Abstract
Background: Fatty acid-CoA ligase 4 (FACL4) has been detected in various types of tumors. However, there is still very limited information about the role of FACL4 in breast cancer. Tissue microarray (TMA) technique analyzes thousands of specimens in a parallel fashion with minimal damage to the original blocks. This study was designed with the application of TMA to analyze the FACL4 status in breast cancer. Materials and Methods: Archival tissue specimens from 102 patients with primary invasive breast cancer were selected and FACL4 expression was analyzed by immunhistochemical staining with TMA. The data of primary tumor staging, age, estrogen receptor status, lymph node status, histological grading and TNM staging were also collected. Results: As shown my multivariate analysis, TNM stage was significantly related to the overall five-year survival rate. Nevertheless, FACL4 expression failed to have any significant relationship to overall five-year survival. Conclusion: Immuno-histochemical staining with TMA was convenient and feasible for the analysis of FACL4 expression status in breast cancer. Our preliminary results showed that FACL4 expression had no significant prognostic value in breast cancer.
Clinically, breast cancer is a heterogeneous disease that may influence patients with similar clinicopathological manifestations in different ways (1-3). This clinical heterogeneity can be attributed in large to different gene expressions within tumors. Nowadays, identification of the gene expression of tumors is feasible and such information has been applied to either plan the patient's therapeutic options or to offer prognostic information (3, 4).
Human long chain fatty acyl-CoA ligase (FACL) is a central enzyme in controlling the free fatty acid pools and is necessary for usage of free fatty acid as building blocks and energy sources (5-7). There are five FACL isoenzymes present in human tissues (7, 8). Fatty acid-CoA ligase 4 (FACL4) is an arachidonic acid (AA)-preferring isoenzyme of the acyl-CoA ligase family (9). FACL4 has been noted highly expressed in human steroidogenic tissues, such as placenta, brain, testis, ovary, spleen and adrenal cortex, and to be less expressed in the gastrointestinal system, including the liver (5).
The implementation of tissue microarrays (TMA) allows for the rapid immunohistochemical analysis of thousands of tissue samples in a parallel fashion with minimal damage to the original blocks (10, 11).
This study applied TMA with the aim of analyzing FACL4 status in breast cancer in order to elucidate the possible relationship between FACL4 expression and breast cancer.
Materials and Methods
Specimen selection and data collection. Archival tissue specimens from 102 patients with primary invasive breast cancer were selected from the pathology files of Kaohsiung Chang Gung Memorial Hospital at between January 1994 and December 1998. All the 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) (12). The data of primary tumor staging, age, estrogen receptor status (13-15), lymph node status, histological grading and TNM staging were also collected. The hematoxylin-eosin-stained slides of the paraffined-embedded tumor specimens were reviewed by our pathologists to confirm the accuracy of the histological diagnoses and lymph node status.
TMA assembly. Representative areas of both tumor and non-tumorous parts for each case were selected and circled to match the blocks for the TMA. Then the blocks matching the circled slides were retrieved to prepare the recipient block for the microarray. Three areas each for both tumor and non-tumorous parts per case were used 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 (16).
Immunohistochemical analysis. Rabbit polyclonal antibody against human FACL4 was purchased from Abnova (PAB 2504), Taipei, Taiwan and was diluted 1:400 in phosphate-buffered saline (PBS). Five-micrometer sections were cut from the recipient blocks of the tissue microarray, incubated overnight in a 37°C oven, de-waxed in xylene, and dehydrated in a series of graded alcohols. The sections were then treated with 3% hydrogen peroxide for 10 minutes to exhaust 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 FACL4 antibody for 1 h followed by PBS wash. Horseradish peroxidase (HRP)/Fab polymer conjugate (Ultra Tek HRP anti-polyvalent kit; Scy Tek, Utah, USA) was then applied to the sections for 30 min. After washing, the sections were incubated with peroxidase substrate diaminobenzidine for 5 min and counterstained with hematoxylin.
Scoring of FACL4 immunoreactivity. The staining intensity for FACL4 was scored as follows. Score 0 was defined as the absence of staining in tumor cells; score 1 as faint or focal (less than 10% cells) cytoplasmic staining in tumor cells; score 2 as intermediate staining between score 1 and score 3 in tumor cells; and score 3 as strong and diffuse (more than 90% cells) cytoplasmic staining in tumor cells (Figure 1).
FACL4 immunostaining was mainly localized in the cytoplasm but not the nuclei of tumor cells of the breast carcinomas.
Patients and follow-up. All of patients were women from 26 to 76 years old, with a mean age of 49±11 years. The mean follow-up was 68±28 months (range, 5 to 98 months). 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, follow-up became annual. Chest radiography, serum alkaline phosphatase level, and detailed physical examination were usually performed at follow-up. Annual mammography or breast sonography (for the younger patient) were performed. Radionuclide bone scan, abdominal sonography or other image 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 outpatients clinics or by telephone, or both.
Statistical analyses. Comparisons between groups were performed using Fisher's test. For survival analyses, the end-point was overall survival. Survival differences were compared using the log-rank test. To assess the relative influence of the potential prognostic variables on survival, all clinicopathological and genetic variables were entered into the final Cox's proportional hazards model for multivariate analysis. Statistical analyses were conducted using SPSS software (version 17.0; SPSS, Chicago, IL, USA). Statistical significance was set at p<0.05. All p-values were estimated from two-sided tests.
Results
There were 12 patients (12%) with FACL4 expression score 0, 44 patients (43%) with score 1, 37 patients (36.0%) with score 2 and nine patients (9.0%) with score 3 (Table I). Using the Chi-square test, comparisons between groups were performed. There was no significant relationship between FACL4 expression and age (p=0.492), estrogen receptor status (p=0.061), histological grading (p=0.984), primary tumor staging (p=0.204), or lymph node status (p=0.162). FACL4 expression was found to be significantly related to TNM staging (p=0.026).
For survival analyses, the end-point was overall survival. The overall five-year survival rates for different categories are listed in Table II. By multivariate analysis, TNM stage was significantly related to the overall five-year survival rate (Table III, p<0.0001). Nevertheless, FACL4 expression failed to show any significant relationship to the overall five-year survival (p=0.689).
Discussion
Production of acyl-CoA by FACL4 is an essential reaction in mammalian cells. Free AA entering cells exogenously or released endogenously is rapidly converted to AA-CoA esters by the catalytic action of FACL, particularly by the AA-preferring FACL4 (5, 17). Certain lines of evidence show that the level of free AA in cells regulates apoptosis (18, 19). Furthermore, overexpression of FACL4 prevents AA-induced apoptosis by reducing the level of intracellular free AA (20). FACL4 has been reported to be overexpressed in human hepatocellular carcinoma, as well as colon adenocarcinoma, and thus FACL4 may play a role in the carcinogenesis of these neoplasms (21, 22). There is still very limited information concerning FACL4 in breast cancer.
An array-based high-throughput technique has recently been described which can facilitate analysis of very large numbers of tumors at once, either at the DNA, RNA, or protein levels (23). Up to 1,000 cylindrical tissue biopsy specimens from individual tumors can be arrayed in a single TMA block. The TMA technique is able to analyze thousands of specimens in a parallel fashion with minimal damage to the original tissue blocks (24, 25). When compared to immunohistochemical analyses on large sections, TMA provides a high level of standardization for immunohistochemical staining, since all specimens are managed under exactly the same conditions. Being different from the reading of large sections which always is an attempt to integrate the observations in 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 interpretation methods are thus much easier to set up between the individual samples on the array and to compare among different observers (24, 25).
However, the use of TMA with small specimens (diameter 0.6 mm) has been challenged regarding whetter the results can really be representative of their donor tumors. Moch et al. indicated 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 results obtained from TMA with results 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 (24). The reliability of TMA in detecting protein expression and gene amplification in breast cancer has been confirmed (26, 27).
In the present study, TMA was used to analyze FACL4 expression in 102 patients with a mean follow-up of 68±28 months (range, 5 to 98 months). We found there was no significant relationship between FACL4 and other basic clinicopathological parameters except for TNM staging (Table I). By multivariate analysis, FACL4 did not have any meaningful prognostic value and was not significantly related to the overall five-year survival rate (p=0.689, Table III). This is probably due to the retrospective nature of the study and the relative small number of patients in this study. A prospective study with a larger number of patients is warranted for further evaluation and elucidation. To the best of our knowledge, this is probably the first report with long-term follow-up concerning FACL4 expression in patients with breast cancer analyzed by TMA.
In summary, immunohistochemical staining with TMA was convenient and feasible for the analysis of FACL4 expression status in breast cancer. Our preliminary results show that FACL4 expression failed to have any significant prognostic value regarding breast cancer. A larger prospective study may be warranted for further evaluation.
Acknowledgements
This study was supported by CMRPG83042 from Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taiwan.
We would like to thank Chang Gung Medical Foundation Kaohsiung Chang Gung Memorial Hospital Tissue Bank (CLRPG8B0031) for technical support.
Footnotes
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↵* These Authors contributed equally to this study and share first authorship.
- Received November 9, 2013.
- Revision received December 4, 2013.
- Accepted December 6, 2013.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved