Abstract
Background: The creation of a tissue microarray (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion. This study applied TMA in order to analyse the topoisomerase II alpha status in breast cancer and to elucidate its relationship in breast cancer biology. Patients and Methods: Archived tissue specimens from 94 patients with primary invasive breast cancer were selected and the topoisomerase II alpha expression was analysed by TMA. Data concerning age, oestrogen receptor status, histological grading and TNM staging were also collected. Results: There were 20 patients (21.3%) with 1+ expression in topoisomerase II alpha, 33 patients (35.1%) with 2+ expression and 41 patients (43.6%) with 3+ expression. By multivariate analysis, oestrogen receptor status, histological grading and TNM staging were found to be significantly related to the overall five-year survival rate. Conclusion: Topoisomerase II alpha expression failed to demonstrate prognostic value in patients with breast cancer.
Breast cancer is a clinically heterogeneous disease that may affect patients with similar clinicopathologic manifestations differently (1, 2). This clinical heterogeneity can be attributed largely to different gene expression within tumours. Identification of gene expressions of tumours is now feasible and such profiles can be used to either design the patient's therapeutic plan or to provide prognostic information (1, 2).
Topoisomerase II alpha (IIa) is a DNA-modifying enzyme that binds to the double helix to release torsional stress and create double-strand breaks that allow replications to occur. Anthracyclines have been reported to interfere with topoisomerase II alpha and act by binding covalently with topoisomerase II alpha after double-strand breaks have occurred, inducing lethal cellular damage by inhibition of religation (3, 4). Elevation in topoisomerase II alpha is associated with sensitivity to anthracyclines, both in cell lines and tumours (3, 4).
The creation of a tissue microarray (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion with minimal damage to the original blocks (5, 6).
This study applied TMA in order to analyse the topoisomerase II alpha status in breast cancer, aiming to elucidate the possible relationship between topoisomerase II alpha expression and breast cancer.
Patients and Methods
Specimen selection and data collection. Archived tissue specimens from 94 patients with primary invasive breast cancer were selected from the pathology files of Chang Gung Memorial Hospital, Kaohsiung, Taiwan between January 1994 and December 1998. All patients 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) (7). Data concerning primary tumour staging, age, oestrogen receptor status (8-13), lymph node status, histological grading and TNM staging were also collected. The haematoxylineosin-stained slides of the paraffined-embedded tumour specimens were reviewed by the group's pathologists to confirm the accuracy of the histological diagnoses and lymph node status.
Tissue microarray assembling. The representative areas of both tumour and non-tumour parts for each case were selected and circled to match the blocks for the tissue microarray. The blocks matching the circled slides were retrieved to prepare the recipient block for the microarray. To assure the representation of the selected cores, three areas, each for both tumour and non-tumour parts, per case, were determined for assembling 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 (14).
Immunohistochemical analysis. The rabbit polyclonal antibody against topoisomerase II alpha (ab45175), obtained from Abcam Plc (Cambridge, UK), was diluted 1:150 in phosphate-buffered saline (PBS). Five-micrometer sections were cut from the recipient blocks of the tissue microarray, incubated overnight at 37°C, dewaxed in xylene and dehydrated in a series of graded alcohols. The sections were then treated with 3% hydrogen peroxide for 10 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 topoisomerase II alpha antibody for 1 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 minutes. After washing, the sections were incubated with the peroxidase substrate diaminobenzidine for 5 minutes and counterstained with haematoxylin.
Grading for topoisomerase II alpha immunoreactivity. Topoisomerase II alpha immunostaining was observed mainly in the cytoplasm with focal nuclear staining in the tumour cells. In order to evaluate the immunoreactivity of topoisomerase II alpha, the staining was classified according to the following four-grade scale: 0, absence of staining in tumour cells; 1+, faint or focal (fewer than 10% cells) cytoplasmic staining in tumour cells; 2+, an intermediate staining intensity between 1+ and 3+ in tumour cells; and 3+, strong and diffuse (more than 90% cells) cytoplasmic staining in tumour cells (Figure 1).
Patients and follow-up. All of the patients were women with an age range from 29 to 76 years and a mean age of 49.8±10.1 years. The mean follow-up period was 68.7±25.8 months (range: 5 to 98 months). Follow-up was normally performed every 3 months for the first 2 years and then every 6 months for the next 3 years. After 5 years, follow-up was performed annually. Chest radiography, measurement of serum alkaline phosphatase levels and detailed physical examination were usually performed at follow-up. Annual mammography or breast sonography (for the younger patients) were performed. Radionuclide bone scan, abdominal sonography or s CT scan were performed if specific symptoms, signs or elevated serum alkaline phosphatase levels were noted. Data regarding patient survival, clinical status, and clinicopathological factors were obtained from medical records and from contact with the patients at the outpatient clinics or by telephone, or from both.
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 by the chi-square test and Fisher's exact tests, as 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 hazards 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 20 patients (21.3%) with 1+ expression of topoisomerase II alpha, 33 patients (35.1%) with 2+ expression and 41(43.6%) with 3+ expression. By using the chi-square test, comparisons among groups were performed. As shown in Table I, there was no significant relationship between topoisomerase II alpha expression and the following parameters: age (p=0.118), histological grading (p=0.256), primary tumour staging (p=0.545), lymph node status (p=0.368) and TNM stage (p=0.519). There was a significant relationship between topoisomerase II alpha expression and oestrogen receptor status (p=0.015, Table I). For the survival analyses, the end point was overall survival. The overall 5-year survival rates for various categories are listed in Table II. By multivariate analyses, the topoisomerase II alpha expression failed to show any significant relationship to the overall 5-year survival rate (p=0.227, Table III).
Discussion
Kononen et al. (15) recently described an array-based high-throughput technique that facilitates analysis of very large numbers of tumours at once, either at the DNA, RNA, or protein level. As many as 1000 cylindrical tissue biopsy specimens from individual tumour can be arrayed in a single TMA block. The power of the TMA technique is its capability to perform a series of analyses of thousands of specimens in a parallel fashion with minimal damage to the original blocks (5, 6, 15). In contrast to immunohistochemical analyses on large sections, TMA allows a high level of standardisations for immunohistochemical staining, because all tumours are pretreated and stained under exactly the same conditions. Being different from the reading of large sections which always is an attempt to integrate the observations in multiple 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 TMA. The criteria for diagnostic decisions are therefore much easier to establish among the individual samples on the array and to compare among different observers (5, 6, 15).
Nevertheless, critique about TMA arises as to whether small specimens (diameter 0.6 mm) are really representative of their donor tumours. It has been reported that some tissue alterations are not detected if the analysis of heterogeneous tumours is restricted to samples measuring 0.6 mm (16). However, Moch et al. (5) pointed out that the TMA approach has been designed to examine tumour populations and not to survey individual tumours. They analysed the impact of tissue heterogeneity on TMA data, compared results obtained from TMA with results from large sections in multiple different studies and found that the results did indeed show heterogeneity within tumours but suggested that this heterogeneity did not influence the identification of prognostic parameters. The reliability of TMAs in detecting protein expression and gene amplification in breast cancer has been confirmed (17, 18). The present study analysed topoisomerase II alpha expression in breast cancer by immunohistochemical staining with TMA and the results were obtained reliably. By multivariate analysis, TNM stage, oestrogen receptor status and histological grading were related significantly to the overall five-year survival rate (Table III). Nevertheless, the topoisomerase II alpha expression failed to demonstrate prognostic value in this study.
Arriola et al. (19) measured the gene copy of topoisomerase II alpha by chromogenic in situ hybridisation (CISH) and reported that topoisomerase II alpha amplification predicted a better overall survival and disease free survival (p=0.028 and 0.026, respectively). Rody et al. (20) investigated the expression level of topoisomerase II alpha mRNA and found that it is an independent prognostic factor in oestrogen receptor-positive breast cancer and could be used for risk assessment. In the present study, immunohistochemical analysis of topoisomerase II alpha expression with TMA was performed and surprisingly, topoisomerase II alpha expression failed to demonstrate prognostic value in patients with breast cancer (Table III). The following two reasons may offer an explanation for this finding: (i) there is no correlation between topoisomerase II alpha gene copy number and topoisomerase II alpha protein expression levels in breast tumours (21), and (ii) the number of cases in the present study is still small, and a further larger group study may provide a more firm conclusion. To the best of the Authors' knowledge, this is the first report with long-term follow-up about topoisomerase II alpha expression in breast cancer analysed by using TMA.
In summary, topoisomerase II alpha expression by immunohistochemical staining with TMA failed to demonstrate prognostic value in patients with breast cancer.
Acknowledgements
This work was supported by Grant CMRPG83042 from Chang Gung Memorial Hospital, Kaohsiung, College of Medicine, Chang Gung University, Taiwan.
- Received March 6, 2010.
- Revision received May 1, 2010.
- Accepted May 6, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved