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

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          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.

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          HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues.

          Marilyn A. Owens, Bruce Horten, Moacyr M Da Silva (2004)
          To analyze HER2 amplification in a large cohort of diagnostic breast cancer specimens, fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed on the same specimens with use of Food and Drug Administration&approved products. Procedures were standardized following the manufacturers' recommendations. Of 116,736 IHC specimens, 20% were positive for HER2. In 16,092 FISH specimens, 22.7% showed HER2 amplification. In the subset of 6556 tissues analyzed with IHC and FISH, however, 59% were positive on IHC and 23.6% were amplified on FISH. The increased frequency of positive test results is skewed by more frequent reflex FISH testing. In general, expression and amplification trended together, with the least amplification (4.1%) seen in IHC-negative cases, 7.4% amplification seen in IHC 1+ cases, 23.3% amplification seen in IHC 2+ cases, and 91.7% amplification seen in IHC 3+ cases. When FISH amplification ratios were stratified, the low FISH ratios (2.0-2.2) were most frequently seen in specimens with negative IHC results, high ratios (>5.0) were seen in IHC 3+ specimens, and intermediate levels of amplification were similar for all levels of IHC. The effect of changing the cutoff point was analyzed: removing cases with a ratio of exactly 2.0 decreased the FISH positivity rate to 22.2% in the combined IHC and FISH cohort. Sequentially moving the cutoff point to 2.2 and 2.5 affected cases at all IHC expression levels. Each change removed approximately 2% from the apparent positivity. This large database provides the distribution frequency of HER2 protein expression and gene amplification in invasive ductal and lobular breast cancer. The relationship between level of HER2 amplification and clinical outcome will require reanalysis of pivotal trial data.
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            Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.

            Gary M. Bokoch, T Rudel (1997)
            Apoptosis of Jurkat T cells induced the caspase-mediated proteolytic cleavage of p21-activated kinase 2 (PAK2). Cleavage occurred between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, which generated a constitutively active PAK2 fragment. Stable Jurkat cell lines that expressed a dominant-negative PAK mutant were resistant to the Fas-induced formation of apoptotic bodies, but had an enhanced externalization of phosphatidylserine at the cell surface. Thus, proteolytic activation of PAK2 represents a guanosine triphosphatase-independent mechanism of PAK regulation that allows PAK2 to regulate morphological changes that are seen in apoptotic cells.
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              Subclassification of non-small cell lung carcinomas lacking morphologic differentiation on biopsy specimens: Utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6.

              Sanjay Mukhopadhyay, D A Katzenstein (2011)
              The availability of targeted therapies has created a need for precise subtyping of non-small cell lung carcinomas (NSCLCs). The aim of this study was to assess the utility of immunohistochemical markers in subtyping poorly differentiated NSCLC and to compare the results of immunohistochemical staining on biopsies with the corresponding resections. Thirty-nine cases of NSCLC that could not be further classified on biopsy and had subsequent resection specimens were identified. Classification of the tumor was based on the resection specimen using the World Health Organization criteria. All biopsies and resections were stained with CK7, TTF-1, napsin A (novel aspartic proteinase of the pepsin family), p63, CK5/6, and 34βE12. The specimens included 20 adenocarcinomas (ACs), 15 squamous cell carcinomas (SCCs), and 4 large-cell carcinomas (LCCs). TTF-1 was positive in biopsies from 16 of 20 ACs, 2 of 4 LCCs, and none of the SCCs. p63 was positive in all 15 SCCs, 2 of 20 ACs (both were also positive for TTF-1 and napsin A), and none of the LCCs. CK5/6 was positive in 11 of 15 SCCs (all p63 positive) but none of the ACs or LCCs. Napsin A stained 11 of 19 ACs (all TTF-1 positive) but none of the other tumors. Staining for CK7 was present in 19 of 19 ACs and 9 of 15 SCCs. 34βE12 stained both SCCs (15 of 15) and ACs (12 of 20). The combination of TTF-1, napsin A, p63, and CK5/6 allowed an accurate classification of 30 of39 (77%) cases. Of 232 pairs of slides (biopsy and resection) stained with immunohistochemical markers, 12 (5%) showed discrepancies in immunohistochemical staining between biopsies and their corresponding resections. Immunohistochemical staining using a combination of TTF-1, napsin A, p63, and CK5/6 allows subclassification of poorly differentiated NSCLCs on small lung biopsies in most cases. Discrepancies in immunohistochemical staining between biopsies and resections are uncommon.
              Article 0 comments Cited 68 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Mol Cell Proteomics
                Journal ID (iso-abbrev): Mol. Cell Proteomics
                Journal ID (hwp): mcprot
                Journal ID (pmc): mcprot
                Journal ID (publisher-id): MCP
                Title: Molecular & Cellular Proteomics : MCP
                Publisher: The American Society for Biochemistry and Molecular Biology
                ISSN (Print): 1535-9476
                ISSN (Electronic): 1535-9484
                Publication date (Print): September 2013
                Publication date (Electronic): 7 May 2013
                Publication date PMC-release: 7 May 2013
                Volume: 12
                Issue: 9
                Pages: 2615-2622
                Affiliations
                [1]From *Translational Research Sciences, Pharma Research & Early Development, F. Hoffmann - La Roche AG, Basel, Switzerland;
                [2]§Translational Research Sciences, Pharma Research & Early Development, Roche Diagnostics GmbH, Penzberg, Germany;
                [3]¶Oncology, Pharma Research & Early Development, Roche Diagnostics GmbH, Penzberg, Germany;
                [4]‖Department of Biochemistry and Protein Profiling, NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany
                Author notes
                ** To whom correspondence should be addressed: Thomas Schindler, Tel.: +41-61-6874573; E-mail: thomas.schindler@ 123456roche.com .

                ‡ The authors contributed to this work equally.

                Article
                Publisher ID: M112.023051
                DOI: 10.1074/mcp.M112.023051
                PMC ID: 3769334
                PubMed ID: 23653450
                SO-VID: 31fb4377-bc82-4030-b862-ded5985cae9d
                Copyright © © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
                License:

                Author's Choice—Final version full access.

                History
                Date received : 20 August 2012
                Date revision received : 9 April 2013
                Categories
                Subject: Technological Innovation and Resources

                ScienceOpen disciplines: Molecular biology
                Data availability:
                ScienceOpen disciplines: Molecular biology

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