68Ga-PSMA and 68Ga-DOTA-RM2 PET/MRI in Staging of High-risk Prostate Cancer Patients: a Prospective Pilot Trial

Purpose: The aim of the present study is to investigate the synergic role of 68Ga-PSMA PET/MRI and 68Ga-DOTA-RM2 PET/MRI in PCa staging. Methods: Fifteen patients with biopsy-proven PCa underwent both 68Ga-PSMA PET/MRI and 68Ga-DOTA-RM2 PET/MRI within one month. TNM classication based on image ndings was performed and semi-quantitative PET and quantitative MRI parameters were collected for each scan. Finally, DICE score between regions of interest manually segmented on the primary tumor on 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and on T2 MRI was computed. Results: All imaging modalities detected the primary PCa in 15 patients, with slight differences regarding the multifocality of intra-prostatic ndings. Two patients presented seminal vesicles involvement on MRI, one of these was also detected by 68Ga-PSMA, with no uptake on 68Ga-DOTA-RM2 images. Regarding extra prostatic disease, 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and MRI resulted positive in 6, 2 and 4 patients at lymph-nodal level, respectively, and at bone level in 2, 0 and 1 patients, respectively, with 68Ga-PSMA PET detecting more lesions compared to 68Ga-DOTA-RM2 PET. The different ndings detected by 68Ga-PSMA PET and 68Ga-DOTA-RM2 PET might reect the complementary role of these radiotracers, as they detected different foci of intraprostatic disease, seminal vesicle invasion, lymph nodal and bone involvement. Conclusion: These preliminary results suggest a synergic role of 68Ga-PSMA PET, 68Ga-DOTA-RM2 PET and mpMRI in PCa characterization during the staging phase. 68 Ga-DOTA-RM2 SUVmax, SUVmean50 and SUVmean60 correlated with PSA level at diagnosis (respectively, ρ = -0.55, -0.53, -0.53 and p value = 0.04, 0.04, 0.04). 68 Ga-PSMA PET and MRI parameters did not correlate with any of the considered clinical data (p value ≥ 0.05).


Introduction
Prostate cancer (PCa) is one of the worldwide leading causes of cancer-related death. Approximately 15% of men present with high-risk PCa, which is characterized by an increased risk of extracapsular extension, locally advanced disease, and/or bone metastases [1]. Hence, at diagnosis, a whole body staging for high-risk PCa patients is strongly recommended regardless of the surgical or radiation-based treatment decision [2].
The current staging of intermediate and high-risk PCa includes imaging of abdomen and pelvis performed by using Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) and bone scan to evaluate potential sites of metastatic spread.
The current EAU-ESTRO-SIOG guidelines report that Positron Emission Tomography/CT (PET/CT) is a sensitive imaging modality that might be considered in men with high-risk disease undergoing initial staging. However, as no randomized-control trials demonstrating survival bene t are available yet, its role in guiding therapeutic decisions must be cautious [3].
Multi-parametric MRI (mpMRI) is a well-established imaging modality for PCa assessment and it is used to detect the primary tumor, guide biopsies and de ne the local extent of the disease; its usefulness for local staging has been largely reported, although local staging with MRI might be associated with limited sensitivity [4][5][6].
Molecular imaging with PET represents a valid imaging approach in PCa staging, with new PET tracers other than Choline having a relevant role in improving diagnoses, staging and follow-up of PCa [7][8][9][10].
In this regard, prostate-speci c membrane antigen (PSMA), a transmembrane protein with a signi cantly increased expression in PCa cells, is an imaging probe that has been introduced in clinical practice, with recent data demonstrating good accuracy in PCa staging [11,12].
Gastrin releasing peptide receptor (GRPR) is a G-protein coupled receptor overexpressed in different types of cancer including PCa [13,14]. 68 Ga-DOTA-RM2 is a GRPR antagonist used as a PET imaging probe that has demonstrated promising, but still limited results in PCa imaging [15][16][17].
Hybrid PET/MRI allows for the simultaneous acquisition of metabolic, structural, and functional imaging information regarding PCa status in a whole-body single session examination, thus representing an innovative imaging approach capable to overcome the pitfalls of conventional imaging and, potentially, helping clinicians in the management of PCa. Only few and preliminary studies have compared 68 Ga-PSMA and 68 Ga-DOTA-RM2-PET radiotracers in PCa by using PET/CT or PET/MRI so far, with promising results in both patients presenting with biochemical recurrence and in those with newly diagnosed intermediate-or high-risk prostate cancer [15,18,19].
The aim of the present study is to report our experience on the synergic use of 68 Ga-PSMA PET/MRI and 68 Ga-RM2 PET/MRI in prostate cancer staging.

Patients
In this prospective pilot clinical study, 15 patients with biopsy proven PCa have been enrolled from 1 September 2020 to 31 March 2021 at IRCCS San Raffaele Scienti c Institute.
Inclusion criteria were age greater than 18 years at the time of PET/MRI scan, biopsy proven high-risk PCa (de ned as PSA > 20 ng/ml and/or clinical stage ≥ cT2c and/or biopsy ISUP grade ≥ 4, according to European Association of Urology guidelines [3]) in men referred to prostatectomy and pelvic lymphadenectomy. Exclusion criteria were inability to complete the required imaging examinations (i.e. severe claustrophobia), medical condition possibly interfering and signi cantly affecting study compliance, all contraindications to undergo MRI scan (i.e. metallic/conductive or electrically/magnetically active implants without MR-safe or MR-conditional labelling) and evidence of metastatic disease on conventional imaging contraindicating the surgical procedure.
All recruited patients underwent both 68 Ga-PSMA PET/MRI and 68 Ga-DOTA-RM2 PET/MRI in two different days (> 48 hours) and within one month one from the other, for staging purpose before radical prostatectomy.
This study was approved by the Institutional Ethics Committee of IRCCS San Raffaele Scienti c Institute (EudraCT: 2018-001034-18) and all patients gave written informed consent to participate to the study. 68 Ga-PSMA PET/MRI acquisition protocol 68 Ga-PSMA-11 was synthesized by a fully automated synthesis module (Neptis Mo-saic-RS, ORA, Neuville, Belgium) connected to a 68 Ge/ 68 Ga generator (1.85 GBq Galli Ad, IRE ELiT, Fleurus, Belgium) and equipped with a disposable single-use cassette kit (ABX GmbH, Radeberg, Germany). A standardized labelling sequence with 15 µg (15 nmol) of unlabelled PSMA 11 (ABX GmbH) was used. The nal product was sterilely ltered over 0.22 µm PVDF lters. For quality control, 68 Ga-PSMA 11 was analysed by radio analytic high-performance liquid chromatography on a modular system (Waters) equipped with a diode array detector and a radio detector using an RP-18 column (ACE 5µm C18, 150 x 3 mm, Advanced Chromatography Technologies Ltd, Aberdeen, Scotland). A gradient elution over 13 minutes at a ow of 1.5 mL/min from 90%A to 30%A and again 90%A was employed, where Solvent A was Water + 0,1% TFA and Solvent B was CH3CN + 0,1% TFA. Other quality controls performed before release included TLC on iTLC strips with MeOH/1M AcONH4, pH measurement and radionuclidic purity. Residual HEPES determination, Ethanol quanti cation and Microbiological purity were assessed on decayed product. Uncorrected radiochemical yield was over 70% with a radiochemical purity > 91%.
PET images were reconstructed using a Bayesian penalized likelihood reconstruction algorithm [20] with a reconstructed FOV of 60 cm and image matrix of 192x192. The algorithm includes a Point Spread Function and Time of Flight information.
Attenuation Correction (AC) of PET data was performed using MR AC technique based on the processing of the LAVA-Flex sequences acquired simultaneously with the PET data. 68 Ga-DOTA-RM2 PET/MRI acquisition protocol 68 Ga-DOTA-RM2 was synthesized by a kit-like procedure developed for the radio-labeling with GalliAd® generator (IRE Elite).
Brie y, the eluate from the 68 Ge/ 68 Ga generator (1.85 GBq Galli Ad, IRE ELiT, Fleurus, Belgium) was added to a sterile vial containing 40 µg of DOTA-RM2 (Life Molecular Imaging, Fribourg, Germany) in format buffer and ascorbic acid. Reaction vial is placed in a pre-heated thermostat at 115°C for 10 minutes. Successively, vial is left to cool down for 10 minutes at room temperature. No puri cation step was needed. The solution is sterile ltered over sterile 0.22 µm PVDF membrane and dispensed as injectable solution. For quality control, 68 Ga-DOTA-RM2 was analysed by radio analytic high-performance liquid chromatography on a modular system (Waters) equipped with a diode array detector and a radio detector using an RP-18 column (ACE 5µm C18, 150 x 3 mm, Advanced Chromatography Technologies Ltd, Aberdeen, Scotland). A gradient elution over 16 minutes at a ow of 1.0 mL/min from 80%A to 20%A and again 80%A was employed, where Solvent A was Water + 0,1% TFA and Solvent B was CH3CN + 0,1% TFA. Unbound gallium was quanti ed by iTLC strips with MeOH/1M AcONH4 while ionic gallium with TLC strips with 0.1 M sodium citrate pH5. pH of the nal solution was 3.2-3.8. Radionuclidic purity was assessed before release, microbiological purity was assessed on decayed product. Uncorrected radiochemical yield was over 60% with a radio-chemical purity > 91%. 68 Ga-DOTA-RM2 68 Ga-DOTA-RM2 PET/MRI scan was performed within one month (mean: 3 days, range: 2-16 days) from 68 Ga-PSMA PET/MRI.
As for 68 Ga-PSMA PET/MRI, fasting condition was requested on the day of the examination and the same PET/MRI scanner was used.
The 68 Ga-DOTA-RM2 PET/MR examination protocol included a HS scan (20 minutes), covering a single bed position, that was simultaneously acquired to the following MR sequences: an axial T2 weighted sequence with large FOV (32x32 cm 2 ), an axial 3D T2 sequence with small FOV, a T1-Lava Flex sequence of the whole pelvic region. Following the single bed acquisition, a TB PET scan (5-6 FOVs, 4min/FOV) was then simultaneously acquired with a TB axial Lava Flex sequence and a TB sagittal STIR sequence on the spine. Reconstruction and attenuation correction of PET images were performed by using the same algorithms and parameters used for 68 Ga-PSMA PET images.
PET/MR image analysis 68 GA-PSMA and 68 Ga-DOTA-RM2 image read-out has been performed on the Advantage Workstation (AW, General Electric Healthcare, Waukesha, WI, USA) on which PET, MRI and fused PET/MRI images could be visualized in axial, coronal and sagittal planes. HS PET acquisition bed on the pelvic region and TB PET examination of both 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET images were qualitatively interpreted by two experienced (more than 10 years of experience) Nuclear Medicine physicians, with knowledge of all the available patients' clinical and imaging information.
For the primary tumor assessment, HS and TB pelvic PET images were qualitatively evaluated for both 68 Ga-PSMA and 68 Ga-DOTA-RM2. The presence of 68 Ga-PSMA and 68 Ga-DOTA-RM2 increased uptake was considered suspicious for malignancy with the anatomical site being de ned on the basis of MRI anatomy, except for those areas of physiologically increased uptake [21,22]. Regions of interest (ROIs) on the primary tumor, showing 68 Ga-PSMA and 68 Ga-DOTA-RM2 uptake on HS PET images were semi-automatically de ned on transaxial PET images. Furthermore, the following semi-quantitative parameters have been calculated for the primary tumor on HS PET images for both radiotracers: maximum standardized uptake value (SUVmax), mean SUV (SUVmean, using different thresholds, namely 40%, 50%, 60% of the maximum value -SUVmean40, SUVmean50, SUVmean60) and metabolic tumor volume (MTV) calculated at different thresholds (MTV40, MTV50, MTV60).
In addition, to determine the volume and the location of 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET primary tumor uptake, one experienced Nuclear Medicine physician manually segmented the primary tumor slice-by-slice using 3D Slicer software (revision 29402) [23] on both 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET images. Afterwards, all segmentations were co-registered and brought to a common reference volume (the one of the rst PET study). To do that, the MRAC of the 68 Ga-DOTA-RM2 PET study was rstly co-registered to the one of the 68 Ga-PSMA PET, by means of 3D Slicer, and then the obtained transformations were applied to the 68 Ga-DOTA-RM2 PET images and the corresponding ROI segmentations.
After the evaluation of the primary prostatic tumor, the whole-body distribution pattern of both 68 Ga-PSMA and 68 Ga-DOTA-RM2 were qualitatively assessed, and the presence of extra-prostatic 68 Ga-PSMA and 68 Ga-DOTA-RM2 increased uptake was considered suspicious of malignancy, with the exception of areas of physiologically increased uptake. The number and the site of lymph nodal involvement were reported, as well as the presence of suspect distant metastases. The anatomical site was de ned on the basis of MRI anatomy.
In case of suspect bone metastasis in PET images, the whole-body MRI sequences were screened to con rm the spreading of the disease.
MR images acquired during HS 68 Ga-PSMA PET were initially processed using AW software: small FOV DWI with b values of 50-800 were used to generate ADC maps. Volumetric ROIs of lesions visible to T2 and ADC images were created using 3D Slicer to obtain the following quantitative parameters: lesion volume, mean ADC (ADCmean) and minimum (ADCmin).
Qualitative and quantitative comparison of 68 Ga-PSMA, 68 Ga-DOTA-RM2 and MRI A qualitative comparison between 68 Ga-PSMA and 68 Ga-DOTA-RM2 intra-prostatic uptake and morphological ndings detected on MR images was performed in order to describe the possible concordances and discrepancies between metabolic and morphologic imaging. Moreover, a qualitative comparison has been also performed in terms of number and sites of lymph nodal and distant metastases for all patients, considering all the three different imaging modalities.
Finally, DICE score between the ROIs manually segmented on the primary tumor on 68 Ga-PSMA PET and 68 Ga-DOTA-RM2 PET and on MRI was computed in order to evaluate the correspondence of the intra-prostatic ndings referable to the site of primary tumor across modalities.

Correlations between PET semi-quantitative and MRI quantitative imaging parameters
To provide an improved characterization of the primary tumor, a correlation between multitracer PET and MRI parameters was performed. In particular, a Spearman correlation was calculated between the semi-quantitative PET parameters measured on HS 68 Ga-PSMA PET and HS 68 Ga-DOTA-RM2 PET images (SUVmax, SUVmean40, SUVmean50, SUVmean60, MTV40, MTV50, MTV60), the quantitative parameters measured on MRI (lesion volume, ADCmin, ADCmean) and clinical data (PSA, Gleason Score, ISUP score). A p value < 0.05 was considered statistically signi cant.

Patients
Fifteen men (mean age: 68 years; range 52-80) with biopsy proven high-risk PCa have been enrolled so far in this prospective study. All patients had a Gleason score ≥ 7 on biopsy, with a mean PSA at time of diagnosis of 5.90 ng/mL (range: 3.03-11.13).
Patients' characteristics are reported in Table 1. All patients safely completed both 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET/MRI scan with a good rate of compliance and no major discomfort. PET/MRI ndings An example of whole-body biodistribution of 68 Ga-PSMA PET and 68 Ga-DOTA-RM2 PET is reported in Fig. 1. Physiological high 68 Ga-PSMA uptake can be visualized in the salivary and lacrimal glands, liver, spleen, small intestine, kidneys, urinary bladder and ureters (Fig. 1A), while 68 Ga-DOTA-RM2 showed physiological high uptake in the pancreatic gland and urinary bladder.  Table 2.  Table 3.  Table 4.   Table 2.
In 3 patients 68 Ga-PSMA PET showed a bifocal prostatic uptake (n.4, n.11, n.13, Table 2), while a focal lesion was present on 68 Ga-DOTA-RM2 PET images. Similarly, MRI also detected a single lesion in these 3 patients.
Regarding distant metastases, 68 Ga-PSMA showed increased pathological uptake at bone level in 2 patients (n.2, n.6, Table 2), with one of them (patient n. 6, Table 2) being positive also on MRI. 68 Ga-DOTA-RM2 did not detect any pathological uptake at bone level. (Fig. 5) DICE score was computed to quantitatively assess the overlap between the volume of the primary intra-prostatic lesions  Table 6. (Fig. 6).

Discussion
The present pilot study reports our preliminary experience on the use of 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET/MRI imaging in high-risk prostate cancer staging.
Few studies have investigated prostate cancer by using both 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET so far, both in the staging [15] and restaging setting of the disease [18, 19] In our cohort of patients, differently to all the other published papers, all subjects have been studied by using a hybrid PET/MRI scanner both for 68 Ga-PSMA and 68 Ga-DOTA-RM2 radiotracers [15,18,19].
In fact, among the few published studies that investigated the role of this peculiar multitracer approach in PCa, PET/MRI and PET/CT have been used alternatively for 68 Ga-PSMA and 68 Ga-DOTA-RM2 PET scans [18,19] or PET/CT have been adopted as the only hybrid imaging modality [15].
The use of a PET/MRI scanner in the staging phase of PCa allows to perform a diagnostic MRI on the pelvic region, thus obtaining all the necessary morphological and multiparametric information for an accurate identi cation and characterization of the primary tumor. Moreover, the possibility to simultaneously acquire a PET scan with two different radiotracers assessing different metabolic pathways provides additional information regarding primary tumor characteristics, together with a wholebody evaluation of the disease. Differently from other groups that investigated the dual tracer approach of 68 Ga-PSMA and 68 Ga-DOTA-RM2 in PCa staging, or restaging, using a PET/CT scanner [15,18,19], one of the most relevant patients' advantage in the present study relies on the possibility to have received a diagnostic MRI simultaneously acquired to the PET image acquisition.
In fact, MRI is expected to increase the diagnostic accuracy of PET imaging for local staging (ECE and SVI) [25], and the information derived from both modalities could be incorporated into clinical nomograms to signi cantly enhance the preoperative staging accuracy [26,27]. Moreover, MRI shows excellent diagnostic performance in the detection of bone metastases [28]. If used in combination with PET, MRI could provide complementary information on bone disease when PET ndings are equivocal or when metastatic lesions do not show signi cant PSMA uptake. Finally, WB-MRI could be of added value in monitoring the response to loco-regional or systemic treatments [29,30].
In our cohort of patients, the primary PCa was detected in all patients by all three imaging modalities, with slight differences regarding the multifocality of intra-prostatic ndings. Even if the comparison with histological examination was not yet available for the patients included in the study, the different intra-prostatic ndings detected by 68 Ga-PSMA and 68 Ga-DOTA-RM2 might re ect the complementarity of these radiotracers; the same consideration might be applied to lymph nodal and bone localizations. These results enlightening a synergic role of 68 Ga-PSMA and 68 Ga-DOTA-RM2 in prostate cancer are in line with previous published data [15,18,19]. PET/MRI, concluded that the qualitative ndings of PET scans could provide combined relevant information. In their study, both radiotracers partially showed the same tumor region and, in some cases, different tumor parts, thus providing a better PCa characterization and re ecting the heterogeneous and sometimes polyclonal behaviour that characterize PCa [15]. Similarly, the results reported by Baratto  PET. Furthermore, concerning the correlation between imaging parameters and clinical data, an inverse correlation was found between 68 Ga-DOTA-RM2 SUVmax, SUVmean40, SUVmean50 and PSA level at diagnosis. All the other tested correlations resulted non-signi cant and this is in line with what is reported by Fassbender et al. [15]. These results have to be interpreted with great caution and more evidence is needed before speculating on the clinical utility of these ndings, since our sample was small and quantitative analyses might be susceptible to lack of statistical power. Future studies, with larger samples, will allow to unravel the possible association between semi-quantitative PET, quantitative MRI parameters and clinical data.
Some limitations should be pointed out regarding the present study. First of all, histopathological correlation with the postsurgical specimen was not performed because only a minority of patients have undergone radical prostatectomy so far.
Therefore, being the present analysis a pilot study with only a preliminary evaluation of the PET/MRI data, we decided to only consider prostatic biopsies as standard of reference. This aspect, will be certainly improved as soon as all histological data will be available, with a detailed co-registration between imaging and histopathological data and subsequent data analysis.
Another limitation of this study is the low number of patient population. However, besides the fact that the few papers already published on PCa staging and using both 68 Ga-PSMA and 68 Ga-RM2 PET radiotracers included a number of patients even lower than the one presented in the present paper [15], we consider that these preliminary data are interesting to underlie the potential complementary and synergic role of the two different PET radiotracers together with mpMRI.
To conclude, based on the results of the present study, a potential complementary role of 68 Ga-PSMA and 68 Ga-DOTA-RM2 in PCa staging can be enlightened, in view of the different ndings detected by the two imaging modalities in some of the patients