 68Ga chloride from the generator (IRE-elit, Fleurus, Belgium) according to the general manufacturing practice (GMP). The radiochemical purity of the labeled precursor was more than 90% when analyzed by thin layer chromatography (TLC) and was approved by the National Institute of Drug Control, ensuring the absence of colloidal form of 68Ge in the final product.
PET/MRI imaging process. 68Ga-PSMA-11 PET/MRI imaging was performed using integrated PET/MRI equipment (Biograph mMRI, Siemens Healthineers, Erlangen, Germany), fifty to sixty minutes after the administration of 68Ga-PSMA-11 at the activity of 1.25 MBq per kilogram of body weight of the patient. Data acquisition started with the imaging of the pelvic region with a single PET acquisition for 10 minutes; magnetic resonance was performed during this period containing the T2 weighted images using TSE T2 in axial and sagittal planes. Thereafter, diffusion weighted imaging, using an echoplanar sequence with b values of 50, 400 and 800 mm2/s, followed by dynamic postcontrast imaging using T1 gradient echo VIBE (volume interpolated breath-hold examination) sequence in the transverse plane after the uniform dosing of 4 milliliters of 2 molar gadolinium of the contrast agent (gadobutrol, Gadovist, Bayer Pharma, Berlin, Germany) was performed. The imaging was completed after the end of the targeted pelvic data acquisition using the whole trunk PET/MRI imaging with VIBE-Caipirinha in the axial and coronary planes and motion corrected diffusion weighted imaging in the transverse planes. PET images were calculated with the attenuation correction according to the tissue modelling based on the Dixon-derived tissue model.
Analysis. All examinations were analyzed using the commercially available software Syngovia mMR, including the multimodality fusion of the MRI and PET. The evaluation of maximum standardized uptake values (SUVmax) and the analysis of minimum ADC values (ADCmin) within the region of interest were performed. The pharmacokinetic analysis was performed using the Syngovia module Tissue4D to create parametric maps of transfer constant (Ktrans), rate constant (kep), extracellular volume (ve), and the initial area under the curve (iAUC). The analysis of the additional image characteristics mentioned above was performed within the region of interest defined by the elevated accumulation of 68Ga-PSMA-11 to calculate the minimum value of ADC (ADCmin) and values of Ktrans, kep,ve, and iAUC.
Immunochemistry staining was performed targeting the PSMA molecule by a standardized procedure following the initial hematoxylin-eosin staining. The analysis of the intensity of a PSMA specific staining was evaluated semi-quantitatively by a very experienced histopathologist specialized in prostate carcinoma, a member of the iSUP board. The level of staining positivity was given a value between 0 and 4 within the following regions (if they were all present): normal glandular tissue, Gleason pattern 3, 4, and 5.
Association between 68Ga-PSMA-11 accumulation in SUVmax, the rate of PSMA specific staining in histopathology, ADCmin, Ktrans, kep,ve, and iAUC, serum levels of PSA, proPSA and PHI were described (Spearman correlation coefficient, Spearman Correlation Coefficient prob > |r| under H0: Rho=0). A significance level of alpha 0.05 was applied. Statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA).
Results
The following Gleason patterns in the prostate resection samples were found: pattern 3+3 in 5 cases, 3+4 in 15 cases, 4+3 in 9 cases, 4+4 in 5 cases, 4+5 in 2 cases, 5+3 in 2 cases, and 5+5 in 5 cases. The correlation of the PSMA expression, evaluated using PSMA-specific immunohistochemistry with the accumulation of 68Ga-PSMA-11, was found using the Spearman correlation coefficient (p=0.0011). A better correlation was found in patients where the Gleason score pattern was 3 or 4 (p=0.06). The overall 68Ga-PSMA-11 accumulation within the tumor or non-tumor tissue was found to be significant (p=0.0157). Table I summarizes the results and Gleason score. Due to the smaller representation of Gleason pattern 5 (only 5 cases), it was impossible to perform the analysis with significant results, but the portion of the non-accumulating tumors with Gleason 5 pattern was higher than those with Gleason pattern 3 and 4. When the pharmacokinetics parameters of DCE-MRI were assessed according to the accumulation of 68Ga-PSMA-11, a significant relation was only found with the elimination constant Kep; this was stronger in Gleason pattern 4 than pattern 3. A weaker correlation was found between the accumulation of 68Ga-PSMA-11 and Ktrans in Gleason pattern 4. No relation between 68Ga-PSMA-11 and iAUC, Ve, ADCmin, size of the tumor, PSA level, or proPSA levels were detected. Other than 68Ga-PSMA-11 accumulation, the correlation between the ADCmin parameter, PSA levels and proPSA levels with the Gleason pattern and size of the tumor were tested: the only significant relation was between ADCmin and Gleason pattern 3 and 4 (p=0.0074). The total size of the tumors correlated with the levels of proPSA (p<0.0001) and the tumor extra-prostatic extensions correlated with the levels of proPSA (p<0.0001). The correlation of PHI with the ADCmin was found to be weak (p=0.0391).
Results of ligand of prostatic specific membrane antigen (PSMA-11) accumulation, microstructure, prostatic specific antigen (PSA) levels, Gleason score, and histopathology classification of prostatic carcinoma in the sample of patients.
Discussion
We conducted a prospective study evaluating the surgically removed prostates with proven carcinoma. The aim was to determine whether 68Ga-PSMA-11 accumulation and/or the assessment of the vascularization can reliably and accurately assess the level of aggressiveness in prostatic carcinoma, as well as to correlate the level of the radiopharmaceutical uptake with the presence of PSMA molecule, which was confirmed using immunochemistry (10, 11). The overall concordance in the PET accumulation of 68Ga-PSMA-11 with the level of PSMA-specific staining was very high, especially when the semiquantitative matter of the assessments, both of SUV and staining, is taken in account. This is the most important finding because it confirms that 68Ga-PSMA-11 accumulation is a stable method for the assessment of the presence of PSMA, enabling us to quantify it approximately (8, 9). The stability of the results across the Gleason patterns shows that the main advantage of measuring 68Ga-PSMA-11 PET is its assistance in identifying tumor tissue. It is rather improbable that the tumor could be missed despite performing only a 68Ga-PSMA-11 accumulation assessment. Those statements are true for Gleason patterns 3 and 4, with only a few exceptions. The crucial question is whether the level of PSMA expression in prostate carcinoma is relevant to its aggressiveness. The findings have revealed an important point: poorly differentiated or anaplastic tumors with a Gleason pattern 5 only have a weak correlation with 68Ga-PSMA-11 accumulation. Why do the most aggressive tumors show low 68Ga-PSMA-11 accumulation in some cases? The answer to this question might be that the highly-altered cells stop expressing PSMA as they return to a more primitive cell membrane in the de novo developed anaplastic tumors. On the other hand, the explanation could lie in the different organization of the vascular bed in those de novo created anaplastic tumors, which might form the so-called vascular mimicry. Thus, the penetration of the 68Ga-PSMA-11 macromolecule into the individual cells could be problematic (11). While Gleason 3 and 4 exhibit a changed, but present, glandular pattern, the vascular bed of those tumors could be more highly organized and include the development of neovascularization (11).
Nevertheless, it is typical for Gleason pattern 5 tumors to lose their glandular architecture and subsequently their extracellular space as well, resulting in the restriction of free molecule movement. It was no surprise that a correlation was found between the ADCmin level and the Gleason pattern, given that the lowest ADC levels were found in Gleason pattern 5.
It is interesting to note how the DCE-MRI parameters correspond to 68Ga-PSMA-11 accumulation. The statistically significant correlation between kep and 68Ga-PSMA-11 is further evidence that the vascular bed organization plays a key role in the 68Ga-PSMA-11 accumulation. Because the vascular bed of the malignant tumor exhibits an increased flux of molecules across the endothelium into the extracellular extravascular space (and vice versa), the size of the transfer constant Ktrans seems to reflect the extravascular escape of the molecules. The rate constant, or elimination constant kep, seems to be equal to the molecular release backwards into the intravascular space. The elimination constant kep therefore reflects the relatively established function of microcirculation (1, 9).
Importantly, an independent parameter showing the local aggressiveness of prostate carcinoma was found. ProPSA was a very good indicator of enlarged tumors, as well as of the extra-prostatic extension. This parameter can help support the findings of the imaging methods and confirm the presence of an extra-prostatic extension previously determined by whole-mount sections.
Conclusion
68Ga-PSMA-11 accumulation correlates well with the expression of PSMA. Gleason pattern 3 and 4 had a higher correlation with 68Ga-PSMA-11 levels than did Gleason pattern 5. The results support the use of 68Ga-PSMA-11_PET/MRI in the assessment of the tumor tissue aggressiveness. The results highlight the possibility to guide the biopsy according to the accumulation levels in the prostatic tissue.
Acknowledgements
This study was supported by the COOPERATIO project of Charles University and Conceptual support of the development of research organization of Czech Ministry of Health 00669806 - University Hospital Pilsen.
Footnotes
Authors’ Contributions
J. Ferda, E. Ferdová, H. Mirka: concept, imaging, data analysis, manuscript preparation; O. Hes: concept, histopathology, manuscript review; M. Hora: surgery, manuscript review; J. Pernický, V. Rudnev: image analysis; V. Pecen: statistics; O. Topolcan: immunochemistry.
Conflicts of Interest
There are no conflicts of interest.
- Received August 29, 2022.
- Revision received October 10, 2022.
- Accepted October 14, 2022.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
