Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Hanno Langen,Berthold Gierke,Tiantom Jarutat,Alexandra Farfsing,Miro Venturi,Ewa Breitinger,Michael Pawlak,Markus F Templin,Laurent Essioux,Susanne Arbogast,Thomas Schindler,Hubert Paul,Jens Lamerz,Maziar Assadi

doi:10.1074/mcp.m112.023051

Abstract

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Highlights

From *Translational Research Sciences, Pharma Research & Early Development, F
It would be highly desirable if protein-specific epitopes could be quantitatively extracted and analyzed from formalin-fixed and paraffin embedded (FFPE) tissue, as this would make the majority of clinical specimens accessible for mechanistic protein-based research
To assess whether the analysis of FFPE tissue lysates would result in biologically meaningful data, we analyzed FFPE breast cancer tissue samples by Reverse-phase protein arrays (RPPAs) for the expression of human epidermal growth factor receptor 2 (HER2) and compared it to HER2 assessment by the gold standard used in clinical practice, which is based on immunohistochemistry (IHC)

Summary

Technological Innovation and Resources

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays□S. Because of the high amount of effort involved in the use of liquid nitrogen for sample preservation, in almost all hospitals worldwide formalin fixation and paraffin embedding is the preferred method for tissue preservation It would be highly desirable if protein-specific epitopes could be quantitatively extracted and analyzed from formalin-fixed and paraffin embedded (FFPE) tissue, as this would make the majority of clinical specimens accessible for mechanistic protein-based research. To assess whether the analysis of FFPE tissue lysates would result in biologically meaningful data, we analyzed FFPE breast cancer tissue samples by RPPA for the expression of human epidermal growth factor receptor 2 (HER2) and compared it to HER2 assessment by the gold standard used in clinical practice, which is based on immunohistochemistry (IHC).

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

AC SCC