Abstract
The integrin αvβ3 is a key player in angiogenesis and metastasis. Our aim was to study the uptake patterns of the αvβ3-selective PET tracer 18F-galacto-RGD in invasive ductal breast cancer. Methods: Sixteen patients with primary (n = 12) or metastasized breast cancer (n = 4) were examined with 18F-galacto-RGD PET. Standardized uptake values (SUVs) were derived by region-of-interest analysis, and immunohistochemistry of αvβ3 expression was performed (n = 5). Results: 18F-Galacto-RGD PET identified all invasive carcinomas, with SUVs from 1.4 to 8.7 (mean ± SD, 3.6 ± 1.8; tumor-to-blood and tumor-to-muscle ratios, 2.7 ± 1.6 and 6.2 ± 2.2, respectively). Lymph-node metastases were detected in 3 of 8 patients (mean SUV, 3.3 ± 0.8). SUVs in distant metastases were heterogeneous (2.9 ± 1.4). Immunohistochemistry confirmed αvβ3 expression predominantly on microvessels (5/5) and, to a lesser extent, on tumor cells (3/5). Conclusion: Our results suggest generally elevated and highly variable αvβ3 expression in human breast cancer lesions. Consequently, further imaging studies with 18F-galacto-RGD PET in breast cancer patients for assessment of angiogenesis or planning of αvβ3-targeted therapies are promising.
The treatment of breast cancer nowadays requires an individualized approach and determination of the best therapeutic strategy. The integrin αvβ3 is an interesting target for specific therapies in oncology and as a new prognostic factor, as it is highly expressed on activated endothelial cells during angiogenesis and plays an important role in the regulation of tumor growth, local invasiveness, and metastatic potential (1–3). Specifically in breast cancer, its involvement in metastasis, and especially in migration of tumor cells to the bone, has been demonstrated in preclinical tumor models (4,5). Moreover, anti-αvβ3–targeted drugs, alone or in combination with radioimmunotherapy, have been proven to be effective in breast cancer xenografts (6,7). Histopathologic studies have examined the role of integrin αvβ3 in human breast cancer and have shown contradicting results (8–11). Molecular imaging has the potential to show specific biologic properties of tissues as a whole and also in several tumor sites in the body in one session (12). Therefore, imaging of αvβ3 expression might help to elucidate the complex role of this integrin in human breast cancer patients. We have developed the αvβ3-specific tracer 18F-galacto-RGD for PET (13). It has already been demonstrated that 18F-galacto-RGD PET allows specific imaging of αvβ3 expression and that the uptake of 18F-galacto-RGD correlates with αvβ3 expression in tumor xenografts as well as in patients (14–17). We now report—to our knowledge, for the first time—on the specific use of 18F-galacto-RGD PET in breast cancer patients. The goals of our study were to analyze the uptake patterns of 18F-galacto-RGD in normal breast tissue and in primary as well as metastatic tumor lesions. Our hypothesis was that 18F-galacto-RGD PET allows for identification of αvβ3 expression in primary and metastatic breast cancer lesions with good image quality, which, in consequence, would demonstrate its usefulness for future applications such as noninvasive assessment of angiogenesis and integrin αvβ3 expression in human breast cancer.
MATERIALS AND METHODS
Radiopharmaceutical Preparation
Synthesis of the labeling precursor and subsequent 18F labeling were performed as described previously (18).
Patients
The study was approved by the Ethics Committee of the Technische Universität München, and informed written consent was obtained from all patients. Sixteen patients were included in the study (15 female, 1 male; mean age ± SD, 62.1 ± 11.7 y).
Inclusion criteria consisted of:
Either biopsy-proven or suspected primary breast cancer (Breast Imaging Reporting and Data System [BIRADS] 4 or 5) according to conventional staging (mammography and breast ultrasound, chest radiography, ultrasound of abdomen and bone scintigraphy; additional MRI mammography in 5 patients);
Size of primary lesions scheduled for surgery of at least 1.5 cm in maximum diameter as determined by conventional imaging because of the limited resolution of PET;
Known metastasized breast cancer according to clinical staging (bone scintigraphy and CT in all patients; 18F-FDG PET/CT in 1 patient);
Age > 18 y;
Ability to give written and informed consent.
Exclusion criteria consisted of pregnancy, lactation period, and impaired renal function (serum creatinine level > 1.2 mg/dL).
For details on the patient population, see Table 1.
Patient Data
PET Procedure
Imaging was performed with an ECAT EXACT PET scanner (Siemens Medical Solutions, Inc.) as described previously (17). In each subject, a static emission scan was acquired in the caudocranial direction, beginning on average 63.0 ± 4.1 min (mean ± SD) after injection of 18F-galacto-RGD, covering a field of view at least from the pelvis to the thorax (5–7 bed positions, 5 min per bed position). The imaging time point was chosen according to former results from dynamic and serial static emission scans, showing good tumor-to-background contrast ∼60 min after injection (14).
Image Analysis
Positron emission data were reconstructed using the ordered-subsets expectation maximization (OSEM) algorithm using 8 iterations and 4 subsets. The images were corrected for attenuation using the collected transmission data. For image analysis, CAPP software, version 7.1 (Siemens Medical Solutions, Inc.), was used. The static emission scans were calibrated to standardized uptake values (SUVs).
In the static emission scans, circular regions of interest (ROIs) with a diameter of 1.5 cm were placed over the left ventricle, the forearm (muscle tissue activity), breast tissue on the contralateral side to the tumor-bearing breast, liver, spleen, lung, and tumor tissue in 3 adjacent slices by an experienced operator. Polygonal free-hand ROIs were placed over the intestine in areas with visible tracer uptake in the lower abdomen. Results were expressed in mean SUV. In the tumors, the areas with the maximum intensity were chosen for measurements. In tumors with a diameter of ≤2 cm and visible uptake (n = 4), the SUV might be underestimated because of partial-volume effects. Therefore, the diameter of the ROI was adapted to the tumor size, and the SUV in the ROI was corrected for partial-volume effects using the tumor diameter mentioned in the pathology report and the correction tables established by Brix et al. for our PET scanner by phantom studies (19).
For evaluation of the sensitivity of 18F-galacto-RGD for primary staging, lesions with elevated uptake on 18F-galacto-RGD PET were compared with the results of conventional staging, and histopathologic staging served as the standard of reference. For evaluation of metastatic lesions, the findings of clinical staging served as the standard of reference. A maximum of 10 lesions was evaluated per patient, as 2 patients had disseminated metastatic disease to the liver and bone.
Collection of Tissue Samples and Immunohistochemistry of αvβ3 Expression
The mean time interval between PET and surgery was 2.4 ± 2.3 d. In the operating room, tissue samples from the tumors were obtained from tumor regions with maximum tracer uptake. The specimens were snap-frozen in liquid nitrogen and stored at −70°C until staining was performed.
For immunohistochemical investigation, frozen tumor tissues were sectioned (6 μm) and stained using the biotinylated monoclonal anti-αvβ3 antibody LM609 (1:100; Chemicon Europe). Sections were processed by peroxidase staining (peroxidase substrate AEC [3-amino-9-ethylcarbazole] kit; Vector Laboratories).
Analysis of staining of microvessel and tumor cells was performed by a senior pathologist, who was unaware of the results of the SUV measurements.
Statistical Analysis
All quantitative data are expressed as mean ± 1 SD. The correlation between quantitative parameters was evaluated by linear regression analysis and by calculation of the Pearson correlation coefficient R. Statistical significance was tested by using ANOVA. All statistical tests were performed at the 5% level of statistical significance, using MedCalc statistical software (version 6.15.000).
RESULTS
Static Emission Scans
During the observation period of ∼3 h after tracer injection until the end of scanning, no adverse reactions were noted in our patient population.
The results of the SUV measurements are summarized in Figure 1. The mean tumor-to-blood ratio was 2.9 ± 1.7, and the mean tumor-to-muscle ratio was 6.6 ± 1.9. The SUVs of the primary tumors (n = 12) did not correlate with the tumor diameter (r = 0.116, P = 0.719).
Box plot diagram of mean SUVs of tumors and normal tissue (breast, muscle, blood pool, liver, spleen, lungs, intestine) from static emission scans. Tumor uptake was very heterogeneous but higher than uptake in background tissue, resulting in good image contrast.
Comparison with Clinical Staging
In the static emissions scans, all primary tumors could be identified (n = 12/12). No false-positive findings occurred in the tumor-bearing or the contralateral breast. One patient was scanned after completion of neoadjuvant chemotherapy with no residual viable tumor cells in histology (ypT0) and no elevated signal on the 18F-galacto-RGD PET scan. One patient with a suggestive finding according to mammography and MRI mammography (BIRADS 4) showed no elevated uptake on the 18F-galacto-RGD PET scan, and no tumor was found on histopathology. Lymph-node metastases were detected in 3 of 8 patients with a mean SUV of 3.3 ± 0.8. Although the mean size of the missed metastases was small—5.8 ± 7.2 mm—not only microscopic but also macroscopic lymph-node metastases were missed (size, 15 and 20 mm). Eleven of 24 evaluated distant metastases were detected by 18F-galacto-RGD PET and SUVs were heterogeneous (mean, 2.9 ± 1.4; range, 1.0–3.8). Osseous metastases were depicted successfully by 18F-galacto-RGD PET on a per-patient basis (n = 4); however, some osseous metastases in the same patients did not show tracer uptake. Also, multiple metastases to the liver were not identified in 1 patient. One small lung lesion was detected by 18F-galacto-RGD PET, which was not detected by conventional staging, and which was confirmed by CT (Fig. 2).
A 70-y-old patient with invasive ductal breast cancer of left breast (arrow, open tip), axillary lymph-node metastases on left side (arrow, open tip, dotted line), an osseous metastasis to the sternum (arrow, closed tip), and a pulmonary metastasis on right side (arrow, closed tip, dotted line). Maximum-intensity projection of 18F-galacto-RGD PET (A) and planar images (B, axial plane; C, coronal plane; D, axial plane) show primary tumor, lymph-node metastases, and osseous metastasis with good tumor-to-background contrast. High tracer retention is notable in urogenital tract, predominantly due to renal tracer elimination. Intermediate uptake is notable in liver, spleen, and intestine. One small pulmonary lesion was notable in nonattenuation–corrected images (E) and confirmed by CT (F). In the context of metastasized breast cancer, this was considered to be highly suggestive of a pulmonary metastasis.
Immunohistochemistry of αvβ3 Expression
αvβ3 expression could be confirmed by immunohistochemistry in all tumor specimens. αvβ3 expression was identified predominantly on the endothelium of newly formed blood vessels. In 3 specimens, αvβ3 expression could also be seen on the tumor cells themselves in addition to staining of the neovasculature (Fig. 3).
Comparison of 18F-galacto-RGD PET (A and D) and immunohistochemistry of αvβ3 expression (B, C, E, and F). (Upper row) Patient with large invasive ductal carcinoma of left breast and low 18F-galacto-RGD uptake (A, arrow). Corresponding immunohistochemistry shows negative staining of most parts of tumor (C, low magnification) and only faint positive staining of single vessels (D, high magnification; arrow). (Lower row) Patient with invasive ductal carcinoma of left breast and intense 18F-galacto-RGD uptake (D, arrow). Corresponding immunohistochemistry (E, low magnification; F, high magnification) shows intense staining of tumor vessels (arrow).
DISCUSSION
In this study we could demonstrate elevated but highly variable αvβ3 expression in primary and metastatic breast cancer by using 18F-galacto-RGD and PET, with the integrin being expressed predominantly on endothelial cells and, to a lesser extent, on tumor cells. 18F-Galacto-RGD PET performed well for primary tumor detection but did not show sufficient sensitivity for lymph-node staging.
Our results demonstrated 18F-galacto-RGD uptake in all primary tumor lesions, which was very heterogeneous and did not depend on tumor size. Tracer uptake in metastases also was very heterogeneous. This suggests generally elevated but widely varying levels of αvβ3 expression in human breast cancer. This information contributes to the growing literature about integrin expression in human breast cancer, as results from former histopathologic studies were contradictory (8,11). Up to now, only 1 other study has examined αvβ3 expression in humans with imaging methods using the αvβ3-specific SPECT (single-photon CT) tracer 99mTc-NC100692 (20). Our results illustrate that noninvasive imaging of αvβ3 expression with PET is feasible with good quality in breast cancer patients. However, the small number of patients studied is a limitation, and further prospective studies using 18F-galacto-RGD in a larger patient population to evaluate the biologic and clinical significance of this tracer in more detail are warranted.
The immunohistochemistry of αvβ3 expression in primary tumors in our study revealed αvβ3 expression predominantly on endothelial cells as well as on tumor cells. Therefore, the signal from 18F-galacto-RGD PET represents a mixture of tracer binding on neovasculature and on tumor cells. This is in accordance with clinical as well as preclinical reports that describe αvβ3 expression on neovasculature and on breast cancer cells (8). Consequently, αvβ3 imaging for assessment of angiogenesis alone should be interpreted carefully in the context of breast cancer as part of the signal might be attributed to tumor cells and not to neovasculature alone. Quantification of αvβ3 expression in tumor specimens and correlation with tracer uptake was not performed because of the low number of samples, which is a limitation of our study. However, a significant correlation of αvβ3 expression in immunohistochemistry and 18F-galacto-RGD uptake has been demonstrated previously in murine tumor models as well as in patients using static emission scans such as in the current study (16,17).
18F-Galacto-RGD PET also showed good results relating to the identification of primary lesions with no false-positive results in breast cancer. However, it must be noted that only lesions with a diameter of 15 mm or larger were included in our study, because visualization of smaller lesions might be problematic due to the limited resolution of a clinical PET scanner, which is in the range of 5–6 mm (12). Moreover, primarily invasive ductal carcinomas were examined in our study, so no conclusions can be drawn about the efficacy of 18F-galacto-RGD PET for imaging of lobular carcinoma. The sensitivity for lymph-node metastases or distant metastases in our small patient sample was not adequate for use in N or M staging in clinical routine. This still must be evaluated in more detail in future comparative studies.
CONCLUSION
Imaging of αvβ3 expression in invasive ductal breast cancer with 18F-galacto-RGD PET is possible with good image quality, with αvβ3 expression being elevated but highly variable in primary tumors as well as in metastases.
Acknowledgments
We thank the Cyclotron and PET team—especially Michael Herz, Gitti Dzewas, Coletta Kruschke, and Nicola Henke for excellent technical assistance—and the Münchner Medizinische Wochenschrift and the Sander Foundation for financial support.
Footnotes
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COPYRIGHT © 2008 by the Society of Nuclear Medicine, Inc.
References
- Received for publication July 20, 2007.
- Accepted for publication October 12, 2007.
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