The authors have declared that no competing interests exist.
Conceived and designed the experiments: QQC QJM SG. Performed the experiments: QQC MLC BC QW. Analyzed the data: BC QW. Contributed reagents/materials/analysis tools: BJ FW BTS. Wrote the paper: QJM MLC SG.
This study aimed to explore the diagnostic performance of single photon emission computed tomography / computerized tomography (SPECT/CT) using a new radiotracer 99mTc-RGD-BBN for breast malignant tumor compared with 99mTc-3P4-RGD2.
6 female patients with breast malignant tumors diagnosed by fine needle aspiration cytology biopsy (FNAB) who were scheduled to undergo surgery were included in the study. 99mTc-3P4-RGD2 and 99mTc-RGD-BBN were performed with single photon emission computed tomography (SPECT) at 1 hour after intravenous injection of 299 ± 30 MBq and 293 ± 32 MBq of radiotracers respectively at separate day. The results were evaluated by the Tumor to non-Tumor ratios (T/NT). 99mTc-RGD-BBN and 99mTc-3P4-RGD2 SPECT/CT images were interpreted independently by 3 experienced nuclear medicine physicians using a 3-point scale system. All of the samples were analyzed immunohistochemically to evaluate the integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) expression. The safety, biodistribution and radiation dosimetry of 99mTc-RGD-BBN were also evaluated in the healthy volunteers.
No serious adverse events were reported in any of the patients during the study. The effective radiation dose entirely conformed to the relevant standards. A total of 6 palpable malignant lesions were detected using 99mTc-RGD-BBN SPECT/CT with clear uptake. All malignant lesions were also detected using 99mTc-3P4-RGD2 SPECT/CT. The results showed that five malignant lesions were with clear uptake and the other one with barely an uptake. 4 malignant cases were found with both αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) and 1 case with only integrin αvβ3 positive expression (GRPR negative).
99mTc-RGD-BBN is a safe agent for detecting breast cancer. 99mTc-RGD-BBN may have the potential to make up for the deficiency of 99mTc-3P4-RGD2 in the detection of breast cancer with only GRPR positive expression (integrin αvβ3 negative). The preliminary application of 99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.
Breast cancer is the most frequent malignancy in women all over the world. In the United States, a Cancer Journal for Clinicians estimates that 234,580 women will be diagnosed with breast cancer in 2013 and expected to account for 30% of all female new cancers. Also, the incidence rate is increasing year by year in China [
X-ray mammography (XMM) and ultrasound (US) are now employed as conventional tools for breast tumor screening. The wide use of them in early detection of tumor has saved thousands of lives. With the advent of molecular imaging era,nuclear medicine techniques is considered promising in early detection of tumor through a functional perspective. Of them, scintimammography (SMM) with various targeted probes have become a major interest in this area [
Expression of cell-surface receptors by cancer cells can be heterogeneous and inhomogeneous. Breast cancer cells, for example, over-express two receptors [gastrin-releasing peptide receptor (GRPR) in 70% breast cancer cells and integrin αvβ3 in 58% breast cancer cells]. It is difficult to identify all breast cancer cells with just one target-based cancer imaging. Therefore, it is desirable to develop a new type of radiotracers that can target not just one but several different receptors simultaneously. Of course, the receptors need to be more specific peptide vs integrin receptors.
Recently, our laboratory designed and synthesized a dual integrin αvβ3 and GRPR targeted peptide Glu-c(RGDyK)-bombesin (RGD-BBN) that contained dual RGD and BBN motifs in one molecule [
6 patients (mean age 59 ± 10yr) with breast malignant tumor diagnosed by fine needle biopsy (FNAB) at least 7 days prior to surgery were included in the study. The healthy volunteers consisted of 3 males and 3 females aged between 25 and 51 years (
Volunteer No./Sex | Age (y) | Height (cm) | Weight (kg) | Body mass index (kg/m2) | Injected Activity (MBq) |
---|---|---|---|---|---|
1/M | 51 | 175 | 73.5 | 24.00 | 1076.7 |
2/M | 30 | 174 | 70.0 | 23.12 | 952.75 |
3/M | 27 | 173 | 77.0 | 25.73 | 1061.9 |
4/F | 25 | 155 | 41.0 | 17.07 | 1128.5 |
5/F | 41 | 161 | 58.0 | 22.38 | 954.6 |
6/F | 35 | 158 | 52.0 | 20.83 | 995.3 |
Mean±SD | 34.83±9.81 | 166±8.99 | 61.92±13.99 | 22.19±2.99 | 1028.29±71.75 |
All patients were sent to the 99mTc-3P4-RGD2 and 99mTc-RGD-BBN SMM on an individual basis. The time interval between two imaging procedures was 48–54 hr. Finally, 99mTc-RGD-BBN and 99mTc-3P4-RGD2 SMM results were compared with each other. To be included, a patient had to be without any other history of breast disease. Exclusion criteria included pregnancy, lactation and a body weight greater than 80 kg.
A final diagnosis of the specimens obtained by the surgical procedure was made by histopathology. The most representative samples were submitted to immunohistochemical evaluation. For the resected lesions, the largest dimension of the tumor was considered the pathologic size. Results of SPECT/CT were compared with histological findings.
The 3P4-RGD2 and RGD-BBN were generously provided by the Medical Isotopes Research Center of Peking University in freeze-dried kits form. Na99mTcO4 was obtained from a commercial 99Mo/99mTc generator (Beijing Atom High Tech Co., Ltd.). The kit for preparation of 99mTc-3P4-RGD2 was formulated by containing, per millilitre, 20 μg of HYNIC-3P4-RGD2, 5 mg of TPPTS, 6.5 mg of tricine, 40 mg of mannitol, 38.5 mg of disodium succinate hexahydrate and 12.7 mg of succinic acid. Radiolabeling and quality control procedures for 99mTc-3P4-RGD2 were performed as described previously 4. The kit for preparation of 99mTc-RGD-BBN was formulated by containing, per millilitre, 20 μg of HYNIC-RGD-BBN, 6 mg of TPPTS, 10 mg of tricine, 40 mg of mannitol, 38.5 mg of disodium succinate hexahydrate and 12.7 mg of succinic acid [
SPECT: Emission images were acquired using a dual-head, large field-of-view scintillation camera (Precedence, Philips Healthcare), equipped with a low-energy, high-resolution and parallel-hole collimator. In all healthy volunteers, planar images in both anterior and posterior views were acquired at 10 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h post-injection and were stored digitally, using 99mTc with a 20% energy window centered on 140 keV and 128×128 matrix. The velocity of scanning was 15 cm/min. SPECT images in all the patients were acquired over 360° (180°per head) in supine position with raised arms during the imaging. Imaging with both radiotracers was performed using 6°angular steps in a 20 s time frame, with a 64×64 matrix size. Distance between the breast and detector was minimized. The position of the patient was recorded in detail and kept consistent to the greatest extent in the second SPECT scan to make sure that the two different SPECT images could be fused to the CT images.
CT images from neck to abdomen were acquired sequentially in a non-dedicated 3rd-generation scanner installed in the SPECT camera gantry, with a 10 mm slice thickness, a maximum current of 2.5 mA and a 140 kV potential. The patients just had one CT scan since 99mTc-3P4-RGD2 and 99mTc-RGD-BBN SPECT were undertaken on the same device.
All images were interpreted qualitatively by three experienced nuclear medicine radiologists who were unaware of the clinical history and other test results of all patients. Visual analysis was performed on a per-lesion basis and in a blinded fashion. A 3-point scale system was adopted to describe the uptake degree for breast lesions. The rules for classification were as follows: grade 1, no abnormal increased uptake (not higher than contralateral or peripheral normal breast tissue); grade 2, mildly increased uptake (lower than mediastinum or equivalent to mediastinum); grade 3, definite focal increased uptake (higher than mediastinum). Homogeneous uptake in both breasts was classified as grade 1. SMM was considered positive for malignancy if the visual score was ≥2. There was no disagreement between three radiologists in our study.
The immunohistochemistry of αvβ3 and GRPR expression of the breast tissue sample was performed as described previously with some modifications [
The 99mTc-RGD-BBN with a range of 786.7 ± 55.8 MBq (19.1–24.2 mCi) was injected into the antecubital vein of all healthy volunteers with a rapid bolus, followed by a 10 mL saline flush. Measurements of vital signs (body temperature, systolic and diastolic blood pressure and pulse rate), laboratory safety tests (renal and liver function chemistry, hematology, and blood coagulation parameters) and 12-lead electrocardiogram were recorded before and after tracer injection.
1.5 mL venous blood sampling was collected via an indwelling catheter throughout the imaging period specifically at 1, 3, 5, 10, 15, 30, 60 and 120 minutes post-injection. Samples were weighed and counted in a γ-counter (Wallac 1470–002,Perkin Elmer,Finland). Decay corrected time-activity curve was expressed as percentage of injected dose per gram (%ID/g).
A urine sample was collected at the following hourly intervals after tracer injection: 0 to 2, 2 to 4, 4 to 8, 8 to 12 and 12 to 24 hours. Samples had been weighing the volume. Samples’ radioactivity was measured with the gamma counter.
Visual analysis was applied to determine the integral biodistribution of the tracer and transient and intersubject stability. For each subject, regions of interest (ROIs) were delineated over the identified organs including: lung, heart, liver, kidneys, spleen, intestine, urinary bladder and a background region near the body on the anterior image. The mirror ROIs were applied to the posterior images of each organ. The mean counts of each organ on planar images were measured. The results were expressed as percentage of initial injected activity after decay-correction.
Fitted residence time functions were plotted and multiplied by the exponential decay functions for 99mTc. These functions were then integrated analytically to determine the area under the curve (AUC) to yield the residence time of each organ. Then, these residence times were input in OLINDA/EXM 1.0 software (Vanderbilt, University, Nashville, TN) to calculate equivalent organ doses and the effective dose (ED) based on the 70-kg reference adult phantom in International Commission on Radiological Protection (ICRP) publication 60 [
By using SPSS 13.0 statistical analysis software, data are expressed as mean ± SD. A two-tailed Student’s
No clinically significant abnormalities or abnormal clinical chemistry were reported in any of the patients during the study. The results of measurements of vital signs (body temperature, systolic and diastolic blood pressure and pulse rate), laboratory safety tests (renal and liver function chemistry, hematology, and blood coagulation parameters) and 12-lead electrocardiogram were normal before and after the study.
Error bars indicate standard deviations.
The concentration of radioactivity in the urine was shown in
Error bars indicate standard deviations.
(a) Anterior planar whole-body images. (b) Posterior planar whole-body images.
By measuring ROIs drawn on both anterior and posterior images, the quantitative tracer uptakes in major organs were presented in
A summary of dosimetric parameters for various organs and whole body is given in
Target Organ | Dosimetric data(×10–3 mSv/MBq) | ||
---|---|---|---|
Male(n = 3) | Female(n = 3) | Total(n = 6) | |
Adrenal glands | 3.53±0.40 | 4.16±0.92 | 3.85±0.66 |
Brain | 1.11±0.19 | 0.86±0.26 | 0.99±0.22 |
Breasts | 1.08±0.12 | 1.17±0.27 | 1.12±0.18 |
Gallbladder wall | 5.09±0.79 | 5.22±1.13 | 5.15±0.80 |
Lower region of colon | 19.40±2.18 | 27.37±7.33 | 23.38±5.95 |
Small intestine | 3.08±0.28 | 3.86±0.76 | 3.47±0.61 |
Stomach wall | 2.66±0.21 | 2.93±0.56 | 2.80±0.37 |
Upper colon | 2.50±0.24 | 3.04±0.64 | 2.77±0.48 |
Heart wall | 3.57±0.49 | 3.77±0.54 | 3.67±0.43 |
Kidneys | 23.60±4.24 | 27.50±7.78 | 25.55±5.48 |
Liver | 5.06±0.51 | 5.45±1.29 | 5.26±0.82 |
Lungs | 3.96±0.41 | 4.48±1.05 | 4.22±0.70 |
Muscle | 1.65±0.14 | 1.88±0.40 | 1.77±0.27 |
Ovaries | - | 4.54±0.95 | 4.54±0.95 |
Pancreas | 9.68±3.03 | 11.43±1.36 | 10.56±2.11 |
Red marrow | 1.88±0.20 | 2.19±0.47 | 2.03±0.33 |
Osteogenic cells | 3.80±0.42 | 4.28±1.01 | 4.04±0.68 |
Skin | 0.91±0.10 | 0.99±0.23 | 0.95±0.15 |
Spleen | 14.93±1.96 | 9.59±4.21 | 12.26±3.78 |
Testis | 1.48±0.05 | - | 1.48±0.05 |
Thymus | 1.60±0.20 | 1.71±0.37 | 1.66±0.25 |
Thyroid gland | 14.16±4.71 | 14.57±1.17 | 14.37±2.81 |
Urinary bladder wall | 12.25±2.99 | 16.50±5.89 | 14.38±4.37 |
Uterus | - | 4.14±0.77 | 4.14±0.77 |
Whole body | 2.02±0.19 | 2.31±0.49 | 2.17±0.34 |
All underwent surgery within 1 wk. A total of 6 palpable malignant lesions in 6 patients were described in the standard of truth including 4 invasive ductal carcinomas (IDC) and 2 ductal carcinoma in situ (DCIS) (
(b) CT scan demonstrates a mass in the right breast. (c) 99mTc-RGD-BN SMM demonstrates high uptake observed in the lesion. (d) Histopathology staining indicated a ductal carcinoma in situ. ×40 (e) Immunohistochemistry demonstrates barely αvβ3 expression in tumor vessels and tumor cells. ×400. (f) Immunohistochemistry demonstrates intense GRPR expression in tumor vessels and tumor cells. ×400.
Patients | Pathology | Location | Lesion size(cm) | Visual uptake grading | RGD (T/N Ratio) | Visual uptake grading | RGD-BBN (T/N Ratio) | Immunohistochemistry | |
---|---|---|---|---|---|---|---|---|---|
RGD | RGD-BBN | αvβ3 | GRPR | ||||||
1 | DCIS | R | 2.3 | 1 | 1.04 | 3 | 3.31 | - | + |
2 | DCIS | L | 2.0 | 3 | 3.09 | 3 | 2.72 | + | + |
3 | IDC | L | 5.1 | 3 | 3.12 | 3 | 3.06 | + | + |
4 | IDC | R | 2.1 | 3 | 2.56 | 3 | 2.45 | + | - |
5 | IDC | L | 5.8 | 3 | 4.43 | 3 | 4.17 | + | + |
6 | IDC | L | 6.3 | 3 | 3.12 | 3 | 3.03 | + | + |
SPECT/CT with 99mTc-RGD-BBN and 99mTc-RGD: 3-grade scale, where grade 1 = no abnormal increased uptake; grade 2 = mildly increased or heterogeneous uptake; grade 3 = definite focal increased uptake. IDC = infiltrative ductal carcinoma; DCIS = ductal carcinoma in situ.
For 6 malignant samples, 4 cases were found with dual αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) (
The SMM is less widespread than XMM and ultrasound US, but this does not mean that it has to remain in the background, as it is of great diagnostic value in breast tumor, especially after the emergence of SPECT/CT and the constant development of new tracers offers great potential for SMM. The combination of SMM and CT implies a huge progress due to its high diagnostic performance and ability to detect tumor pathological conditions. As non-invasive and sensitive imaging methods they have been widely used for diagnosing diseases in the clinic [
The emergence of various probes has become an indispensable factor for SMM. In previous studies, several one-target based radiotracers have been developed, such as a series of arginine-glycine-aspartic acid (RGD) containing peptides and radiolabeled BBN analogue which can specifically bind to integrin αvβ3 and GRPR respectively[
No clinically significant abnormalities or abnormal clinical chemistry were reported in any of the patients during our study. This showed that 99mTc-RGD-BBN had good security and stability for human use. The pharmacokinetic results were also satisfactory. There was a very sharp decline of radioactivity in the circulation and the radioactivity in the urine kept increasing with a total cumulative recovery of (73.56 ± 2.04) % of original dose at 24 h. The biodistribution of 99mTc-RGD-BBN in normal subjects indicated a renal-urinary excretion of the tracer.
The effective radiation dose to the body of 99mTc-3P4-RGD2 and 99mTc-RGD-BBN were 1.15 ± 0.13 mSv and 0.65 ± 0.07 mSv respectively 9. According to the 2007 Recommendations of the International Commission on Radiological Protection (ICRP) [
All of the 6 malignant lesions were clearly detected by 99mTc-RGD-BBN SPECT/CT imaging with intense uptake, which is consistent with the recent report of 18F, 64Cu and 68Ga labeled RGD-BBN PET imaging [
It is well-known that a realistic strategy for the reduction of breast cancer mortality rates and timely treatment is to detect the disease as early as possible. The most common screening method for early breast cancer is mammography. Our previous research of 99mTc-3P4-RGD2 imaging has shown promise as an additional tool to mammography to avoid unnecessary biopsies [
In this study, our findings are of a preliminary nature and need to be further corroborated. It is necessary to investigate the effects of linkers of different lengths, solubility, lipophilicity, and flexibility on the in vitro and in vivo behaviors of the dual integrin αvβ3 and GRPR targeted peptide to make it possible to bind to both receptors simultaneously, which may result in enhanced targeting efficacy and higher uptake of 99mTc-RGD-BBN by the tumor. This may help to provide new idea in the development of tumor targeted therapy. The design of heteromultimeric tracers that recognize other tumor targets is also worth further investigation for tumor-targeted imaging and therapy.
There are several limitations to this study that call for further discussion. First, as a combined probe, the sensitivity of 99mTc-RGD-BBN SPECT/CT in detecting breast malignant tumor should be improved, but the specificity may be also affected. In our study, there was no false positive or false negative case by 99mTc-RGD-BBN SPECT/CT imaging. Such a high accuracy might be related to the small numbers of patients included in this study and all of them were with breast malignant tumor, which was chosen with a subjective wish. Further research is needed to include a larger patient population. Second, although the immunohistochemistry of integrin αvβ3 and GRPR expression were conducted in this study, quantification and correlation with tracer uptake was not performed due to the heterogeneous and inhomogeneous expression of cell-surface receptors by cancer cells.
The dual integrin αvβ3 and GRPR targeting 99mTc-RGD-BBN showed an excellent ability to detect breast cancer without clinically safety problems. 99mTc-RGD-BBN may have the potential to make up for the deficiency of 99mTc-3P4-RGD2 in the detection of breast cancer with only GRPR positive expression (integrin αvβ3 negative). The preliminary application of 99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.