Correlation between Beta 1 integrin expression and prognosis in clinically localized prostate cancer

ARTICLE INFO _________________________________________________________ ___________________ Vol. 39 (3): 335-343, May June, 2013 doi: 10.1590/S1677-5538.IBJU. 2013.03. 06 IBJU | beta1 integrin in prostate cancer 336 the discovery of new markers is necessary to identify those patients with worse prognoses and increased likelihood of tumor recurrence more effectively, which can improve the management of PC patients. Many aspects of oncogenesis involve changes in intercellular adhesion, and it was shown that altered expression of the cell adhesion molecule (CAM) correlates with invasion and progression in PC and other neoplasms (5-7). Integrins are transmembrane glycoprotein receptors that regulate cell-matrix interactions, thereby functioning as sensors of the environment. These CAMs form heterodimers composed of one α and one β subunit. Currently, 18 α and 8 β subunits have been identified, and different combinations of these subunits are known to dictate both their specificity for the extra cellular matrix (ECM) proteins and their signaling properties. Integrins have several functions in cell homeostasis that involve the adhesion of cells to the ECM proteins as well to other cells. As receptors, integrins mediate the anchoring and migration of cells via the recognition of various ECM molecules. Moreover, intracellular signals generated by integrins influence gene expression and thus regulate growth, proliferation and survival (8). The existence of alternative splicing forms of the messenger RNA of some integrins further increases the diversity within the integrin family. Despite the existence of previous research examining β1 integrin in PC, no study to date has evaluated β1 integrin in clinical specimens as a prognostic marker for a clinical outcome. Most prior studies in PC have primarily focused on describing integrin expression rather than on examining its correlations to tumor recurrence or biochemical free survival (9-11). The aim of this case-control study was to assess the expression profile of β1 integrin in surgical specimens of clinically localized PC through the use of immunohistochemistry by utilizing a tissue microarray (TMA), and to evaluate the association between integrin expression and biochemical recurrence following RP. MATERIALS AND METHODS Case selection We retrospectively evaluated 954 patients with clinically localized PC who underwent RP with a curative intention between January 1994 and April 2000, all performed by the same surgeon. For this case-control study, we selected 51 patients with low, intermediate or high risk PC that had biochemical recurrence after surgery (case group). Then, 60 patients without biochemical recurrence ten years after surgery and matched to the first group, according to the PC stratification of risk, were selected as controls. We stratified the PC patients into three groups of risk according to a combined evaluation of pre operatory PSA serum level, pathologic stage and surgical specimen ́s Gleason score. Low risk PC patients was characterized by PSA level lower than 10ng/mL, Gleason score ≤ 6 and pT2a; the risk was intermediate when PSA level was 10 to 20ng/mL, Gleason score 7 and pT2b and higher risk when PSA level was ≥ 20ng/mL, Gleason score ≥ 8 and pathologic stage was higher than pT2c. This risk stratification is similar to that one proposed by D’Amico; the difference is the adoption of pathologic stage and Gleason score in our study, which are more reliable in terms of prognostic strength, when compared to the clinical parameters employed in D’Amico criteria (12). The median follow-up time was 123 months. Regarding race, 97.3% of the patients were Caucasian and 2.7% were Asian. The demographic and clinical data according to recurrence are shown in Table-1. Tumor recurrence was defined as a PSA level exceeding 0.2ng/mL during follow-up. The tumor-node-metastasis (TNM) staging designations were assigned according to the TNM 2010 classification. All subjects provided informed consent to participate in the study and to allow their biological samples to be analyzed. Approval for the study was given by the Institutional Board of Ethics (no1074/04). Immunohistochemistry Histological examination of the prostatectomy specimens were performed in formalin-fixed and totally paraffin-embedded sections stained with hematoxylin and eosin. The slides containing the primary tumor for each patient were selected by sampling the area representative of the final pathologic Gleason score. Two areas from each IBJU | beta1 integrin in prostate cancer 337 tumor were marked with permanent ink; these areas were included in the tissue microarray assay (TMA) (13). The samples underwent a heat antigen retrieval process using citrate buffer (1mM, pH 6.0). The slides were incubated overnight at 4oC with the anti-β1 integrin monoclonal antibody (Dako Cytomation, CA) in a 1:50 dilution. The LSAB system was used for the immunostaining (Dako Cytomation, CA). Color was developed through a reaction with a 3.3’diaminobenzidine substrate-chromogen solution followed by counterstaining with Harris hematoxylin. The slides were dehydrated, coverslipped and observed under a light microscope. β1 integrin expression was evaluated as positive or negative through immunohistochemistry in a TMA containing two samples from each case. We employed optical microscopy and the expression was considered positive when it was strong and diffusely present in the tumor tissue (i.e., +++). In order to confirm the integrity of the antigen determinants, we also evaluated the expression of PSA and cytokeratin 18 (CK18) in the same TMA. All of the cases were evaluated by a single uropathologist (KRML). Table 1 Demographic and clinical data: recurrence vs. no recurrence. Biochemical recurrence (n = 51) No biochemical recurrence