Evaluation of Radiation dosimetry of 99mTc-HYNIC-PSMA and imaging in prostate cancer

To date, several 99mTc-labelled PSMA inhibitors have been developed for PCa detection, including 99mTc-MIP-1404[9],99mTc-MIP-1405[9], 99mTc-PSMA-I&S[14] and 99mTc-EDDA/HYNIC-iPSMA[15]. Compared with other imaging techniques, such as CT and MRI, SPECT/CT using 99mTc-labelled PSMA has demonstrated great potential for detecting PCa metastasis and guiding the treatment of targeted lesions, thus benefiting patients[7,10].

The present study conducted a clinical safety evaluation of 99mTc-HYNIC-PSMA according to a method that we developed[16]. Our results suggest that 99mTc-HYNIC-PSMA was excreted mainly through the urinary system and that its absorbed doses in organs, including the brain and heart, were low. The kidneys showed the highest absorbed dose, which had a value of 2.87E-02±1.53E-03 mGy/MBq. In other organs, including the red bone marrow, thyroid and adrenal gland, the absorbed doses were lower. The effective dose of 99mTc-HYNIC-PSMA was 3.72E-03±4.50E-04 mSv/MBq, so it is 2.75±0.33 mSv when the administered activity was 740 MBq. This effective dose value is similar to that of 99mTc-EDDA/HYNIC-iPSMA[15] (3.73E-03 mSv/MBq) but lower than that of 99mTc-MIP-1404[9] (8.8E-03 mSv/MBq) and 99mTc-MIP-1405[9] (7.9E-03 mSv/MBq). The total effective dose in the body is also much lower than that of 68Ga-labelled and 18F-labelled PSMA-targeted tracers, including 68Ga-PSMA-11[17] (2.36E-02 mSv/MBq), 68Ga-PSMA-617[18] (2.1E-02 mSv/MBq), 18F-PSMA-1007[19] (2.2E-02 mSv/MBq) and 18F-DCFPyL[20] (1.39E-02 mSv/MBq). This is because 68Ga and 18F have a dual-photon property and emit gamma radiation with higher energy than 99mTc. Furthermore, the effective dose of 99mTc-HYNIC-PSMA is also lower than that of conventional 99mTc-labelled radiopharmaceuticals used in SPECT/CT scans, such as MDP (5.68E-03 mSv/MBq)[21] and MIBI (7.83E-03 mSv/MBq)[21]. Of note, because the main excretory pathways for the tracer are the kidneys and bladder, which is in agreement with the results shown in our current study, we suggest that patients drink plenty of water or take diuretics after the scan to reduce the absorbed dose.

The Dosimetry Toolkit supports 3 different scenarios, including multiple whole-body SPECT/CT scenarios,
multiple whole-body planar scenarios and a hybrid planar-SPECT/CT scenario. In order to avoid the long scan
time required by multiple whole-body SPECT imaging, which could cause tracer metabolic difference between segments for each scan, the hybrid imaging scenario was chosen. Conventionally, for organ segmentation, it is
very difficult to manually utilize information from 2D planar images, particularly for those regions with low tracer uptake. Unlike the conventional technique, this computer-assisted semiautomatic method provides a more consistent and convenient method for image processing.

For the tracer imaging feasibility study, Table 3 showed that the 99mTc-HYNIC-PSMA was able to be accumulated at primary lesion sites and metastatic lesion sites (bone, soft tissue and lymph nodes). The average
tumor-to-background ratio for 99mTc-HYNIC-PSMA at 2 h was 9.42 2.62, which is moderately higher than
that observed for 99mTc-MIPs (range 3.8–6.2)[9]. When the PSA is very low (Patient #3 from Table 3 as an example, with PSA of only 0.56), the 99mTc-HYNIC-PSMA image can detect the uptake of the malignant lesion, while the anatomical imaging of the patient did not show any corresponding lesions (Fig. 4).

The main limitation of this study is the small sample size (5 patients were included in the dosimetry investigation and 10 patients were used for the validation of the usefulness of 99mTc-HYNIC-PSMA), but the small standard deviation of the effective dose suggests that our analysis is reproducible for the dose calculation of 99mTc-HYNIC-PSMA. Another limitation is due to the imaging protocol which has an experimental period of 8 h for each patient, so it is very difficult to guarantee a consistent scanning position, which would likely lead to tiny organ shifts. Additionally, there are also limitations for the hypotheses which were proposed based on the dose estimation process. Firstly, according to the method established by MIRD, radioactivity is supposed to be evenly distributed throughout the body instantly after administration. Secondly, any unmeasured radioactivity is assigned to the other organs in the body.

In this study, 99mTc-HYNIC-PSMA was shown a high specific uptake (with a tumor-to-background ratio of 9.42 ± 2.62) in the malignant lesions of PCa patients, and it was found the urinary system to be its main excretory pathway. Our dosimetry study also showed that, at the routine clinical dose (740 MBq), the effective dose of 99mTc-HYNIC-PSMA was 2.75 ± 0.33 mSv (range: 2.27–3.19 mSv) which is similar to the effective doses from
other PSMA inhibitors, such as 99mTc-EDDA/HYNIC-iPSMA published in the literature, so it indicates that could be a safe SPECT tracer.

All studies were approved by the Fudan University Shanghai Cancer Center ethics committee, and all the procedures in the studies involving human participants were performed in accordance with the ethical standards of the institution. For the dosimetry study, each subject signed a written informed consent form prior to participating in the 99mTc-HYNIC-PSMA dosimetry study. For the imaging feasibility study, a retrospective review on patients was conducted, and its results did not influence further therapeutic decision-making, so the ethics committee approved that informal consent was not required.

Subjects
From May to June 2018, 5 PCa patients (mean age ± SD, 60 ± 6 years; age range, 48–68 years)
underwent 99mTc-HYNIC-PSMA single-photon emission computed tomography/computed tomography
(SPECT/CT) scanning. Unlike conventional PCa patients, in order to mimic the conditions in healthy subjects,
the five patients selected in this study did not show obvious tracer accumulation in tumor lesions or metastatic
sites in the SPECT/CT scans. The biodistribution of radiopharmaceuticals throughout their bodies were similar
to that in healthy adult males. Two patients had made only their first-visit, so were untreated, while the other
three had received a radical prostatectomy. One of these three patients had experienced a biochemical recurrence. None of the patients received any radiation therapy or chemotherapy treatment. For the imaging feasibility study using 99mTc-HYNIC-PSMA, which went from January to May 2019, 10 patients aged 52 to 77 years (mean age ± SD, 68 ± 8 years) were enrolled, and all patients were histologically diagnosed with PCa. In addition, all lesions detected in the imaging feasibility study were confirmed by biopsy or clinical follow-up.

99mTc-HYNIC-PSMA synthesis and administration
99mTc-HYNIC-PSMA was synthesized using a method developed by our team[11].. In this method, 10 μg of HYNIC-Glu-Urea-A, 0.5 mL of EDDA, 0.5 mL of Tricine and 25 μg of SnCl2 solution were allowed to react with 1110–4440 MBq of Na99mTcO4. The reaction was carried out in a water bath that was boiled for 10 min and then cooled to room temperature. The results showed that a labelling yield of more than 99% was achieved, and no additional purification was needed. In addition, endotoxin tests and retrospective analysis of the bacterial cultures all showed negative results. All patients were asked to void their bladders before receiving intravenous injections of 99mTc-HYNIC-PSMA (mean activity ± SD, 740 ± 74 MBq).

Image acquisition
Whole-body planar images (anterior, ANT; posterior, POST) were collected at 0.5 h, 1 h, 2 h, 4 h and 8 h after the injections, respectively, to estimate the dosimetry of 99mTc-HYNIC-PSMA. A 99mTc-HYNIC-PSMA reference source with a known activity (37 MBq) was prepared and placed 10 cm above each patient's vertex during each scan for the conversion of the count per minute (cpm) to activity. Data were acquired using a dual-detector SPECT/CT instrument (Discovery NM/CT 670, GE, Milwaukee) with low-energy, high-resolution, parallel-hole collimators. The patients were asked to void their bladders before each scan made at 2 h, 4 h and 8 h respectively. For the whole-body planar images, the scanning parameters were chosen as follows: the main energy window was 140 keV ± 10%, the scatter energy window was 120 keV ± 5%, the matrix size was 256x1024 and the scan speed was 15 cm/min. The SPECT/CT scan was performed immediately following the whole-body planar image scan performed after 2 h; the patients' positions remaining unchanged. Scans were conducted from each patient's apex pulmonis to his pubic symphysis and included the chest, abdominal cavity and pelvic cavity. SPECT scans were first performed using the following acquisition parameters: the matrix size, 128x128; Zoom, 1; acquisition was over 360 degrees in 6 degree increments taking a total of 30 minutes. Then, conventional low-dose CT was conducted.

For the study involving the validation of the usefulness of 99mTc-HYNIC-PSMA in 10 PCa patients, SPECT/CT images were acquired at 2 h after radiopharmaceutical administration. The SPECT/CT scan protocol was the
same as that stated above. ROIs were drawn around tumors, and for the background, a circular ROI with a diameter of 2 cm was drawn within the obturator muscle.

Image processing
The ANT and POST images were processed into geometric mean (GM) images with scatter correction. The collected SPECT data were reconstructed using an iterative algorithm with resolution recovery, attenuation, decay and scatter corrections. The volumes of interest (VOIs) were delineated both manually in the CT imaging (for low uptake areas and overlapping organs such as lungs, liver and spleen) and automatically in the SPECT imaging (for high uptake areas and organs or regions that did not overlap such as the kidneys, salivary glands, heart and the total body) by a medical physicist and a radiologist using the Dosimetry Toolkit. The whole-body GM images from other time points were automatically registered to the GM image collected at 2 h. Then, the organ segmentations VOIs were projected onto the GM images.

TIACs and absorbed dose calculations
At each time point, the percentage of injected dose (ID%) of each source organ was assessed according to the following formula:

where %IDsource organ (t) represents the %ID of the source organ measured at time t after injection; Asource organ (t), Ainjected dose and Areference source represent the activity of the source organ at time t after injection, the injected dose, and the activity of the reference source at the injection time (the time of the prepared reference source is as the same with the injection time), respectively; cpm(t) represents the counts per minute (cpm) of the reference source or source organs at time t; and T1/2 is the physical half-life of the nuclide 99mTc.
cpmsource organ (t) and cpmreference source (t) were extrapolated from the projected images at each time point of 0.5 h, 1 h, 2 h, 4 h and 8 h. In general, the measured kinetic data (with the exclusion of that for the urinary bladder) could be represented as mathematical expression with one or more exponential terms, and the OLINDA/EXM 1.1 code (Vanderbilt University, Nashville, TN, USA) allowed the user to enter kinetic data for each source organ (%ID at different times) and fit it to one or more exponential terms[22].. The time-%IDsource organ (t) curves were fitted by least-squares analysis using the EXM module in the OLINDA/EXM 1.1 application. The areas under the fitted time-%IDsource organ (t) curves, which represent the TIACs, were calculated based on the physical T1/2 of the radioisotope and the integral of the fitted time-activity curve from a time of zero to infinity. In this study, the curves for the 99mTc-HYNIC-PSMA time-%ID data for the source organs was fitted with a single exponential equation ID%(t) = C ⋅ exp(−ct), and the TIAC was given by the integration of the curve, the result of which was C/c.

The total urine excretion UE8h at 8 h after the tracer injection was estimated from the acquired images as follows (Eqn. (3)):

where − A′ WB 8h is the whole body activity corrected according to the decay at 8 h after the administration and A0 is the injection activity. The 2-phase exponential association curve was then fitted to the cumulative urine activity using the following formula (Eqn. (4)):

where U(t) is the accumulated urine at time t, A and B are regression parameters, TA1/2is the A phase half-life and TB1/2 is the B phase half-life. The fraction of the tracer excreted through the bladder and the corresponding T1/2 value were assessed according to the accumulated excreted activity calculated according to the whole body time-%ID curve.

To assess the TIAC of urinary bladder content, UE8h/A0 ⋅ [A/(A + B)], UE8h/A0 ⋅ [B/(A + B)], TA1/2and TB1/2 were used in the bladder-voiding model in OLINDA/EXM 1.1, with a voiding interval of 2 h. The TIAC of the rest of the body was obtained by the following formula (Eqn. (5)):

where TIACrest is the TIAC of the rest of the tissue, TIACWB is the TIAC of whole body, TIACsource is the TIAC of the source organs (lungs, kidneys, liver, spleen, salivary glands and heart) and TIACbladder is the TIAC of the urinary bladder.

The absorbed doses were estimated using the organ TIAC data described above, by the OLINDA/EXM 1.1 program22–24. The absorbed dose of the salivary glands was calculated based on the mass and S-values obtained from Liu et al.25. The effective dose (ED) is the sum of the weighted doses of each of the organs, which were calculated by multiplying the absorbed doses for the individual organ doses by a stochastic risk weighting factor (ICRP 103). The radiation transport phantom selected from OLINDA/EXM 1.1 was the hermaphroditic phantom, which is defined by Cristy and Eckerman as a 73.7 kg adult phantom.

Acknowledgements
The authors would like to thank GE Healthcare China for technical assistance. This study was carried out with financial support from the General Project of the Shanghai Municipal Health and Family Planning Commission Foundation, China (Grant No. 20170425) and Shanghai Engineering Research Center of Molecular Imaging Probes, China (Grant No. 19DZ2282200).

Author contributions
Jianping Zhang and L.Q. Shi conceived and designed the study; Xiaoping Xu synthesized the radiopharmaceutical; Jiangang Zhang created the imaging database; Jiangang Zhang and Linjun Lu acquired imaging data; Jingyi Cheng, Shaoli Song, Silong Hu, Chang Liu, Yingjian Zhang performed delineation and visual interpretation the data; Jianping Zhang provided the radiation dosimetry calculation; Chang Liu, Jingyi Cheng, Shaoli Song and Yingjian Zhang provided the patient data; Jianping Zhang wrote the manuscript; Jianping Zhang and L.Q. Shi, Jiangang Zhang, Jingyi Cheng, Shaoli Song and Yingjian Zhang provided edits; all authors read and approved the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
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