Application of Reverse Phase Protein Lysate Array (RPPA) to Formalin-Fixed, Paraffin-Embedded Tissues.
Russell Broaddus, Su-Su Xie, Caimiao Wei, Elizabeth Euscher. M.D. Anderson Cancer Center, Houston, TX
Background: RPPA is a high-throughput analysis in which dilutions of protein lysate are spotted on nitrocellulose slides (maximum 1,152 spots per slide). Each slide is probed with a different antibody. A DAKO-catalyzed signal amplification system is used for signal detection. RPPA requires less protein than Western blots. An advantage over immunohistochemistry is that RPPA is quantitative rather than qualitative. A current limitation of RPPA is that protein is typically derived from cell lines or frozen tissues, restricting its use to research settings. The RPPA platform incorporates many antibodies directed to phosphorylated proteins; these same antibodies typically do not work well for immunohistochemistry using formalin-fixed, paraffin-embedded (FFPE) tissues. It would therefore be advantageous if RPPA could be adapted to FFPE tissues.
Design: We examined FFPE uterine carcinosarcoma (CS; n=52). Uterine CS is a rare but highly malignant type of endometrial cancer with limited availability of frozen tissue. Fifty micrograms of protein were extracted from FFPE uterine CS, one paraffin block per case. Integrity of the protein was verified by Western. RPPA was performed using a panel of 225 antibodies directed towards proteins and phosphorylated proteins important in cancer cell growth regulation. ANOVA was used to calculate differences in protein expression between different subgroups of uterine CS.
Results: Protein differences were noted in the uterine CS cases with heterologous sarcomatous differentiation (presence of rhabomyosarcoma or chondrosarcoma). Twenty-eight proteins were differentially expressed in CS with heterologous components, including nine phosphorylated proteins (pARAF, pSRC, pSTAT3, pATM, pER, pX4EBP1, pYB1, pFAK, and pNFKB). Several of the differentially expressed proteins, EGFR, ER, HER2, VEGFR2, AKT, and SRC, are key components of cell signaling pathways that can be targeted by drugs currently in clinical trials.
Conclusions: The application of RPPA is feasible using FFPE tissues. RPPA therefore has the potential to be used in the clinical laboratory for routine cancer patient care. Using a combination of traditional H&E based light microscopic examination and RPPA, we have shown that uterine CS with heterologous sarcomatous elements differentially express transcription factors, DNA repair proteins, and cell growth regulatory proteins. These proteins may be important for targeted therapy of uterine CS as well as deciphering the molecular pathogenesis of these clinically aggressive neoplasms.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, February 28, 2011 1:00 PM
Poster Session II # 252, Monday Afternoon