10.1245/s10434-006-9275-6
Annals of Surgical Oncology 14:1237-1242 (2007)
© 2007 Society of Surgical Oncology
Expression of Tumor Antigens and Heat-Shock Protein 70 in Breast Cancer Cells After High-Intensity Focused Ultrasound Ablation
Feng Wu, MD, PhD,
Zhi-Biao Wang, MD, PhD,
You-De Cao, PhD,
Qiang Zhou, MD,
Ying Zhang, MD,
Zhong-Lin Xu, MD and
Xue-Qiang Zhu, MD
Clinical Center for Tumor Therapy of 2nd Affiliated Hospital and Institute of Ultrasonic Engineering in Medicine, Chongqing University of Medical Sciences, 1 Medical College Road, Chongqing 400016, China
Correspondence: Address correspondence and reprint requests to: Feng Wu, MD, PhD, HIFU Unit, The Churchill Hospital, Headington, Oxford, OX3 7LJ, UK; E-mail: mfengwu{at}yahoo.com
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ABSTRACT
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Background: Previous results have shown that high-intensity focused ultrasound (HIFU) ablation can potentially activate a host antitumor immunity. The goal of this study was to investigate whether the tumor antigens expressed on breast cancer cells may be preserved after HIFU treatment, and to explore the potential mechanisms regarding the enhanced antitumor response.
Methods: The primary lesion in 23 patients with biopsy-proven breast cancer were treated with HIFU, then submitted to modified radical mastectomy. By using biotin-streptavidin-peroxidase immunohistochemical technology, a variety of cellular molecules expressed on breast cancer cells, including tumor antigens and heat-shock protein 70 (HSP-70), were stained in all breast specimens. A complete absence of staining was recorded as negative, and immunoreaction of the tumor cells was considered to be positive for antigen expression.
Results: Nuclear positivity of breast cancer cells for proliferating cell nuclear antigen, estrogen receptor, and progesterone receptor was detected in 0%, 9%, and 9% of the treated samples, respectively. The positive rate of cytoplasmic staining for matrix metalloproteinase 9, carbohydrate antigen 15–3, vascular endothelial growth factor, transforming growth factors ß1 and ß2, interleukin 6, and interleukin 10 was 0%, 52%, 30%, 57%, 70%, 48%, and 61% in the treated cancer cells, respectively. The positive rate of cellular membrane staining for epithelial membrane antigen, CD44v6, and HSP–70 was 100%, 0%, and 100% in the zones of treated cancer cells, respectively.
Conclusions: After HIFU ablation, some tumor antigens remained in the tumor debris. This could provide a potential antigen source to stimulate antitumor immune response.
Key Words: High-intensity focused ultrasound • Breast cancer • Immune • Tumor antigens • Heat-shock protein • Thermal ablation
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INTRODUCTION
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Selective recognition and destruction of tumor cells by components of the host immune system is a major role of antitumor immunity. To achieve this effect requires tumor antigens to be expressed by tumor cells. Tumor antigens are a variety of proteins that can elicit immune responses specific to the tumor cells, including protective and therapeutic effects. However, in most patients with cancer, the immune system fails to control the development and growth of initial cancer, and to prevent local recurrence and metastasis after conventional therapies. There are several mechanisms by which tumors evade the immune system, including poor tumor antigen processing and presenting, and immunosuppressive cytokines released by the tumor. As a result, either the initial cancer cells are insufficient to stimulate the immune response or the stimulated immune response is unable to prevent the initial establishment of cancer in the patients.
As a noninvasive technique, tumor thermal ablation with high-intensity focused ultrasound (HIFU) energy has received increasing interest for the treatment of localized solid malignancies over the past decade.1 Encouraging clinical results have been recently achieved in patients with solid malignancy, including those of prostate,2 breast,3,4 liver,5 kidney,6 bone,7 and pancreas.8 Recently, long-term follow-up survival data were observed in a phase 3 prospective trial in which HIFU was performed as a breast conservation treatment.4 All patients received chemotherapy, radiation, and tamoxifen after HIFU ablation for primary lesions. Five-year disease-free survival and recurrence-free survival were 95% and 89%, respectively. Two of 22 patients developed local recurrence, and 1 patient died during the follow-up period.
In addition to the thermal effect directly on tumors, previous studies reported some interesting findings that HIFU could potentially activate a host antitumor immunity;9–12 this may be of benefit in micrometastatic control and long-term tumor resistance for patients with cancer. To date, the mechanisms involving antitumor immunity enhancement are still unknown. Large amounts of tumor debris in situ can be released and reabsorbed after HIFU ablation. It is unclear whether ablated tumor debris may contain tumor antigens, and as such whether the remaining debris could be a potential antigen source available for inducing antitumor response. On the basis of our previous results indicating complete coagulation necrosis of breast cancer induced by HIFU treatment,13,14 this study used the same histological samples to investigate whether the tumor antigens expressed on breast cancer cells might remain after HIFU treatment, and to explore the potential mechanisms relating to the enhanced antitumor response.
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MATERIALS AND METHODS
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Patients
Twenty-three female patients with histologically proven breast cancer were enrolled onto a prospective clinical trial. Their average age was 46.5 ± 1.7 years. Tumor size ranged from 2 to 4.7 cm in diameter (mean, 3.1 ± .79 cm). According to the tumor, node, metastasis system of classification, 2 patients (8.7%) were classified as having stage I (T1N0M0) disease, and the remaining 21 patients (91.3%) had stage II (T2N0M0, or T2N1M0) disease. Invasive breast carcinoma and noninvasive breast carcinoma were confirmed in 21 patients (91.3%) and 2 patients (8.7%), respectively.
The ethics committee at our university approved the trial. At the time of enrollment, the patients did not receive any intervention, and they signed an informed consent form, in accordance with the specifications stipulated by the Helsinki Committee.
HIFU Treatment
The Model-JC HIFU therapy system (Chongqing Haifu [HIFU] Tech, Chongqing, China) was used in this study to treat all patients, as described previously.13–16 Therapeutic ultrasound energy is produced by a 12-cm-diameter transducer with a focal length of 90 mm, operating at a frequency of 1.6 MHz. All patients received one session of HIFU treatment for primary breast cancer, and the ablated extent included the breast lesion and 1.5 to 2.0 cm of normal breast tissue surrounding the visible tumor. Acoustic focal peak intensities ranged from 5000 to 15,000 W cm–2. Total therapeutic time excluding anesthesia for the patients ranged from 45 minutes to 2.5 hours (median, 1.3 hours).
Immunohistochemical Staining
Modified radical mastectomy was performed in all patients 1 to 2 weeks after the HIFU treatment. Each breast specimen was immediately submitted to the pathology department. Tissue blocks were sampled from the central and peripheral edges of the treated region for assessing the effect of HIFU ablation on the breast cancer. They were fixed in 10% phosphate-buffered formalin (pH 7) and embedded in paraffin.
Before immunohistochemical staining, the specimens were sectioned in 4-µm-thick slices. By using biotin-streptavidin-peroxidase immunohistochemical technology, a variety of biological markers expressed on breast cancer cells including tumor antigens were stained in all breast specimens. They included proliferating cell nuclear antigen (PCNA), cell adhesion molecule CD44v6, epithelial membrane antigen (EMA), matrix metalloproteinase 9 (MMP–9), estrogen receptor, progesterone receptor, carbohydrate antigen 15–3 (CA15-3), vascular endothelial growth factor, transforming growth factor (TGF) ß1, TGF-ß2, heat-shock protein 70 (HSP-70), interleukin (IL) 6, and IL-10. The primary antibody used for each antigen staining was mouse monoclonal anti-human. In addition to CA15-3 and HSP-70 monoclonal antibodies purchased from Maxim Biotech (San Francisco, CA), we obtained others from Santa Cruz Biotechnology (Santa Cruz, CA).
Sections were incubated at room temperature with each primary antibody, followed by biotinylated second antibody incubation. The chromogen was 3,3-diaminobenzidine tetrahydrochloride (brown). Sections were incubated with phosphate-buffered saline, which served as a negative control instead of the primary antibody. The tumor cells were considered to be positive when there was a homogeneous and clearly visible signal present in tumor cells (brown), and negative when the signal was absent.
Evaluation and Analysis
Immunohistochemical staining was assessed qualitatively for each antigen respectively within each specimen. A complete absence of staining was recorded as negative, and immunoreaction of the tumor cells was considered to be positive for antigen expression.
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RESULTS
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Positive immunostaining for PCNA, estrogen receptor, and progesterone receptor was confined to the nuclei of breast cancer cells; positive staining for EMA, CD44v6, and HSP-70 was confined to the tumor cell membrane; and for MMP-9, CA15-3, vascular endothelial growth factor, TGF-ß1, TGF-ß2, IL-6, and IL-10, staining was predominantly of tumor cell cytoplasm.
Nuclear positivity of breast cancer cells for PCNA, estrogen receptor, and progesterone receptor was detected in 0%, 9%, and 9% of the treated samples, respectively. The positive rate of intense cytoplasmic staining for MMP-9, CA15-3, vascular endothelial growth factor, TGF-ß1, TGF-ß2, IL-6, and IL-10 was 0%, 52%, 30%, 57%, 70%, 48%, and 61% in the treated cancer cells, respectively (Table 1
). The positive rate of EMA, CD44v6, and HSP-70 was 100%, 0%, and 100% in the zones of treated cancer cells, respectively. Correlations among positive immunostaining in nucleus, cytoplasm and cytoplasmic membrane, and clinical stage, histological grade, and lymph node metastasis are listed in Table 2.
Received for publication May 9, 2006.
Accepted for publication October 17, 2006.
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