Abstract
Background: Tissue microarray (TMA) allows the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion. This study was designed to analyze the cortactin status in breast cancer using TMA and to investigate the relationship of cortactin status to breast cancer biology. Patients and Methods: Archival tissue specimens from 99 patients with primary invasive breast cancer were selected. The cortactin expression was analyzed by TMA. Age, estrogen receptor status, histological grading and TNM staging data were also collected. Results: There were 23 patients (23.2%) with low (+) expression of cortactin, 60 patients (60.6%) with intermediate (++) expression and 16(16.2%) with strong (+++) expression. There was no significant relationship between cortactin expression and age, histological grading, primary tumor staging, lymph node status, estrogen receptor and TNM stage. By multivariate analysis, estrogen receptor status and TNM staging were found to be significantly related to the overall five-year survival rate. Conclusion: Cortactin expression failed to demonstrate a prognostic value for patients with breast cancer.
Cortactin was initially identified as one of the major substrates for src kinase (1). It is localized on the cortical actin structure (2), hence its name. Originally, little was known about its function, except that it bound to actin filaments, had an SH3 domain and was phosphorylated in its C-terminus by src kinase (2). Later, the cortactin gene was found to be identical to EMS1 (3), a gene that is frequently overexpressed in human carcinomas due to its presence in the 11q13 amplicon (4). Recent studies have shown that enhancement of cell motility and loss of cell–cell adhesion is essential for tumor progression (5-7). Cortactin is one of the important components among these actin cross-linking proteins (8, 9). Uncovering possible mechanisms promoting tumor cell invasion may be helpful to the design of future new therapeutic options for human malignancy.
Breast cancer is a clinically heterogeneous disease that may affect patients with similar clinicopathological manifestations differently (10, 11). This clinical heterogeneity may be attributed largely to different gene expression within tumors. Nowadays, the identification of gene expressions of tumors is feasible and the resulting expression profiles may be used to design a therapeutic plan as well as providing prognostic information.
The creation of tissue microarray (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion with minimal damage to the original samples (12, 13).
This study was designed to analyze the cortactin status in breast cancer using TMA, with the aim of elucidating the possible relationship between cortactin expression and breast cancer.
Patients and Methods
Specimen selection and data collection. Archival tissue specimens from 99 patients diagnosed with primary invasive breast cancer Kaohsiung between January 1994 and December 1998 were selected from the pathology files of the Chang Gung Memorial Hospital at Kaohsiung. All the patients except those at stage VI underwent modified radical mastectomy due to invasive breast cancer, defined as carcinoma with invasion to or beyond the basement membrane, regardless of histological classification (ductal or lobular) (14). Data regarding primary tumor staging, age, estrogen receptor status (15-21), lymph node status, histological grading and TNM staging were also collected. The hematoxylin-eosin-stained slides of the paraffin-embedded tumor specimens were reviewed by the pathologists of the research group to confirm the accuracy of the histological diagnoses and lymph node status.
TMA assembly. The representative areas of both tumor and non-tumor parts for each case were selected and circled to match the blocks for the TMA analysis. Then the blocks matching the circled slides were retrieved to prepare the recipient block for the TMA analysis. To ensure the representation of the selected cores, three areas, each for both tumor and non-tumor parts for each patient, were determined for the assembly of the recipient blocks. Each target area on the selected blocks was punched to form a 0.6-mm-diameter tissue core and placed consecutively on the recipient blocks of approximately 3 cm × 2 cm with a precision instrument (Beecher Instruments, Silver Spring, MD, USA), as described elsewhere (22).
Immunohistochemical analysis. Rabbit polyclonal antibody against cortactin (ab51073) was obtained from Abcam plc (Cambridge, UK) and was diluted 1:100 in phosphate-buffered saline (PBS). Five-μm thick sections were cut from the recipient blocks of the TMA, incubated overnight in an oven at 37°C, dewaxed in xylene and dehydrated in a series of graded alcohols. The sections were then treated with 3% hydrogen peroxide for ten minutes to deprive the endogenous peroxidase activity and microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. After antigen retrieval, the sections were incubated with diluted cortactin antibody for one hour followed by PBS wash. Horseradish peroxidase/Fab polymer conjugate (PicTure™-Plus kit; Zymed, South San Francisco, CA, USA) was then applied to the sections for 30 min. After washing, the sections were incubated with peroxidase substrate diaminobenzidine for five minutes and counterstained with hematoxylin.
Grading for cortactin immunoreactivity. The immunoreactivity of cortactin was graded with a four-tier system with the following correspondence: 0, absence of staining in tumor cells; 1+, faint or focal (less than 10% cells) cytoplasmic or nuclear staining in tumor cells; 2+, intermediate staining intensity between 1+ and 3+ in tumor cells; and 3+, diffuse (more than 90% cells) and strong staining in tumor cells (Figure 1).
Patients and follow-up. Patient age ranged from 29 to 76 years, with a mean±standard deviation of 49.5±10.1 years. The mean follow-up period was 68.1±25.8 months (range, 5 to 98 months). The follow-up was usually performed every three months for the first two years and then every six months for the next three years. After five years, the follow-up became annual. Chest radiography, measurement of serum alkaline phosphatase levels and a detailed physical examination were usually performed at follow-up. Annual mammography or breast sonography (for the younger patients) was performed. Radionuclide bone scanning, abdominal sonography or other imaging studies were performed if specific symptoms, signs or elevated serum alkaline phosphatase level were noted. Data regarding patient survival, clinical status, and clinicopathological factors were obtained from medical records, contact with the patients at the outpatient clinics and/or by telephone.
Statistics. All analyses were performed using SPSS, release 17.0 (SPSS, Inc., Chicago, IL, USA). Differences of clinicopathological features among groups by immunostaining were assessed with either the chi-squared or the Fisher's exact test, whichever was appropriate. Overall survival was calculated using univariate analysis by the Kaplan-Meier method. Differences were tested using the log-rank test. To control for confounding factors, the Cox proportional hazard model was used. Survival plots were constructed using the Kaplan-Meier method. All tests were two-sided. Statistical significance was set at p<0.05.
Results
There were 23 patients (23.2%) with low (+) expression of cortactin, 60 patients (60.6%) with intermediate (++) expression and 16 (16.2%) with strong (+++) expression. By using the chi-squared test, comparisons between groups were performed. There was no significant relationship between cortactin expression and age (p=0.301), histological grading (p=0.830), primary tumor staging (p=0.633), lymph node status (p=0.666), estrogen receptor (p=0.995) TNM stage (p=0.856, Table I). For survival analysis, the endpoint was overall survival. The overall five-year survival rates for the various categories are listed in Table II. By multivariate analysis, cortactin expression failed to show any significant relation to the overall five-year survival rate (p=0.517, Table III).
Discussion
Kononen et al. (23) recently described an array-based high-throughput technique that facilitates the analysis of very large numbers of tumors simultaneously, at the DNA, RNA, or protein level. As many as 1,000 cylindrical tissue biopsy specimens from individual tumors can be arrayed in a single TMA block. The power of the TMA technique is the ability to perform a series of analyses of thousands specimens in a parallel fashion with minimal damage to the original tissue blocks (12, 13, 23). In contrast to immunohistochemical analyses of large sections, TMA allows a high level of standardizations for immunohistochemical staining because all tumors are pretreated and stained under exactly the same conditions. Being different from the reading of large sections, which is always an attempt to integrate the observations from multiple different regions of a tissue section, the morphological classification and interpretation of immunoreactivity are based on the findings within one small, highly defined tissue area in TMAs. The criteria for diagnostic decisions are, therefore, markedly easy to establish between the individual samples in the TMA and to compare among different observers (12, 13, 23).
Nevertheless, a criticism of TMA arises is whether the small specimens used, with a diameter of 0.6 mm, are really representative of their donor tumors. It has been reported that some alternations are not detected if the analysis of heterogeneous tumors is restricted to samples measuring 0.6 mm (24). However, Moch et al. (12) noted that the TMA approach has been designed to examine tumor populations and not to survey individual tumors. They analyzed the impact of tissue heterogeneity on TMA data, comparing the results obtained from TMA with those obtained from large sections in multiple different studies and found that the results did show heterogeneity within tumors but suggested that this heterogeneity did not influence the identification of prognostic parameters (12). The reliability of TMAs in detecting protein expression and gene amplification in breast cancer has been confirmed (25, 26). The present study analyzed the cortactin expression in breast cancer by immunohistochemical staining with TMA. This is, to the Authors' knowledge, the first report with long-term follow-up regarding cortactin expression in breast cancer analyzed by using TMA.
Immunostaining with the cortactin antibody on the tissue microarray slides of breast tumors. A representative 3+ case reveals strong cytoplasmic and nuclear immunoreactivity to cortactin in the tumor cells. Original magnification, ×200.
Cortactin expression level in relation to clinicopathological variables.
Overall 5-year survival rate for each category.
Multivariate analysis for overall 5-year survival rate.
Cortactin is a ubiquitously expressed actin filament (F-actin)-binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the actin-related protein 2/3(Arp2/3) complex. Overexpression of cortactin in cancer cells stimulates cell migration, invasion and experimental metastasis; however, the real mechanism of cortactin involvement in tumor progression is not yet fully understood (27).
The amplification of chromosome locus 11q13 is a common feature of several human carcinomas, including squamous cell carcinomas of the esophagus, lung and head and neck as well as bladder cancer and breast cancer (28). There are two candidate oncogenes within the locus, namely CCND1, which encodes the cell-cycle regulatory protein, cyclin D1, and EMS1, which encodes a v-src substrate, cortactin (1). Hui et al. (29) measured EMS1 (cortactin) mRNA levels in breast cancer samples and analyzed the relationship between EMS1 mRNA and other clinicopathological parameters or survival outcomes in 234 patients with a median follow-up period of 74 months (range, 25-107 months). No association was found between EMS1 mRNA levels and disease-free or overall survival in the population studied (29). In the present study, TMA was used to analyze the cortactin status in 99 patients with a mean follow-up period of 68.1±25.8 months (range, 5 to 98 months). Likewise, it was found that cortactin levels in breast cancer are not associated with overall survival (Table III). The patient number in the present study is still limited and, therefore, further larger group studies may provide more conclusive results than this study.
In summary, cortactin expression by immunohistochemical staining with TMA failed to demonstrate a prognostic value for patients with breast cancer.
Acknowledgements
This work was supported by a grant (ref: CMRPG83042) from Chang Gung Memorial Hospital, Kaohsiung, College of Medicine, Chang Gung University, Taiwan, R.O.C.
- Received October 20, 2010.
- Revision received November 23, 2010.
- Accepted November 24, 2010.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
