Advances in Brief Clinicopathological Significance of Fragile Histidine Triad Transcription Protein Expression in Breast Carcinoma

The fragile histidine triad (Fhit) gene, which is frequently lost in many cancers, was identified as a candidate tumor suppressor gene at chromosome 3p locus 14.2. Loss of Fhit expression is an important step in tumor progression from premalignancy, to in situ, to invasive breast carcinoma. To determine whether the absence of Fhit protein correlates with other established pathological-clinical parameters or prognosis, we assessed Fhit expression using immunohistochemistry in 166 invasive breast carcinomas. Lost or significantly decreased Fhit protein expression was identified in 70 cases (42.2%). Fhit expression was inversely correlated with histological grade (P < 0.0001), negative estrogen receptor status (P 0.0016), p53 overexpression (P 0.0040), and tumor proliferation activity (P 0.0006). Survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that reduced expression of Fhit was associated with a poor outcome (P 0.0086, by log-rank test). Multivariate analysis using the stepwise Cox proportional hazard model showed that lymph node metastasis was related to poor survival rates; in addition, patients with loss of Fhit expression still tended to have poor survival (P 0.0563). Therefore, loss of Fhit expression is associated with higher malignant phenotypes and appears to be a prognostic factor in breast carcinoma. Introduction Recent advances in molecular biology have led to a concept that carcinomas arise from the accumulation of a series of genetic alterations involving activation of proto-oncogenes, inactivation of tumor suppressor genes, and inactivation of DNA repair genes in a single cell. The Fhit gene has been identified in fragile locus, FRA3B, at 3p14.2 (1) and has been reported to be deleted in a number of human tumors (2, 3). Stable Fhittransduced clones expressing exogenous wild-type Fhit, isolated after transfection of various epithelial cell lines carrying inactivated endogenous Fhit, show reduced colony formation efficiency in vitro and inhibition of tumor development in nude mice, indicating that Fhit acts as tumor suppressor gene (2). The Fhit gene belongs to the histidine triad superfamily and encodes a cytoplasmic Mr 16,800 protein with diadenosine triphosphate (Ap3A) hydrolase activity (4). Preliminary studies have revealed marked reduction or absence of Fhit mRNA and/or protein expression in lung, bladder, renal, colon, cervical, endometrial, and breast carcinomas. However, correlation between Fhit expression and other clinicopathological parameters, particularly prognosis, is controversial (5–9). Whether Fhit expression has some prognostic role for breast carcinoma is still unknown. In the present study, we investigated Fhit expression in a large number of breast carcinomas to determine whether abnormal expression of Fhit gene is an independent prognostic marker for breast cancer. This is the first report describing the clinicopathological significance of Fhit expression in a large series of Asian patients with sporadic breast carcinoma. Patients and Methods Patients and Samples. Paraffin-embedded tissue was obtained from 166 patients who underwent surgery in Affiliated Kihoku Hospital of Wakayama Medical Universtiy between 1985 and 1995. Information about the patients’ clinical history was obtained from the patients’ medical records and from our institution’s adjuvant chemotherapy database and pathology data files. Age at diagnosis was considered as the patient’s age. All had histological evidence of invasive breast carcinoma, and none had a family history in first-degree relatives, as judged by questioning at the time of admission for surgery. Patient and tumor characteristics are shown in Table 1. None of the patients had been treated with neoadjuvant chemotherapy, hormonal therapy, or irradiation prior to tumor excision. The patients had received subcutaneous glandectomy with axillary lymph node dissection or mastectomy with axillary lymph node dissection. The size of the primary tumor was considered to be the largest tumor diameter observed after surgical excision. Lymph node Received 6/5/01; revised 8/29/01; accepted 8/31/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by in part by Grants 014563, 12770657, and 10045073 from the Ministry of Education, Culture, Sports, Science and Technology, Government of Japan. 2 To whom requests for reprints should be addressed, at Second Department of Pathology, Wakayama Medical College, 811-1, Kimiidera, 641-0012 Wakayama City, Japan. Phone: 81-73-441-0635; Fax: 81-73446-4825; E-mail: [email protected]. 3 The abbreviations used are: Fhit, fragile histidine triad; ER, estrogen receptor. 3869 Vol. 7, 3869–3873, December 2001 Clinical Cancer Research Research. on April 14, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from status was determined with histological evidence of metastatic breast carcinoma. Histological typing and histological grading were done according to the WHO classification (10) and the Nottingham scheme (11), respectively. ER levels were determined by standard biochemical methods. Hormone receptor levels 10 fmol/mg were considered positive. All of our patients received postoperative adjuvant therapy consisting of combination chemotherapy with cyclophosphamide, epirubicin, and fluorouracil. The patients also received tamoxifen therapy, regardless of ER status or axillary lymph node status. None of them had postoperative radiotherapy. Immunohistochemical Studies. For the immunohistochemical study, 4m-thick sections were cut from paraffin blocks that contained representative histology of the breast carcinoma. Paraffin sections on silane-coated slides were dewaxed with xylene and rehydrated through a graded alcohol series. Then, endogenous peroxidase activity was blocked in absolute methanol solution containing 1% hydrogen peroxide for 35 min, and the slides were washed in 10 mM PBS (pH 7.4). For antigen retrieval, the slides were immersed in 1 mM citrate phosphate buffer and microwaved at 100°C for 15 min. After the buffer had cooled, normal horse serum was reacted with the slides for 15 min to eliminate nonspecific immunostaining. The slides were reacted with various primary antibodies overnight at 4°C in a humidified chamber. The details of the primary antibodies used in this study were as follows: polyclonal rabbit IgG antibody to Fhit (ZR44; Zymed Laboratories, Inc., San Francisco, CA) with a dilution of 1:200, monoclonal antibody DO7 for p53 protein with a dilution of 1:300 (Novocastra Laboratories, Ltd., Newcastle, United Kingdom), and monoclonal antibody for MIB-1 with a dilution of 1:75 (Immunotech, Marseilles, France). After reaction with a mouse biotinylated secondary antibody, antigen-antibody reactions were visualized using a streptavidin-horseradish peroxidase conjugate (DAKO LSAB kit; DAKO, Los Angeles, CA) with diaminobenzidine as the chromogen. All slides were counterstained with hematoxylin. Staining without antibody was performed as a negative control. For immunohistochemical evaluation of Fhit, cytoplasmic labeling of tumor cells was classified as either negative (if no staining or positive staining was present in 10% of tumor cells) or positive (if 10% of tumor cells stained positively). With the method described in our previous studies (12), nuclear staining of neoplastic cells was scored as positive for p53 and MIB-1. Tumors were considered positive for p53 when 10% of the tumor cells demonstrated positive staining. MIB-1 scoring was as follows: 5% (score 0); 5–25% (score 1); 25–45% (score 2); and 45% (score 3). Statistical Analysis. Descriptive statistics comparing Fhit expression with conventional markers of tumor aggressiveness were analyzed by standard 2 tests, or, when appropriate, Fisher’s exact test. Estimates of disease-free survival were calculated by the Kaplan-Meier product-limit method, and the differences were assessed by the log-rank test. Probabilities of survival were calculated from the date of breast carcinoma diagnosis to either the date at which relapse from breast carcinoma was clinically identified or the date of last contact. Multivariate survival analysis using Cox’s proportional hazard regression model was carried out to assess the independent contribution of each variable to survival. Overall survival was not analyzed because of the small number of disease-related deaths (8 patients died of recurrent breast carcinoma). All Ps were two-tailed, and the 0.05 level was considered statistically significant. A computer program package (StatView 5.0; Abacus Concepts, Berkeley, CA) was used for all statistical testing and management of the database.