64Cu-DOTA-trastuzumab PET imaging and HER2 specificity of brain metastases in HER2-positive breast cancer patients
- Hiroaki Kurihara1Email author,
- Akinobu Hamada2,
- Masayuki Yoshida3,
- Schuichi Shimma2,
- Jun Hashimoto4,
- Kan Yonemori4,
- Hitomi Tani1,
- Yasuji Miyakita5,
- Yousuke Kanayama6,
- Yasuhiro Wada6,
- Makoto Kodaira4,
- Mayu Yunokawa4,
- Harukaze Yamamoto4,
- Chikako Shimizu4,
- Kazuhiro Takahashi6,
- Yasuyoshi Watanabe6,
- Yasuhiro Fujiwara4 and
- Kenji Tamura4
© Kurihara et al.; licensee Springer. 2015
Received: 24 October 2014
Accepted: 5 January 2015
Published: 12 March 2015
The purpose of this study was to determine whether brain metastases from HER2-positive breast cancer could be detected noninvasively using positron emission tomography (PET) with 64Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-trastuzumab.
PET was performed on five patients with brain metastases from HER2-positive breast cancer, at 24 or 48 h after the injection of approximately 130 MBq of the probe 64Cu-DOTA-trastuzumab. Radioactivity in metastatic brain tumors was evaluated based on PET images in five patients. Autoradiography, immunohistochemistry (IHC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were performed in one surgical case to confirm HER2 specificity of 64Cu-DOTA-trastuzumab.
Metastatic brain lesions could be visualized by 64Cu-DOTA-trastuzumab PET in all of five cases, which might indicated that trastuzumab passes through the blood-brain barrier (BBB). The HER2 specificity of 64Cu-DOTA-trastuzumab was demonstrated in one patient by autoradiography, immunohistochemistry, and LC-MS/MS.
Cu-DOTA-trastuzumab PET could be a potential noninvasive procedure for serial identification of metastatic brain lesions in patients with HER2-positive breast cancer.
KeywordsHER2-positive breast cancer Trastuzumab PET 64Cu Brain metastasis
Many novel molecular targets for anticancer treatment have been discovered. Targeting of HER2 with the monoclonal antibody trastuzumab is a well-established therapeutic strategy for HER2-positive breast cancer in neoadjuvant , adjuvant , and metastatic settings . Thus, it is important to examine HER2 expression in primary tumor and metastatic sites to determine whether anti-HER2 therapy is indicated.
Although HER2 expression is routinely determined using immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) , technical problems can arise when lesions are not easily accessible by core needle biopsy . In addition, HER2 expression can vary during the course of the disease  and even among tumor lesions in the same patient . To overcome these problems, a novel molecular imaging technique using positron emission tomography (PET) with 124I-, 89Zr-, or 64Cu-labeled antibodies has been studied for the noninvasive evaluation of HER2 expression [8-10].
In a previous study, we reported the production of 64Cu-labeled trastuzumab, specifically 64Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-trastuzumab and that 64Cu-DOTA-trastuzumab PET imaging could detect primary HER2-positive breast cancer and metastatic lesions . This imaging technique can be used to serially monitor HER2 tumor status during HER2-targeting treatment and also to evaluate patients with metastatic brain tumors that are not easily accessible by core needle biopsy.
In this study, we demonstrated that 64Cu-DOTA-trastuzumab PET imaging could visualize metastatic brain lesions and confirmed the HER2 specificity of 64Cu-DOTA-trastuzumab by means of autoradiography, IHC, and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The patients included in this study had histologically confirmed invasive HER2-positive (IHC 3+ or FISH-positive) breast carcinoma with at least one site of measurable brain metastasis, Eastern Cooperative Oncology Group performance status (PS) of 0 to 1, adequate organ function (neutrophil count ≥1,500/μL, platelet count ≥75,000/μL, hemoglobin concentration ≥9.0 g/dL, serum bilirubin ≤1.5 mg/dL, AST and ALT ≤100 IU/L, serum creatinine ≤1.5 mg/dL, baseline left ventricular ejection fraction (LVEF) >60%) and were aged between 20 and 75 years. The main exclusion criteria were congestive heart failure, uncontrolled angina pectoris, arrhythmia, symptomatic infectious disease, severe bleeding, pulmonary fibrosis, obstructive bowel disease or severe diarrhea, and symptomatic peripheral or cardiac effusion.
Preparation of 64Cu-DOTA-trastuzumab and PET/CT protocol
64Cu-DOTA-trastuzumab was prepared as described previously . Briefly, the 64Ni (p, n) 64Cu nuclear reaction was performed with 12-MeV proton irradiation using a small medical cyclotron (HM-12S, Sumitomo Heavy Industries Ltd., Tokyo, Japan). The beam current used was approximately 20 μA (3 h). After purification of trastuzumab IgG (Herceptin®; Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) by ultrafiltration (Amicon Ultra 0.5 mL 50 k) with phosphate-buffered saline (PBS), the trastuzumab in PBS was added to DOTA mono N-hydroxysuccinimide ester (Macrocyclics Inc., Dallas, TX, USA) and dissolved in water. After incubation at 40°C for 3 h, crude DOTA-trastuzumab was purified with PBS by using a PD-10 column. The PBS buffer, including DOTA-trastuzumab (100 μg, 0.7 nmol), was exchanged for a sodium acetate buffer (100 mM, pH 6.5) by filtration. 64Cu-DOTA-trastuzumab was prepared by adding 64CuCl2 to the acetate buffer solution containing DOTA-trastuzumab and incubating the solution for 1 h at 40°C. The reaction mixture was sterilized by filtration through a 0.22-μm Millex GV filter (Merck Millipore, Billerica, MA, USA). The radiolabeling results revealed that the specific activity and radiochemical purity were approximately 350 GBq/μmol and 98%, respectively. Approximately 500 MBq of the final product was obtained by a single radiosynthesis.
PET/CT studies were performed at 1, 24, and 48 h after 64Cu-DOTA-trastuzumab injection with a Discovery 600 (GE Healthcare, Pewaukee, WI, USA), as described previously . PET image evaluation and quantification of the maximum single-voxel standardized uptake value (SUVmax) were performed using AW Volume Share 4.5 software (GE Healthcare). Regions of interest (ROIs) were delineated within the tumor on the PET/CT images, and the SUVmax was determined. For the background uptake, ROIs were placed within the opposite side of normal brain, and the SUVmax was measured.
Autoradiography, IHC, and LC-MS/MS
One patient (patient no. 5), who had a solitary brain metastasis in the left cerebellum and had planned to undergo surgery, agreed to participate in this study. 64Cu-DOTA-trastuzumab injection was performed 1 day prior to PET imaging and subsequent surgical resection. A 20-μm-thick frozen section was immediately prepared from the tumor specimen and used as a sample for autoradiography and IHC, and a 10-μm-thick frozen section was prepared for LC-MS/MS analysis. The residual tissue was fixed and used for routine histopathological diagnosis.
For autoradiography, the frozen section was placed on an imaging plate (BAS IP SR 2025, GE healthcare) for 48 h at 4°C to detect the location of radioactivity in the specimen, and the exposed imaging plate then was read with a FLA 9000 laser scanner system (GE Healthcare). IHC staining was also performed on the cryosection adjacent to that used for autoradiography to assess the spatial distribution of 64Cu-DOTA-trastuzumab. The HercepTest™ kit (DAKO, Glostrup, Denmark) was used for detection of HER2-positive tumor cells. Digitization and registration of corresponding images from the autoradiogram and IHC were performed using a microcomputer system and ImageQuant TL Analysis Toolbox software (GE Healthcare).
All chemical reagents were obtained from commercial sources. This study was conducted according to a protocol approved by the institutional review board and independent ethics committee of the National Cancer Center Hospital. All patients signed a written informed consent form.
Results and discussion
History of trastuzumab treatment
Number of lesions ( φ <1 cm) detected by MRI/CT/ 64 Cu
Number of lesions ( φ >1 cm) detected by MRI/CT/ 64 Cu
The mean and standard deviation of the administrated mass of 64Cu-DOTA-trastuzumab was 74.4 ± 6.8 μg (range, 67.8 to 80.6 μg). The mean administered activity was 141 ± 8 MBq (range, 133 to 148 MBq).
The administration of 64Cu-DOTA-trastuzumab was well tolerated by all subjects. No infusion- or drug-related adverse events were reported in any of the patients. No clinically important trends indicative of safety issues were noted in the laboratory parameters, vital signs, or electrocardiogram parameters.
Accumulation of 64 Cu-DOTA-trastuzumab in brain tumor
The balance between the uptake in the tumor, blood clearance of injected 64Cu-DOTA-trastuzumab, and the radioactive decay of 64Cu indicate the plausible imaging time of 48 h after the injection. Compared to an optimal imaging time of 4 to 5 days after 89Zr-trastuzumab injection , imaging time of 48 h after 64Cu-DOTA-trastuzumab injection seems to be more acceptable in clinical practice. Because of its relatively longer half-life, 89Zn-trastuzumab provides clearer images as the patient can be imaged at longer time points; however, it induces higher radiation exposure. On the other hand, the shorter half-life of 64Cu induces lower radiation exposure; however, it provides images with non-specific activity in the blood . One possible approach to decrease the high uptake of the probe by the liver could be to use cold trastuzumab with 64Cu-DOTA-trastuzumab injection. However, adequate interval from pre-dosing of the cold trastuzumab should be determined in the future study.
HER2 specificity of 64Cu-DOTA-trastuzumab in a human subject
This study accumulated safety data and demonstrated clinical cases of brain metastases visualized by 64Cu-DOTA-trastuzumab PET imaging. We confirmed the HER2 specificity of 64Cu-DOTA-trastuzumab by autoradiography, IHC, and LC-MS/MS in one case of metastatic brain tumor resection.
In clinical settings, it is not possible to obtain metastatic brain tissue samples without surgery. However, brain metastasis is documented in 10% to 16% of patients with metastatic breast cancer during the course of their disease [11,12] and in 40% of patients during the course of herceptin treatment . The IHC profiles of metastatic brain tumors from breast cancer have been reported to differ from the primary site in a certain frequency . Some cases of HER2-positive breast cancer may have HER2-negative brain metastasis, and the converse is also true. Furthermore, since brain metastases generally occur late in the course of metastatic breast cancer, one result of the improving overall survival rate in these patients is that the incidence of brain metastasis will likely increase . Knowing the HER2 status of a metastatic brain tumor would assist in identifying the best additional systemic treatment. 64Cu-DOTA-trastuzumab PET imaging is a potential solution for noninvasively and serially evaluating brain tumor HER2 status.
Although it is generally considered that trastuzumab poorly penetrates the blood-brain barrier (BBB), we were able to visualize brain lesions in this study. Therapeutic antibodies such as trastuzumab are thought to be too large or hydrophilic to cross the BBB [16,17]. In addition, penetration of the tumor by monoclonal antibodies may be hindered by increased intratumoral interstitial pressure [18-20]. In this study, however, high CNS penetration of 64Cu-DOTA-trastuzumab was demonstrated by LC-MS/MS with an 11-fold higher CDR analyte count in the tumor cell region than the non-tumor cell region and by PET imaging with a TNR of 5 to 12 at 48 h after injection. Another study using 89Zr-trastuzumab PET imaging also demonstrated CNS penetration in patients with metastatic brain tumors, with an 18-fold higher uptake in brain tumors than in normal brain tissue at 4 to 7 days after injection . Some researchers have reported that this increased penetration is likely due to disruption or compromise of the BBB at the site of brain metastasis by the metastatic tumor itself or by cancer therapies such as radiotherapy [21,22], though one report suggested that the BBB in HER2-positive tumors was disrupted less than in triple-negative or basal-type breast cancer . Another potential mechanism for transporting trastuzumab across the BBB is the FCR for immunoglobulin G, which expressed on vessels in the brain . However, further studies are needed to clarify the mechanisms involved in transportation across the BBB in tumor sites.
PET imaging with 64Cu-DOTA-trastuzumab has the potential to characterize HER2 status using a potent quantitative biomarker that can predict the biological effect of anti-HER2 antibodies. This information might help clinicians determine the optimal HER2 inhibitor therapy for each individual. In this study there was high accumulation in the normal liver. The SUVmax in the liver at 24 h and 48 h post-injection ranged 5.1 to 8.2 and 5.2 to 8.0, respectively, which may interrupt to detect occult liver metastasis. The high accumulation in normal liver is likely due to high expression of FCR on liver sinusoidal endothelial cell . Another research group reported that 50 mg of trastuzumab reduced liver uptake of 64Cu-DOTA-trastuzumab in HER2-positive metastatic breast cancer . These findings demonstrate that 64Cu-DOTA-trastuzumab PET/CT might be applicable not only for breast cancer, but for other kinds of HER2-positive malignancies as well.
In this study, HER2 specificity of 64Cu-DOTA-trastuzumab was demonstrated for one case. The HER2-positive status of the tumor was confirmed by IHC, high radioactivity was shown in the area of HER2-positive tumor cells by autoradiography, LC-MS/MS revealed an 11-fold higher trastuzumab CDR amount in the tumor cell region than in the non-tumor region, and the average TNR of 64Cu-DOTA-trastuzumab PET imaging at 48 h was 9.8 ± 3.3. These results strongly suggest the HER2 specificity of 64Cu-DOTA-trastuzumab imaging. However, for the other four cases, we could not confirm the HER2 status of metastatic brain tumors because they did not undergo surgery. This is a potential weakness of our study, but in general, it is difficult to sample metastatic brain tumors by surgical operation because patients with HER2-positive breast cancer and metastatic brain tumors are usually treated with systemic chemotherapy. To confirm HER2 specificity of 64Cu-DOTA-trastuzumab PET imaging in humans, more cases of both HER2-positive and HER2-negative tumors are required, along with further study comparing the results of PET imaging with HER2 status.
64Cu-DOTA-trastuzumab PET imaging was safe and feasible for outpatients. This technique can be used to visualize metastatic brain lesions in HER2-positive breast cancer patients. HER2 specificity of 64Cu-DOTA-trastuzumab was demonstrated in a surgical resection case by means of autoradiography, IHC, and LC-MS/MS. To evaluate, 64Cu-DOTA-trastuzumab is a promising candidate for the noninvasive and serial evaluation of HER2 status in metastatic brain tumors.
fluorescence in situ hybridization
hematoxylin and eosin
laser capture micro-dissection system
liquid chromatography-tandem mass spectrometry
left ventricular ejection fraction
magnetic resonance imaging
positron emission tomography
Eastern Cooperative Oncology Group performance status
regions of interest
maximum single-voxel standardized uptake value
tumor-to-normal tissue count ratios
The authors thank Mr. Yuzuru Kono, Mr. Kenta Hiroi, and Mr. Natsuki Honda for their technical support. The authors thank Mr. Takayuki Namma and the staff of Sumitomo Heavy Industries for supporting 64Cu-DOTA-trastuzumab production, and Mr. Akira Hirayama and the staff of GE Healthcare for optimizing PET/CT scan. We also thank Ms. Nao Nakamura and Ms. Rumi Koyama for secretarial support during this study. Finally, we thank all the study participants and patients who provided tissue samples for this analysis. This work was supported by a consignment expense for the Japan Advanced Molecular Imaging Program (JAMP) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japanese Government [10103935 to K.T.].
- Buzdar AU, Ibrahim NK, Francis D, Booser DJ, Thomas ES, Theriault RL, et al. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2–positive operable breast cancer. J Clin Oncol. 2005;23:3676–85.View ArticlePubMedGoogle Scholar
- Romond EH, Perez EA, Bryant J, Suman VJ, Geyer Jr CE, Davidson NE, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005;353:1673–84.View ArticlePubMedGoogle Scholar
- Tripathy D, Slamon DJ, Cobleigh M, Arnold A, Saleh M, Mortimer JE, et al. Safety of treatment of metastatic breast cancer with trastuzumab beyond disease progression. J Clin Oncol. 2004;22:1063–70.View ArticlePubMedGoogle Scholar
- Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol. 2009;27:1323–33.View ArticlePubMedGoogle Scholar
- Lebeau A, Turzynski A, Braun S, Behrhof W, Fleige B, Schmitt WD, et al. Reliability of human epidermal growth factor receptor 2 immunohistochemistry in breast core needle biopsies. J Clin Oncol. 2010;28:3264–70.View ArticlePubMedGoogle Scholar
- Xiao C, Gong Y, Han EY, Gonzalez-Angulo AM, Sneige N. Stability of HER2-positive status in breast carcinoma: a comparison between primary and paired metastatic tumors with regard to the possible impact of intervening trastuzumab treatment. Ann Oncol. 2011;22:1547–53.View ArticlePubMedGoogle Scholar
- Houssami N, Macaskill P, Balleine RL, Bilous M, Pegram MD. HER2 discordance between primary breast cancer and its paired metastasis: tumor biology or test artifact? Insights through meta-analysis. Breast Cancer Res Treat. 2011;129:659–74.View ArticlePubMedGoogle Scholar
- Robinson MK, Doss M, Shaller C, Narayanan D, Marks JD, Adler LP, et al. Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti- HER2 diabody. Cancer Res. 2005;65:1471–8.View ArticlePubMedGoogle Scholar
- Dijkers EC, Oude Munnink TH, Kosterink JG, Brouwers AH, Jager PL, de Jong JR, et al. Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther. 2010;87:586–92.View ArticlePubMedGoogle Scholar
- Tamura K, Kurihara H, Yonemori K, Tsuda H, Suzuki J, Kono Y, et al. 64Cu-DOTA-trastuzumab PET imaging in patients with HER2-positive breast cancer. J Nucl Med. 2013;54:1869–75.View ArticlePubMedGoogle Scholar
- Miller KD, Weathers T, Haney LG, Timmerman R, Dickler M, Shen J, et al. Occult central nervous system involvement in patients with metastatic breast cancer: prevalence, predictive factors and impact on overall survival. Ann Oncol. 2003;14:1072–7.View ArticlePubMedGoogle Scholar
- Lin NU, Bellon JR, Winer EP. CNS metastases in breast cancer. J Clin Oncol. 2004;22:3608–17.View ArticlePubMedGoogle Scholar
- Ono M, Ando M, Yunokawa M, Nakano E, Yonemori K, Matsumoto K, et al. Brain metastases in patients who receive trastuzumab-containing chemotherapy for HER2-overexpressing metastatic breast cancer. Int J Clin Oncol. 2009;14:48–52.View ArticlePubMedGoogle Scholar
- Yonemori K, Tsuta K, Shimizu C, Hatanaka Y, Hashizume K, Ono M, et al. Immunohistochemical profiles of brain metastases from breast cancer. J Neurooncol. 2008;90:223–8.View ArticlePubMedGoogle Scholar
- Leyland-Jones B. Human epidermal growth factor receptor 2-positive breast cancer and central nervous system metastases. J Clin Oncol. 2009;27:5278–86.View ArticlePubMedGoogle Scholar
- Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive and therapeutic targets. Nat Rev Cancer. 2011;11:352–63.View ArticlePubMedGoogle Scholar
- Eichler AF, Loeffler JS. Multidisciplinary management of brain metastases. Oncologist. 2007;12:884–98.View ArticlePubMedGoogle Scholar
- Jain RK. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res. 1990;50 Suppl 3:814–9.Google Scholar
- Weinstein JN, Eger RR, Covell DG, Black CD, Mulshine J, Carrasquillo JA, et al. The pharmacology of monoclonal antibodies. Ann NY Acad Sci. 1987;507:199–210.View ArticlePubMedGoogle Scholar
- Fujimori K, Covell DG, Fletcher JE, Weinstein JN. A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med. 1990;31:1191–8.PubMedGoogle Scholar
- Stemmler HJ, Schmitt M, Willems A, Bernhard H, Harbeck N, Heinemann V. Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood–brain barrier. Anticancer Drugs. 2007;18:23–8.View ArticlePubMedGoogle Scholar
- Mehta AI, Brufsky AM, Sampson JH. Therapeutic approaches for HER2-positive brain metastases: circumventing the blood-brain barrier. Cancer Treat Rev. 2013;39:261–9.View ArticlePubMedGoogle Scholar
- Yonemori K, Tsuta K, Ono M, Shimizu C, Hirakawa A, Hasegawa T, et al. Disruption of the blood brain barrier by brain metastases of triple-negative and basal-type breast cancer but not HER2/neu-positive breast cancer. Cancer. 2010;116:302–8.View ArticlePubMedGoogle Scholar
- Lampson LA. Monoclonal antibodies in neuro-oncology: getting past the blood–brain barrier. MAbs. 2011;3:153–60.View ArticlePubMed CentralPubMedGoogle Scholar
- Muro H, Shirasawa H, Maeda M, Nakamura S. Fc receptors of liver sinusoidal endothelium in normal rats and humans. A histologic study with soluble immune complexes. Gastroenterology. 1987;93:1078–85.PubMedGoogle Scholar
- Mortimer JE, Bading JR, Colcher DM, Conti PS, Frankel PH, Carroll MI, et al. Functional imaging of human epidermal growth factor receptor 2–positive metastatic breast cancer using 64Cu-DOTA-trastuzumab PET. J Nucl Med. 2014;55:23–9.View ArticlePubMed CentralPubMedGoogle Scholar
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