Abstract LB-72: Expression of the BCL2-like 12 (BCL2L12) gene is associated with prolonged survival of breast adenocarcinoma patients.

Abstract

Abstract The objective of this study was to evaluate the prognostic value of the BCL2-like 12 (BCL2L12) splice variants in breast adenocarcinoma. BCL2L12 is a member of the BCL2 family, aberrantly expressed in several human malignancies, including gastrointestinal cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, and chronic lymphocytic leukemia. We have recently cloned ten novel alternatively spliced variants of this apoptosis-related gene, predicted to encode multiple protein isoforms with distinct functions. Up to date, only the classical isoform has been studied. This one was shown to inhibit apoptosis by neutralizing Caspase-3 and Caspase-7 as well as p53 signaling in glioblastoma. Total RNA was isolated from 140 primary breast tumors and adjacent non-cancerous breast tissue specimens, as well as from breast cancer cell lines. After testing the RNA quality, cDNA was prepared by reverse transcription. A common forward primer and a variant-specific reverse primer were designed and used for the PCR-based expression analysis of each distinct BCL2L12 splice variant. A highly specific and sensitive real-time PCR method was also developed for the quantification of the BCL2L12 main transcript (detection limit: 10 mRNA copies / μL), followed by extensive biostatistical analysis. Calculations were made with the comparative CT (2−▵▵CT) method, using HPRT1 as endogenous control gene and the MCF-7 cell line as calibrator. BCL2L12 splice variants 1, 2, 4, 5, 10, 11, 12, and 13 were detected in breast cancer cells. Among all these mRNA isoforms, the main transcript (BCL2L12 v.1) presented the greatest alterations. This variant was significantly lower in breast adenocarcinomas than in their normal counterparts. Moreover, its expression was inversely associated with estrogen receptor (ER) status (P=0.031), tumor size (P=0.032), TNM stage (P=0.021), and presence of distant metastases (P<0.001). Kaplan-Meier survival analysis and univariate Cox regression indicated that BCL2L12 v.1 is a significant prognosticator of prolonged disease-free and overall survival (P<0.001 in both cases). Most importantly, according to the developed multivariate Cox regression models, the favorable prognostic value of BCL2L12 mRNA expression regarding disease-free survival is independent of the tumor grade, TNM stage, and patients' age (P=0.047). Our findings suggest that the expression of the main transcript (BCL2L12 splice variant 1) of the apoptosis-related BCL2L12 gene merits further investigation as a novel, candidate molecular biomarker predicting prolonged disease-free and overall survival in breast adenocarcinoma patients. Acknowledgements: This work was supported by a THALIS grant (#224), co-funded by the European Union (European Social Fund) and National Resources (NSRF 2007-2013, Operational Programme “Education and Lifelong Learning”). Citation Format: Athina Kladi-Skandali, Christos K. Kontos, Alexandros Tzovaras, Maroulio Talieri, Ioannis Missitzis, Alexandros Ardavanis, Andreas Scorilas. Expression of the BCL2-like 12 (BCL2L12) gene is associated with prolonged survival of breast adenocarcinoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-72. doi:10.1158/1538-7445.AM2013-LB-72