VPAC1 receptors for imaging breast cancer: a feasibility study

Abstract

VPAC1 encodes G-protein coupled receptors expressed on all breast cancer (BC) cells at the onset of the disease, but not on benign lesions. Our extensive preclinical studies, have shown that Cu-64-TP3805 has a high affinity for VPAC1, is stable in vivo and has the ability to distinguish spontaneously grown malignant BC masses from benign lesions. Our long term goal is to develop Cu-64-TP3805 as an agent to perform in vivo histology, to distinguish malignant lesions from benign masses noninvasively and thereby avoid patient morbidity and excess economic costs of benign biopsies.

METHOD

F-18-FDG obtained commercially served as a control. Cu-64-TP3805 was prepared by using a sterile kit containing 20 µg TP3805. Radiochemical purity and sterility were examined. Nineteen consenting females with histologically proved BC were given 370 MBq F-18-FDG. One hr later, 6 of these patients were imaged with PET/CT and 13 with PEM (positron emission mammography). Two to seven days later, 6 PET/CT patients received 111±10% MBq (n=2), 127±10% MBq (n=2) or 148±10% MBq (n=2) Cu-64-TP3805 and imaged 2 hr and 4 hrs later. Thirteen PEM patients received 148±10% MBq Cu-64-TP3805, and imaged at 15 min, 1 hr, 2 hr and 4 hr post injection. For PET/CT patients SUV was calculated and for PEM patients PUV/BGV (PEM uptake value/background value) determined. Tumor volume was also calculated.

RESULTS

Radiochemical purity of Cu-64-TP3805 was 97±2% and Specific Activity was 44.4 GBq (1.2 Ci)/µ mol. In 19 patients a total of 24 lesions were imaged (15 IDC, 1 high grade mammary carcinoma, 3 lobular carcinoma, 1 invasive papilloma and 4 sentinel lymph nodes). All lesions were unequivocally detected by Cu-64-TP3805 as well as by F-18-FDG. The average tumor volume with Cu-64-TP3805 as determined by PET/CT was 90.6±16% of that of F-18-FDG PET/CT and SUV values were 92±26.4% of that of F-18-FDG. For PEM, the tumor volume with Cu-64-TP3805 was 113±37% of that of F-18-FDG and PUV/BGV ratio was 97.7±24.5% of that of F-18-FDG.

CONCLUSION

Cu-64-TP3805 is worthy of further investigation in patients requiring biopsy of suspicious imaging findings, to further evaluate its ability to distinguish malignant lesions from benign masses, noninvasively.

INTRODUCTION

Of the approximately 1.6 million breast biopsies which were performed in the United States in 2011, about 288,130 breast cancers (BC) were diagnosed (230,480 invasive and 57,650 in situ) (1, 2), but over 1.3 million of these biopsies resulted in a benign diagnosis. These unnecessary biopsies create significant patient morbidity and potentially unnecessary health care costs. To detect BC, digital mammography, MRI, CT, US, F-18-FDG and Tc-99m sestamibi have made significant advances. However all of these modalities have limited specificity and all continue to produce many false positive and false negative examinations (3–12). At an average cost of $5000–6000 for each biopsy, unnecessary benign biopsies represent a serious health care burden. There is a compelling need for an innovative approach that would decrease the number of unnecessary benign biopsies while still detecting the malignancies.

Recent approaches to drug discovery focus upon understanding of the genesis of diseases and the biomedical pathways that control them at a molecular level. Previous studies have demonstrated that VPAC1 receptors (combined for vasoactive intestinal and pituitary adenylate cyclase activating peptide) are overexpressed in high density on BC (13). VPAC1 receptors encode a G protein involved in cell proliferation, cell differentiation, as well as in survival of BC cells. On stroma, normal cells, and benign masses only a few VPAC1 receptors are expressed (13–18).

We, therefore hypothesized that a radiolabeled biomolecule with a high affinity for VPAC1 receptors would not only image BC early, but would also distinguish malignant lesions from benign masses. In order to validate this hypothesis on a molecular level, a large body of preclinical data was generated (19–27). Based on their high affinity for VPAC1 receptors, we chose four peptide constructs, modified them for radiolabeling with Tc-99m (t½ - 6 hr, γ- 140 keV) and evaluated them for receptor affinity (Kd), receptor specificity, in vivo stability and tissue distribution (20–27). Impetus generated from these results prompted us to label the four molecules with β + (19%, 656 keV) emitting Cu-64 (t½ - 12.8 hr) for positron emission tomography (PET). We used N₂S₂ chelating agent, determined Kd values, performed tissue distribution studies in athymic nude mice bearing T47D human BC, performed receptor blocking studies, determined receptor affinity, and examined their stability in vivo (23–27).

Based on the critical evaluation of these data, we chose a PACAP analog, Cu-64-TP3805, for further evaluation. Cu-64-TP3805 not only imaged all xenografted human BC in athymic nude mice (tumor uptake 6.35±1.28% ID/g at 24 hr post injection), but also localized all (n=8), spontaneously grown BC (visible 5, invisible 1 and metastatic 2) lesions in transgenic MMTVneu mice (n=9) (26). Furthermore, the Cu-64-TP3805 PET images were normal for two lesions that had negligible expression of VPAC1 receptors. These were benign histologically as examined by an independent senior pathologist. These two benign lesions displayed prominent FDG images. Among the eight malignant lesions, only four were imaged by F-18-FDG. All malignant lesions (n=8) were confirmed by histology and expressed VPAC1 receptors as determined by RT-PCR (26). The two histologically determined benign lesions had negligible expression of VPAC1 receptors. Spontaneous growth of BC in MMTVneu mice resembled pathophysiology of human BC. Further evidence of the receptor specificity of Cu-64-TP3805 was obtained by digital autoradiography of human BC specimens in which Cu-64-TP3805 had nearly six times greater uptake of radioactivity (n=5) than that of benign stroma (24). These highly encouraging data, particularly the ability of the probe to image only malignant lesions with high specificity, prompted us to undertake this study in humans.

In this article, we will describe the feasibility of imaging BC in humans with Cu-64-TP3805 using PET and PEM scanners.

MATERIALS AND METHODS

TP3805 Synthesis and Kit Preparation

Briefly, the PACAP analog with a C-terminal diaminodithiol (N₂S₂) chelator was synthesized (25–29) on a Wang resin using an ABI 341A peptide synthesizer (Applied Biosystems, Foster City, CA). Fmoc-Lys (ivDde) was first introduced at the C-terminus of the peptide, followed by 4-aminobutyric acid (y-Aba). The 27-amino acid PACAP sequence was then assembled by standard Fmoc coupling with the final histidyl residue being a t-Boc protected His(Trt) derivative. The capping t-Boc function was necessary to ensure that the N-terminal amino group remained protected during subsequent deprotection and coupling cycles performed at the y-amino group of the C-terminal lysine. The ivDde group at the C-terminal lysine was then selectively removed with 2% hydrazine, followed by the successive additions of di-Fmoc-L-diaminopropionic acid, and S-benzoylthioglycolic acid. The resulting protected diaminedithiol (NS-benzoyl)₂-containing PACAP peptide was cleaved from the resin using trifluoroacetic acid (TFA) water:phenol:thioanisole/ethanedithiol (82.5:5:5:5:2.5) and precipitated with diethyl ether.

The crude peptide was purified to homogeneity by reverse-phase high pressure liquid chromatography (HPLC) (Waters, Milford, MA) on a Vydac C4 column (5 µm, 10 mm × 250 mm). The mass of the analog-chelator construct was confirmed by electrospray mass spectrometry. Following the general synthetic scheme, TP3805 was prepared, purified and characterized by American Peptide Company, Sunnyvale, CA.

Kits were prepared aseptically in a laminar flow hood. All reagents were sterilized including 10 ml glass vials, rubber caps and aluminum sealing caps. All reagents were analytical grade obtained from Fisher Scientific, Inc. (Fair Lawn, NJ) and used without further purification. The reagents added were 100 µg SnCl₂. 2H₂0 (10 mg/ml, 0.05 M HCl) containing 100 µg glucoheptonate (50 mg/ml, H₂0), 20 µg of TP3805 (10 mg/ml, and 0.1 M Na-acetate, pH-5) and 200 µl of 0.2 M glycine buffer pH-9. The mixture was quickly frozen by placing the vials in an acetone/dry ice bath. The vials were then lyophilized for four hours, (Genevac SF50, Genevac, Berkshire, England), sterile N₂ gas was introduced in the chamber, and the vials were sealed, labeled and stored at 4°C until use. Stability of the kits was checked by HPLC and for their ability to label them with Cu-64.

Preparation of Cu-64-TP3805

On the day of preparation, a kit vial was removed and brought to room temperature. The required quantity of Cu-64 solution (Washington University, St. Louis, MO) was added to the vial (usually 6 mCi in <20 µl of 0.1 M HCl), followed by 200 µl of sterile water. The vial was incubated at 50°C for ninety minutes. The solution was then diluted by the addition of 2 ml sterile 0.9% NaCl.

The reaction mixture was analyzed by HPLC, with reverse phase microbond column (Varian, Inc.) eluted with a linear 28 min gradient from 10% acetonitrile in 0.1% aqueous TFA to 90% acetonitrile in aqueous 0.1% TFA. (Labeling efficiency of 95% or greater was considered as the criterion for kit stability). This rendered the specific activity of Cu-64-TP3805 to be 44.4 GBq (1.2 Ci)/µ mol.

Sterility Test

A required activity for patient injection was drawn into a sterile syringe and measured in a calibrated ionization chamber CRC-15R, (Capintec, Ramsey, NJ). Approximately 100 µl of the solution was added to 10 ml tryptic soy broth (TSB) and incubated for seven days in a humidified 5% CO₂ incubator at 37°C. The test tube was observed daily for seven days to detect turbidity or microbial growth.

Patient Inclusion

Exploratory investigational new drug (eIND) number 101550 was assigned by the Food and Drug Administration. Approval was also obtained from the Institutional Review Board. Clinical Cancer Research Review Committee, and Radioactive Drug Research Committee. Non-pregnant women at least 18 years old and with newly diagnosed, histologically proven BC were enrolled. Image guided percutaneous biopsy was performed 2–6 weeks prior to this imaging procedure. All patients signed the Institutional Review Board–approved informed consent form. For this feasibility study, a Thomas Jefferson University biostatistician determined that 6 patients be studied with PET and 13 with PEM. This determination was based on the assumption that Cu-64-TP3805 will detect 80% of the lesions and that there will be 87% power to conclude that this agent is promising.

PET/CT Imaging

Each patient fasted for six hours prior to F-18-FDG injection. Before F-18-FDG injection, blood glucose was monitored. All patients enrolled had glycaemia levels well below 200 mg/DL. Each patient then received 370 MBq (10 mCi) of F-18-FDG through an indwelling intravenous catheter and one hour later underwent a PET/CT or PEM scan. PET/CT images were obtained in supine position with two minute bed time using a Biograph-6 PET/CT scanner (Siemens, Inc. Knoxville, TN).

For Cu-64-TP3805 imaging, patients neither fasted nor were their blood glycemic levels determined. Cu-64-TP3805 was injected intravenously two to thirty days after the F-18-FDG scan. As suggested by the FDA, 111±10% MBq (3±10% mCi) were given to two patients, 127.5±10% MBq (3.5 mCi±10% MBq) for two patients and 148±10% MBq (4±10% mCi) to the two remaining patients. For Cu-64-TP3805 whole body scans, bed time was four minutes and images were obtained at 2 hr and 4 hr post injection. Four min bed time for Cu-64-TP3805 PET/CT imaging allowed to compensate for the low β+ yield (19%) of Cu-64. Two hr and 4 hr whole body imaging time was chosen to determine the optimal imaging time.

PEM Imaging

For PEM (Solo – I, Naviscan, San Diego, CA), imaging, each patient received 148±10% MBq (4±10% mCi) Cu-64-TP3805 or 10±0.8 mCi F-18-FDG intravenously through an indwelling intravenous catheter. Images were obtained for both breasts in MLO (medial lateral oblique) and CC (cranio caudal) positions for 10 minutes per view. Data were collected at 15 min, 1 hr, 2–3 hr and 5 hr post injection to determine the optimal imaging time.

Vital signs for each of the PET/CT and PEM patients were monitored prior to injection and then for every 30 minutes until 4 hr post injection. At the end of the injection, the syringe and the tubing were flushed with 5 ml 0.9% NaCl and the radioactivity remaining was measured. During the course of the PEM study, patients were allowed to drink or eat if they wished.

Image Analysis

All images were read by two board certified nuclear medicine physicians and a board certified breast imaging physician. Image analysis was performed by a nuclear medicine fellow. For six patients with PET/CT images for both agents, SUV (standard uptake value) was calculated for the primary tumor site and metastatic lymph nodes, and compared for the respective radiotracers. Metabolic tumor volume was calculated using 50% isocontour geometry method for comparison, as it is relatively a better parameter, as compared to SUV, and may lead to detection limit, if any by the new agent.

For the thirteen patients with PEM scans, for both agents, PEM uptake values (PUV) and tumor volumes were calculated for comparison. PEM (Max.) uptake value/background intensity (PUV/BGV) and Mean SUV/PUV ratios were also calculated with each radiotracer and compared for evaluation.

RESULTS

Cu-64-TP3805, Radiochemical Purity and Sterility

For all Cu-64-TP3805 preparations, the radiochemical purity, as determined by HPLC averaged 97±2%. Specific activity of the preparations averaged 44.4 GBq (1.2 Ci)/µmol. All preparations were sterile. Total radioactivity remaining in the syringe and the intravenous line was less than 5.5 MBq (150 µCi).

Patient Population

Patients were recruited in a consecutive order as they consented. None of the patients had received any form of therapy for BC. The average age of the six PET/CT patients was 48.7±6.2 years (range 42–59 years). The average age of the thirteen PEM patients was 54±14.2 years (range 26–80 years). Out of the total of nineteen patients, two patients experienced a flushing sensation that resolved without any medication within minutes. The demographic patient data are given in Table 1 together with critical results.

Table - 1.

Patient Demography and Analysis

Patient No.	Patient Initial	Patient Age	MBq Injected		BC Type	ER/PR/ HER2	Max. SUV/PUV/BGV		Mean SUV/PUV/BGV		Tumor Volume (mm3)
			F-18-FDG	Cu-64- TP3805			F-18-FDG	Cu-64- TP3805	F-18-FDG	Cu-64- TP3805	F-18-FDG	Cu-64- TP3805
PET/CT Patients
1	LK	42	370	110.6	IDC	+/+/−	12.8 LN-1.11.0	6.8 LN-1.4.9	6.5 LN-1.5.5	3.48 LN-1.2.4	6084 LN-1.28	5323 LN-1.28
2	RJ	46	370	107.3	IDC	+/+/−	5.5 LN-1.1.8 LN-2.2.3	7.0 LN-1.2.6 LN-2.2.4	3.1 LN-1.3.1 LN-2.0.89	3.8 LN-1.3.4 LN-2.1.2	5028 LN-1.59 LN-2.38	4714 LN-1.49 LN-2.31
3	CR	49	370	125.8	IDC	+/+/−	2.2	2.1	1.2	0.9	113	113
4	SE	52	370	125.8	IDC	−/−/+	5.2 LN-1.5.2	4.7 LN-1.4	2.8 LN-1.1.3	2.3 LN-1.2.1	2614 LN-1.509	1726 LN-1.402
5	MR	44	370	144.3	IDC	+/+/+	1.75	1.9	0.9	0.9	1385	1018
6	TE	59	370	140.6	IDC	+/+/−	5.0	4.4	2.6	2.7	2617	2651
PEM Patients
1	BW	68	370	144.6	IDC	+/+/?	6.7	6.2	5.2	5.6	509	402
2	SD	57	370	140.6	IDC	+/−/+	9.6	7.9	8.5	6.9	3818	4714
3	RS	80	370	144.6	IDC	+/+/−	1.9	3.0	2.3	2.4	141	98
4	LP	46	370	142.5	ILoC	+/+/−	4.3	3.7	4.9	3.4	509	346
5	CC	39	370	159.1	IDC	+/−/?	5.1	4.7	5.7	4.9	785	1357
6	GG	59	370	162.8	IDC	+/+/?	11.6	11.8	13.6	11.9	7812	6912
7	ST	39	370	144.3	IDC	+/+/−	2.9	2.2	3.4	1.8	3178	2828
8	RB	55	370	151.7	ILoC	+/+/−	3.0 3.1	3.3 3.5	2.6 2.5	2.7 2.7	445 395	549 497
9	RW	52	370	159.1	IPaC	+/−/+	7.3	5.8	6.7	6.3	1847	1357
10	MC	58	370	140.6	IDC	+/+/−	6.2	5.4	5.7	4.8	1436	1767
11	ND	55	370	144.3	IDC	+/−/?	2.6	3.1	2.9	3.3	268	381
12	ΒB	68	370	155.4	IDC	+/+/−	2.7	3.1	2.9	2.9	1150	1767
13	PJ	26	370	151.7	HGMC	+/−/−	4.5	2.7	3.8	2.9	696	1150

BGV---->Background Value

PUV---->PEM Uptake Value

SUV---->Standarized Uptake Value

IDC---->Invasive Ductal Carcinoma

ILoC---->Invasive Lobular Carcinoma

IPaC---->Invasive Papilary Carcinoma

HGMC---->High Grade Mammory Carcinoma

LN---->Lymphnode

Image Analysis

In the whole body PET/CT group, all six patients had histologically proven invasive ductal carcinoma (IDC). Five of these were ER+, one ER−, five PR+, one PR− and two, HER₂+ and four HER₂−. The quality for each image, irrespective of the quantity of Cu-64-TP3805 the patients received, was excellent. Within these patients, there were a total of ten lesions detected both by F-18-FDG PET/CT and Cu-64-TP3805 PET/CT. Out of these, six were primary (one lesion in each patient) and four involved lymph nodes (two in one patient (Fig. 1), and one each in the other two).

FIGURE 1.

A 42 Yr old female with histologically proven IDC, (ER+, PR+, HER₂−) had a positive F-18-FDG PET/CT scan (A) which delineated a primary mass in the right breast and a pair of lymph nodes in the axilla. The same lesions are also clearly visible with her whole body Cu-64-TP3805 PET/CT scan (B), obtained 2 hrs after the administration of 140.6 MBq (3.8 mCi) Cu-64-TP3805 (F-18-FDG equivalent 780 µCi).

In the PEM group of thirteen patients, nine had histologically proven IDC, two had invasive lobular carcinoma (ILoC), one had invasive papillary carcinoma (IPaC) and one had high grade mammary carcinoma (HGMC). Of these, all thirteen were ER+ and none were ER−. Eight patients had PR+ lesions and five had PR−. Of these two were HER₂+, seven HER₂−, and for four, HER₂ status was indeterminate. One patient with ILoC had two distinct lesions. These demographic data are given in Table 1.

The primary tumor volume in these six patents as determined by F-18-FDG PET/CT scans ranged from 113 mm³ to 6084 mm³. The corresponding tumor volume as determined by the Cu-64-TP3805 PET/CT scans ranged from 113 mm³ to 5323 mm³. The Cu-64-TP3805 tumor volume was 90.6±16.1% of that of F-18-FDG. The F-18-FDG lymph node volume range for the four nodes was 28 mm³ to 509 mm³ and for that of Cu-64-TP3805 was 28 mm³ to 402 mm³. The node volume as determined by Cu-64-TP3805 was 86.2±9.2% of that found by the F-18-FDG scan.

All six primary lesions and four malignant lymph nodes were unequivocally detected by Cu-64-TP3805 PET/CT imaging. The F-18-FDG PET/CT SUV (max) values for six primary lesions ranged from 1.75 to 12.8, and for the malignant lymph nodes 1.8 through 11.0. The corresponding Cu-64-TP3805 PUV/BGV values were 1.9 through 11.8 for the primary lesions and 2.4 to 4.9 for the lymph nodes. The Cu-64-TP3805 PET/CT SUV (max) values were 92±26.4% of that of F-18-FDG SUV (max) for the primary lesions. Cu-64-TP3805 PET/CT SUV (max) values for the malignant lymph nodes were 89.8±27% of those for F-18-FDG PET/CT.

The whole body images revealed liver uptake of Cu-64-TP3805 (Fig. 1). This was not quantified. The exact nature of this uptake is unknown. However, our preclinical data had indicated that the liver uptake was 25.4±1.74%, of which nearly 60% of the activity had the same molecular weight as TP3805 and that 7.5% of the activity was excreted in feces within 24 hr post injection (26). This preclinical data suggest that the majority of the liver uptake in patients may be due to intact Cu-64-TP3805 and a portion of this may be eliminated through feces.

For the thirteen PEM patients, there were fourteen primary lesions (two in one patient) all of which were unequivocally delineated by Cu-64-TP3805 (Fig. 2). The tumor volume as determined by F-18-FDG PEM scans ranged from 141 mm³ to 3818 mm³. The tumor volume range, as calculated from the Cu-64-TP3805 PEM scans, was 98 mm³ to 6912 mm³, 113±37% of the F-18-FDG values.

FIGURE 2.

The F-18-FDG PEM scan (A, 370 MBq 10 mCi) of the left breast of the 39 yr old female was obtained 1 hr post injection and the Cu-64-TP3805 PEM scan (B, 159 1 MBq 4.3 mCi, F-18-FDG equivalent 860 µCi) was acquired at 15 min post injection. The PUV/BGV ratios for the lesion (IDC, ER+, PR+, HER₂−) were 2.9 for F-18-FDG and 2.2 (lowest of the series) for Cu-64-TP3805.

In PEM imaging, the ratios of PEM uptake value (PUV) to PEM background value, for F-18-FDG ranged from 2.6 to 11.6 and those for Cu-64-TP3805 from 2.7 – 11.8. This is 97.7±24.5% of the F-18-FDG values.

Although the sample size is small, these data are important as they establish the utility of the Cu-64-TP3805 probe for its future clinical applications in patients with BC. The Cu-64-TP3805 tumor uptake as observed in PEM imaging was rapid. The average PUV/BGV ratio calculated for 13 PEM patients from their 15 min PEM images was 3.1±2.15 and remained steady at 3.2±2.2 for 2–3 hr images and 3.3±2.4 at 5 hr post injection images (Fig. 3). These values were not statistically significantly different (p=>0.5).

FIGURE 3.

One hr, F-18-FDG PEM scan (A, 370 MBq, (10 mCi)) and longitudinal Cu-64-TP3805 PEM scans (B, C, D, E, F 162.8 MBq, (4.4 mCi), F-18-FDG equivalent 880 µCi)) PEM images of a 59 yr old female, obtained at 15 min, 1 hr 52 min, 2 hr 10 min, 3 hr 40 min and 4 hr 47 min) post injection. She had IDC of the left breast (ER+, PR+, HER₂ undeterminant). Cu-64-TP3805 PEM images of the normal right breast (G, H) obtained at 2 hr 20 min and 4 hr 25 min post injection are also given. The F-18-FDG PUV was 11.6 and Cu-64-TP3805 PUV/BGV ratio was 11.9 which remained nearly unchanged for all images (C, D, E, F) obtained at various time points after injection.

The tumor volume by F-18-FDG scans most likely represent a total volume of metabolically active malignant cells and that by the Cu-64-TP3805 scans, the volume of the cells that most likely express VPAC1 receptors. The close congruity of tumor volumes (90.6±16.1% and 113±37%) by the two agents indicate that a significantly large number of metabolically active malignant cells, express VPAC1 receptors and vice versa.

By design, the Cu-64-TP3805 received by all nineteen patients ranged from 107.3 MBq to 162.8 MBq. Given that the positron emission of Cu-64 is only 19%, compared to 97% for F-18-FDG, the effective Cu-64-TP3805 dose ranged between 21 MBq to 33 MBq, less than one tenth of that of the F-18-FDG. Despite this small amount of the tracer, the Cu-64-TP3805 image quality was excellent, both for PET/CT images (Fig. 1) and PEM images (Figs. 2, 3). All malignant lesions (n=20), including the malignant lymph nodes (n=4), were clearly delineated by the Cu-64-TP3805.

Chemical Toxicity and Radiation Exposure

Cu-64 is a rapidly emerging β + radionuclide. It is used in humans for PET imaging (ACRIN 6682, Phase II Trial 2012) (28–30). Cu-64 has a t½ (12.8 hr), long enough to be dispatched throughout the country, but not too long to deliver excessive radiation dose to patients after imaging. Cu-64 is produced in large quantities on small cyclotrons and its chemistry is well known. In our PEM studies, 13 patients received 3.87±0.2% mCi and received an estimated dose of 2.52 mSv to the whole body and 36.5 mSv to the liver (target organ, eIND 101550). Table 2 below shows data for other breast cancer (BC) imaging agents, Cu-64-TP3805, ACRIN study and Ga-67 used in humans since 1970. Data demonstrate that Cu-64-TP3805 radiation exposure is less than induced by the well-established radio-tracers and ACRIN-promoted Cu-64-ATSM (Table 2, (30–34)).

Table 2.

Comparative Radiation Dosimetry

Procedure	Effective Dose	Liver Dose	Reference
Cu-64-TP3805 (4 mCi)	2.5 mSv	36.5 mSv	Gingold E, eIND 101550
FDG-PET/CT (F-18 10 mCi)	31.9 mSv	(5.8 mSv)	(31)
Sestamibi Tc-99m (30 mCi)	9.4 mSv	(6.0 mSv)	(32)
FDG-PET/PEM w/o CT (F-18-10 mCi)	7.8 mSv	(5.8 mSv)	(32)
Ga-67 Citrate (10 mCi)	39 mSv	69 mSv	(33)
Cu-64 ATSM (25 mCi)	33.3 mSv	361 mSv	(34)

Lewis et al (34), demonstrated that metal ions in > 6 times the Cu-64 dose we propose, (same manufacturer) are not toxic. In addition, our data in rabbits receiving a 1,000 × dose (adjusted to body weight) of decayed Cu-64-TP3805 did not i) elevate c-AMP, ii) alter blood chemistry or iii) change liver enzymes. No toxicity was observed in any of the 19 patients who received Cu-64-TP3805, (eIND 101550 (MLT)).

DISCUSSION

During the past few decades, advances in imaging BC have contributed extensively to its early detection. Early detection with mammographic screening is proven to decrease breast cancer mortality. However, mammography as well as other breast imaging modalities all produce many suspicious findings of which only about 20% will actually be malignant but all of which will require biopsy for diagnosis. In the USA each year, an estimated 1.6 million biopsies are performed, over 1.3 million of which result in benign pathology yet these produce patient morbidity and cost billions to the healthcare system (2).

A compelling need therefore exists for a biomolecule that will not only detect BC early, but also distinguish these malignancies from benign lesions accurately and noninvasively. Such a biomolecule could minimize the need for invasive biopsies and could significantly reduce healthcare costs.

We hypothesized that VPAC1 oncogene product overexpressed on all BC cells at the onset of oncogenesis and irrespective of their hormonal status, shall serve as an excellent biomarker for early and accurate detection of BC. Furthermore, since VPAC1 receptors are not overexpressed on cells of normal breast tissue or benign masses, the probe targeting VPAC1 receptors shall image only malignant lesions, but not benign masses. We chose to use Cu-64 because of i) relatively inferior quality of Tc-99m-TP3654 (20), ii) poor resolution of gamma camera that may prevent us from imaging lesions <6 mm in diameter and iii) the Tc-99m supply is in question. Furthermore, for Cu-64, PEM Solo II resolution is 2.4 mm and Clear PEM resolution is 1.3 mm. The modern BC imaging camera, Dilon 6800 resolution is 6 mm and discovery NM 750 b resolution, 3.5 mm (20, 35).

Our PEM image quality with Cu-64-TP3805 is much better than Tc-99m-TP3654 images (20). Thus, the Cu-64 availability, elimination of CT radiation in PET/CT, high PEM resolution and better image quality make a compelling case for using Cu-64-TP3805 for PEM. Our preliminary data in MMTVneu mice that spontaneously grew BC and mimicked the pathophysiology of human BC validated our hypothesis in which we had used Cu-64-TP3805 as the specific probe for PET imaging of primary as well as metastatic BC (26). Furthermore, in this study, Cu-64-TP3805 did not accumulate in lesions that were shown by histology to be non-malignant and did not express VPAC1 receptors by RTPCR (26).

In this present human investigation, designed not as an efficacy study, but merely as a feasibility and safety study, Cu-64-TP3805 imaged all twenty primary malignant tumors in nineteen patients (one patient had two lesions and the remaining eighteen had one each). In addition, in whole body PET/CT imaging, four involved sentinel lymph nodes (two in one patient and one each in the other two) were also delineated clearly. Although all lesions were also delineated by F-18-FDG, in large studies F-18-FDG is known to i) be non-specific, ii) miss up to 30% of BC lesions and iii) does not distinguish benign lesions from malignant masses.

In this small patient study, two other observations were made that are noteworthy. First, that the Cu-64-TP3805 uptake was rapid, 15 min post injection. This suggests in principle that, instead of 12.8 hr half-lived Cu-64, we can use a 68 min half-lived, generator-produced Ga-68. The positron emission of Ga-68 is 88%, more than 4 times greater than that of Cu-64. This will permit us to administer < 150 MBq (~ 4 mCi) of Ga-68 without compromising the image quality, yet significantly reducing radiation burden to the subjects. Images can be obtained 15 min post injection without requiring patient fasting or monitoring their blood glycemic level.

Second, we observed that the PUV/BGV ratios of the 15 min post injection images did not alter significantly for up to 5 hrs of imaging, suggesting that Cu-67-TP3805 could be used as a therapeutic agent without alteration in the chemistry of its preparation. Copper-67 (β- 100%, t½ - 2.44d) is considered as a radionuclide of therapeutic importance (36). Although not yet available commercially, many facilities in the nation are capable of producing it.

From this small feasibility study, it is also reasonable to point out two minor, but important observations. One is the tumor volume congruity (F-18-FDG vs. Cu-64-TP3805) which indicates that most metabolically active malignant cells express VPAC1 receptors and vice versa. The other observation suggests that irrespective of their hormonal status (Table 1) all tumors were unequivocally delineated by Cu-64-TP3805. These need to be substantiated in larger clinical studies.

Because the patient population is small, it is premature to comment on the sensitivity and specificity of Cu-64-TP3805. Nevertheless, Cu-64-TP3805, i) detected all primary lesions (100%), ii) delineated all involved sentinel lymph nodes (100%) and iii) is worthy of further investigation in patients recommended for biopsy for suspicious breast lesions, to evaluate its ability to distinguish benign masses from malignant lesions by PET or PEM Imaging (37).

CONCLUSIONS

There is a compelling need for a biomolecule that will distinguish malignant from benign lesions noninvasively. In this feasibility study, Cu-64-TP3805 which targets VPAC1 oncogene receptors, unequivocally detected all malignant breast lesions by PET/CT and PEM and delineated all involved lymph nodes. Cu-64-TP3805 promises to be such an agent, and warrants further studies in patients to validate the hypothesis that Cu-64-TP3805 will help reduce the number of unnecessary biopsies, minimize patient anxiety and reduce healthcare cost.