Abstract 448: Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study
Authors: Rolfo Christian D. , Castiglia Marta , Cabibi Daniela , Calo' Valentina , Piazza Florinda D. , Bazan Viviana , Brocco Fabio , Caruso Stefano , Bruno Loredana , Papadimitriou Konstantinos , Passiglia Francesco , Peeters Marc , Pauwels Patrick , Russo Antonio 
Published: May 2015

Abstract
Introduction There are numerous causes triggering CRC. 25-80% of CRC shown a deregulation in Epidermal Growth Factor Receptor (EGFR) pathway. Two signaling pathways downstream of the EGFR are dysregulated in CRC the mitogen-activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K) pathway. Activating mutations in KRAS and BRAF (MAPK pathway) and PIK3CA affect prognosis and/or response to anti-EGFR MoAb. PTEN is a downstream effector of EGFR pathway and is involved in PI3K pathway. Loss of PTEN protein expression can occur through epigenetic silencing and mutation or allelic loss. Immunohistochemistry (IHC) is the most effective way to assay for loss of PTEN expression. IHC lack of reproducibility and this has lead to discordant results regarding the concordant in paired CRC metastases and primary tumors.The aim of the work was to asses the feasibility of PTEN gene expression analysis from FFPE tissue in CRC samples.
Materials and methods We selected a series of 33 patients, already tested for KRAS mutational status and resulted wild-type. At the time of the study mutational status of NRAS gene has not a role as predictive marker, so was not performed. A retrospective analysis on CRC FFPE tissue was performed. As control samples we have selected specimen of colorectal surgery not tumor-associated (diverticulitis). On these samples BRAF mutational analysis and PTEN gene expression was performed. For BRAF analysis, DNA was extracted by means of QIAamp DNA FFPE tissue kit (Qiagen, USA). BRAF mutational status was assed through direct automated sequencing (3100 Genetic Analyzer, Applied Biosystems) and TaqMan mutation detection assay (ABI PRIM 7900HT, Applied Biosystems). For PTEN gene expression, total RNA was obtained through the miRNeasy FFPE tissue kit (Qiagen, USA), according to manufacturer instructions, and reverse transcribed. PTEN expression was assessed by means of TaqMan probes.
Results According to BRAF mutational status we have found 10/33 (30%) mutated samples. PTEN expression levels were evaluated by the comparison of tumor samples vs diverticula samples, the data obtained were then integrated with those from BRAF mutational analysis. Interestingly we found that all the BRAF mutated samples present also a down-regulation of PTEN. Statistical analyses were conducted through the X2 test with the Yatesha correction (p=0.00023). The p-value was also calculated Exact Fisher Test (p= 0.00021) to confirm the results.
Conclusion The determination of PTEN gene expression level is feasible with real-time PCR using TaqMan probe technology. BRAF analysis might be used as prognostic and/or predictive marker in CRC patients’ treatment. The identification of a validated method for PTEN analysis would establish it as a molecular marker meriting further investigation in large numbers of available primary tumors from patients with CRC. Further analyses are requested to confirm these data.
Citation Format: Christian D. Rolfo, Marta Castiglia, Daniela Cabibi, Valentina Calo', Florinda Di Piazza, Viviana Bazan, Fabio Brocco, Stefano Caruso, Loredana Bruno, Konstantinos Papadimitriou, Francesco Passiglia, Marc Peeters, Patrick Pauwels, Antonio Russo. Molecular analysis of BRAF gene and PTEN gene expression in metastatic colorectal cancer patients: Feasibility study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2014-448