Quantitative Assessment of Change in Protein Phosphorylation as a Function of Ischemic Time before Formalin Fixation
Y Bai, A Gopinath, S Siddiqui, H Cheng, E Pectasides, RL Camp, DL Rimm. Yale Universtiy School of Medicine, New Haven, CT
Background: Post-translational modifications and especially phosphorylation has been shown in preclinical studies to be an excellent method of signaling pathway activation and predicting response to targeted therapies. However, there is evidence that phospho-proteins are dephosphorylated as a function of ischemic time prior to fixation, as is seen in routine processing of surgical pathology specimens. Core Needle Biopsies (CNBs) are typically very rapidly fixed. Here we use this conventional difference in ischemic time to determine the effect of delayed fixation on phospho-specific markers of pathway activation.
Design: Two series of matched CNBs and resections were collected. In series one, 20 cases were examined as a tissue microarray where each specimen was examined in two fold redundancy. The TMA was analyzed by the AQUA method of immunofluorescent analysis using antibodies to Ki67, p53 and Estrogen Receptor (ER) as controls and antibodies to phospho-Erk (p-ERK), phospho-AKT(p-AKT) and phospho-tyrosine (p-tyr) as the test set. The second cohort was analyzed as whole sections where between 5 and 29 20x fields were assessed on each tissue with an antibody to phospho-AKT.
Results: Both ER and p53 as assessed on the TMA cohort showed no overall trend toward decreased or increased expression in the core biopsy vs the resection specimen. However p-AKT, p-ERK, p-tyr and Ki-67 all showed lower expression in resection specimens. To rule out TMA sampling artifact, whole sections were assayed with an average of 12 and 19 fields for biopsies and resections respectively. In each case, there was consistently and significantly lower levels of pAKT in the resection than in the biopsy (Wilcoxon Signed-Ranks test p=0.0069).
Conclusions: This study shows that phospho-proteins are present at decreased levels as a function of ischemic time. This pattern is seen in both TMAs and whole sections. Surprisingly, Ki67 also shows this trend, which could have implications for its use in evaluation of efficacy of neoadjuvant therapy. CNBs appear to be the preferred method for analysis of phospho-proteins for use as a predictor for pathway activation and to assess response to targeted therapies.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, March 9, 2009 1:15 PM
Platform Session: Section G, Monday Afternoon