ANTICANCER RESEARCH 26: 4415-4422 (2006) Cyclin A and E2F1 Overexpression Correlate with Reduced Disease-free Survival in Node-negative Breast Cancer Patients EDITTA BALDINI1, ANDREA CAMERINI1,3, ALESSANDRO SGAMBATO3, TIZIANA PROCHILO1, ALESSANDRA CAPODANNO2, FRANCESCO PASQUALETTI1, CINZIA ORLANDINI1, LETIZIA RESTA4, GENEROSO BEVILACQUA2 and PAOLA COLLECCHI2 1Medical Oncology and 2Division of Surgical Molecular and Ultrastructural Pathology, Department of Oncology, University of Pisa and University Hospital of Pisa, Pisa; 3Istituto di Patologia Generale, Centro di Ricerche Oncologiche "Giovanni XXIII", Catholic University, Rome; 4Dipartimento di Medicina Sperimentale e Farmacologia, Patologia Clinica, University of Messina, Italy Abstract. Background: Available prognostic factors do not cancer patients and support the need for further studies to accurately identify node-negative breast cancer patients at high evaluate their suitability for use in the routine clinical risk of disease recurrence and progression. Patients and management of these patients. Methods: Cyclin A and E2F1 expression levels were evaluated in 75 consecutive node-negative breast cancer patients with a Breast cancer is the leading type of cancer and the second median follow-up of 10 years. Both parameters were tested for leading cause of cancer death in women in the United States. correlation with all the available clinicopathological Adjuvant treatments produce significant benefits in operable parameters and with the clinical evolution of the disease. breast cancer (1). However, despite surgical resection and Results: Cyclin A was overexpressed in 45.3% of patients and subsequent adjuvant therapy, about 20-25% of node-negative significantly related to large tumor size, high Ki67 and high breast cancer patients still develop recurrence and/or distant E2F1 expression levels. No relationship was observed between metastases within ten years from diagnosis (1-3). Available cyclin A and tumor estrogen receptor (ER) status, grading or risk factors, such as tumor size, estrogen receptor (ER) patient age. Seventeen patients relapsed within 5 years from status, grading and age, often fail to clearly identify patients diagnosis. Twelve (71%) of them showed cyclin A at high risk of disease recurrence and progression. Thus, the overexpression in comparison with 22 (38%) out of the 58 who search for new molecular markers continues to be a hot topic did not relapse (p=0.02). Disease-free survival (DFS) was in breast cancer research. significantly shorter in patients with cyclin A-overexpressing Perturbations in cell cycle control mechanisms are tumors compared to non-overexpressing ones (p=0.01). DFS frequently reported in human neoplasms and the was also significantly longer in low vs. high Ki67 expression investigation of their prognostic significance is an (p=0.003) and in low vs. high E2F1 expression (p=0.02). On interesting field of research. On these grounds, cyclin A multivariate analysis, the simultaneous high expression of all seems to be a promising new molecular marker related to three parameters (cyclin A, Ki67 and E2F1) was a strong the human multistep tumorigenesis process. In mammalian independent prognostic factor for shorter DFS (HR 13.4). cells, the cell cycle is tightly regulated by a family of Conclusion: These findings suggest that assessment of cyclin A kinases (cyclin-dependent kinases, Cdks) which are and/or E2F1 expression levels, associated with Ki67, might be activated by different cyclins and in turn regulate cell cycle useful for a better prognostic evaluation of node-negative breast progression. Cyclin A first appears in late G1-phase, is expressed during S- and G2-phases, peaks in late G2 and is degraded when cells enter pro-metaphase. Cyclin A binds to Cdk2 in the G1 to S transition, in the S-phase as well, Correspondence to: Alessandro Sgambato, MD, Istituto di Patologia and with Cdc2 (Cdk1) in the G2/M-phase (4). The specific Generale, Centro di Ricerche Oncologiche "Giovanni XXIII", expression of cyclin A is essential to control the correct Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 sequence of events that brings the cell through S-phase to Rome, Italy. Tel: +39-06-3016619, Fax: +39063012753, e-mail: [email protected] the G2-M checkpoint (5). Given its role in controlling the DNA replication machinery and the G2-M checkpoint, Key Words: Cyclin A, E2F1, Ki67, breast cancer, disease free cyclin A plays an important role in cell proliferation survival. control and, probably, in cancer growth (6). 0250-7005/2006 $2.00+.40 4415 ANTICANCER RESEARCH 26: 4415-4422 (2006) Overexpression of cyclin A accelerates pRb embedded tissue samples, as previously described (26, 27). Briefly, phosphorylation (7), promotes entry into the S-phase (8) representative tumor sections (3 Ìm) were deparaffinized, and is associated with the ability of cells to grow in rehydrated and immunostained using antigen retrieval with a microwave technique at 750W for 15 min in 10 mM citrate buffer suspension (a classic feature of cancer cells) (9); in addition, (pH 6). After cooling at room temperature, the endogenous the cyclin A/Cdk2 complex is a target of the adenovirus E1A peroxidase was blocked with 1% H2O2 in methanol for 5 min. oncoprotein (10). The overexpression of cyclin A has been Sections were incubated for 1 hour at room temperature with anti- found in various human tumors and cancer cell lines (7, 11). human cyclin A (clone BF683, Pharmingen, USA), anti-human E2F1 High levels of cyclin A have been associated with tumor size (clone KH95/E2F, Pharmingen) and anti-estrogen receptor (ER in colorectal and pancreatic cancer (12, 13) and with poor ID5, DAKO, Glostrup, Denmark) monoclonal antibodies diluted differentiation in thyroid carcinomas and pancreatic 1:100 and anti-Ki67 (MIB-1, Immunotech, Marseille, France) monoclonal antibody diluted 1:200. After washing, sections were adenocarcinomas (13). Recently, an increased expression of immunostained using the avidin-biotin-peroxidase complex method cyclin A has been reported to correlate with poor prognosis (Vectastain Elite ABC kit; Vector, Burlingame, CA, USA). The in breast, colorectal and laryngeal squamous cell carcinomas peroxidase activity was visualized using 0.05% 3,3-diaminobenzidine and childhood acute lymphoblastic leukemia (12, 14-17). (Sigma Chemical Co., St Louis, MO, USA) and 0.2% H2O2. The E2F1 transcription factor plays a dual role in cell cycle slides were rinsed with PBS and counterstained with hematoxylin. control acting both as a pro-apoptotic agent (18) and Sections of positive mammary carcinoma were used as positive enhancing the cell proliferation rate (19, 20). E2F1 controls. Negative controls were obtained by omitting the primary antibodies from the procedure. Nuclear staining in the absence of positively regulates cyclin D1, E and A expression levels cytoplasmic background coloration was considered positive. The through a direct interaction with their promoters (21, 22) immunostained nuclei were evaluated by two observers and plays a role in tumor development (23, 24). independently and the discrepant cases were jointly re-evaluated. A In this study, the expression of cyclin A and E2F1 was minimum of 1,000 cells were counted for each tumor and evaluated in a group of operable node-negative breast immunoreactivity was expressed as a percentage of stained cells on cancer patients and their relationship with main biological the total number of tumor cells. A value of 3% of positive cells, and clinical parameters was investigated. corresponding to the median values of cyclin A and E2F1, was used as cut-off point to distinguish high and low expressing tumors. A cut- Patients and Methods off of 10% positive cells was used to identify ER-negative (ER–) and ER-positive (ER+), or high and low Ki67 expressing tumors. No suitable paraffin material was available for immunohistochemical Patient characteristics and tissue samples. Tumor samples were analysis of Ki67 and E2F1 of two tumors, thus, 73 out of the 75 cases obtained from 75 consecutive node-negative breast cancer patients were stained for both parameters. who had undergone surgery at the Santa Chiara University- Hospital in Pisa (Italy) from 1989 to 1995. Patients receiving pre- Statistical analysis. The relationship between cyclin A and other operative chemotherapy or having a family history of breast cancer molecular and clinical parameters were assessed using contingency were excluded. Patients who had undergone quadrantectomy table methods and tested for significance using the Pearson’s chi- received complementary radiotherapy. Chemotherapy and/or square test. Mean values were compared using the Student’s t-test. hormonal therapy were administered according to individual risk Disease-free survival (DFS) was defined as the interval between factors. The percentage of patients who received chemotherapy, surgery and the first documented evidence of disease in the local- hormonal therapy or no therapy were similar in the relapsed and regional area, distant sites, contralateral breast or a combination non-relapsed group. In particular, patients receiving chemotherapy of the above. Relapses were accurately assessed by clinical, were treated either with a combination of methotrexate, radiological and, whenever feasible, histopathological examination. cyclophosphamide and 5-fluorouracil (CMF) or with an DFS curves were calculated using the Kaplan-Meier method and anthracycline-containing regimen at a similar