Abstract 3761: Tissue processing time affects the expression of phosphoproteins after breast cancer resection

Abstract

Abstract Background: Many phosphoproteins are among the targeted pathways of cancer signal transduction and are therefore candidates for cancer biomarkers. However, the collection procedures of biospecimens might affect the expression of the phosphoproteins significantly because the time interval between surgical removal of tissues and fixation, i.e. warm ischemia time, are variable. This study aimed to evaluate the stability of phosphoprotein expression under the differential tissue processing time. Materials and Methods: Specimens were obtained from patients who received mastectomy at National Taiwan University Hospital. Each specimen was cut into 5 pieces, stored at 4oC for 10 minutes, 2, 4, 6, and 24 hours before further processing. At each assigned time point, a small section of the specimen was fixed immediately in 10% buffered formalin, then paraffin embedded for immunohistochemical stains (IHC) of phospho-Akt (p-Akt) and phospho-Erk (p-Erk). The remaining specimens were snap frozen in liquid nitrogen, followed by homogenization in RIPA buffer. Western blot analysis was performed for phospho-Akt (p-Akt), total Akt, phospho-Erk (p-Erk), total Erk, phospho-STAT3 (p-STAT3), and total STAT3 staining. Results: Western blot analysis showed that the expression of p-Erk declined significantly as early as 2 hours. The expression of p-Akt was relatively stable and decreased slightly after 24 hours. The expression of p-STAT3 gave variable results. The expression of total Akt, Erk, and STAT3 remain unchanged at each assigned time point. The immunohistochemical expression of p-ERK declined in a time-dependent manner whereas no obvious p-Akt decrease was detected. Conclusions: Our results indicate that the expression of some phosphoproteins by IHC may be affected by warm ischemia time. Caution should be given to the interpretation of the results of these biomarkers. (The study was supported by the grant NSC 98-3112-B-002-038) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3761.