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

Open AccessPublished:May 07, 2013DOI:https://doi.org/10.1074/mcp.M112.023051
      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.
      Many diseases are characterized by the expression of specific proteins and the activation status of distinct signaling pathways (
      • Hopkins A.L.
      Network pharmacology: the next paradigm in drug discovery.
      ). Thus, protein expression profiling and activation patterns are instrumental for understanding disease, the development of effective treatments, and the identification of patients who will respond to particular therapies. Traditional ways of analyzing protein expression (e.g. Western blot) can be used for these purposes but often are labor intensive, have low throughput, and consume high sample volumes. Reverse-phase protein array (RPPA)
      The abbreviations used are:
      AC
      adenocarcinoma
      FFPE
      formalin-fixed and paraffin embedded
      FISH
      fluorescence in situ hybridization
      HER2
      human epidermal growth factor receptor 2
      IHC
      immunohistochemistry
      NSCLC
      non-small cell lung cancer
      PAK
      p21-activated kinase
      PAK2
      p21-activated kinase 2
      RPPA
      reverse phase protein array
      SSC
      squamous cell carcinoma.
      1The abbreviations used are:AC
      adenocarcinoma
      FFPE
      formalin-fixed and paraffin embedded
      FISH
      fluorescence in situ hybridization
      HER2
      human epidermal growth factor receptor 2
      IHC
      immunohistochemistry
      NSCLC
      non-small cell lung cancer
      PAK
      p21-activated kinase
      PAK2
      p21-activated kinase 2
      RPPA
      reverse phase protein array
      SSC
      squamous cell carcinoma.
      technology is a very promising method that circumvents these issues (
      • Malinowsky K.
      • Wolff C.
      • Ergin B.
      • Berg D.
      • Becker K.F.
      Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays.
      ,
      • Wilson B.
      • Liotta L.A.
      • Petricoin 3rd, E.
      Monitoring proteins and protein networks using reverse phase protein arrays.
      ,
      • Voshol H.
      • Ehrat M.
      • Traenkle J.
      • Bertrand E.
      • van Oostrum J.
      Antibody-based proteomics: analysis of signaling networks using reverse protein arrays.
      ). For RPPA, minute amounts of whole protein lysates from a multitude of samples are spotted onto slides, and individual proteins are detected via protein-specific antibodies. This enables medium- to high-throughput analysis of precious low-volume sample material.
      Lysates for RPPA have so far been generated mainly from cell lines or fresh frozen tissue. However, 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. Therefore, 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.
      In recent years, several research groups have established protocols for protein extraction from FFPE tissue. Common to all of them is the use of high concentrations of ionic detergents, such as sodium dodecyl sulfate, and high temperature. It was shown that these methods even make it possible to extract full-length proteins from FFPE tissue (
      • Becker K.F.
      • Schott C.
      • Hipp S.
      • Metzger V.
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      • Beck R.
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      Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.
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      A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section.
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      Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.
      ,
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      • Selby P.J.
      • Banks R.E.
      Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting.
      ,
      • Chung J.Y.
      • Lee S.J.
      • Kris Y.
      • Braunschweig T.
      • Traicoff J.L.
      • Hewitt S.M.
      A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue.
      ,
      • Shi S.R.
      • Liu C.
      • Balgley B.M.
      • Lee C.
      • Taylor C.R.
      Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.
      ,
      • Becker K.F.
      • Schott C.
      • Becker I.
      • Höfler H.
      Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains.
      ,
      • Addis M.F.
      • Tanca A.
      • Pagnozzi D.
      • Crobu S.
      • Fanciulli G.
      • Cossu-Rocca P.
      • Uzzau S.
      Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues.
      ). The coefficient of variation of the relative extraction efficiency based on Western blot and densitometric assessment of actin typically is below 20% (
      • Nirmalan N.J.
      • Harnden P.
      • Selby P.J.
      • Banks R.E.
      Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using Western blotting.
      ). 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). Successful recovery of HER2 from FFPE tissue should result in concordant HER2 classification between RPPA and IHC.
      In the second part of the study, FFPE samples of non-small cell lung cancer (NSCLC) were examined via RPPA. Samples from two subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC), were analyzed for more than 150 proteins, including two proteins that are known to be differentially expressed between the two subtypes. The objectives of this analysis were to further assess the validity of the approach by confirming the two positive controls and to identify new markers for the differentiation of the two subtypes of NSCLC.

      DISCUSSION

      The quantification of a protein in extracts from tissue depends on two factors: (i) the abundance of the protein of interest in the tissue, and (ii) whether the protein of interest can be quantitatively extracted out of the tissue. The IHC assay for HER2 is currently the gold standard for quantitative assessment of HER2 levels in breast cancer tissue and has been shown to provide clinically important prognostic and predictive information (
      • Navolanic P.M.
      • Steelman L.S.
      • McCubrey J.A.
      EGFR family signaling and its association with breast cancer development and resistance to chemotherapy (review).
      ,
      • Ross J.S.
      • Fletcher J.A.
      • Bloom K.J.
      • Linette G.P.
      • Stec J.
      • Symmans W.F.
      • Pusztai L.
      • Hortobagyi G.N.
      Targeted therapy in breast cancer: the HER-2/neu gene and protein.
      ,
      • Hayes D.F.
      • Thor A.D.
      c-erbB-2 in breast cancer: development of a clinically useful marker.
      ,
      • Masood S.
      • Bui M.M.
      Prognostic and predictive value of HER2/neu oncogene in breast cancer.
      ,
      • Schnitt S.J.
      • Jacobs T.W.
      Current status of HER2 testing: caught between a rock and a hard place.
      ). The scoring system of this assay is based on the receptor number in the membranes of cancer cells: no staining (0), partial membrane staining (1+), light-to-moderate complete membrane staining (2+), and complete membrane staining (3+). Our hypothesis was that a reliable assessment of HER2 levels in breast cancer samples with RPPA should result in classification similar to that obtained with the IHC assay. A very good Spearman correlation coefficient between IHC and RPPA shows that this indeed is the case.
      For a more appropriate comparison of the IHC and RPPA data, an ordinal multinomial logistic regression model was used that utilized the RPPA signal intensity to compute the probability that each sample would have a HER2 score equivalent to one of the IHC scores of 0 to 3+. This model shows a good assignment of RPPA signals to IHC scores of 2+ and 3+, whereas distinguishing between IHC scores of 0 and 1+ by RPPA is error prone. Additionally, when using this model, the agreement between IHC and RPPA for categorization was shown to be highly concordant. This positive finding owes partly to the fact that the model was trained and assessed with the same samples. The concordance of IHC and RPPA was confirmed in an independent validation sample set with high significance. Overall, the κ value was lower in the validation run. The validation sample set was procured from two different suppliers, which might have been a source of additional variability.
      For IHC-based HER2 classification, evaluation by different observers does not result in complete interobserver agreement, despite the standardized scoring system of the HER2 IHC assay (
      • Kay E.W.
      • Walsh C.J.
      • Cassidy M.
      • Curran B.
      • Leader M.
      C-erbB-2 immunostaining: problems with interpretation.
      ). In a study by Thomson et al. with three observers, a κ of 0.7 was obtained with this test (
      • Thomson T.A.
      • Hayes M.M.
      • Spinelli J.J.
      • Hilland E.
      • Sawrenko C.
      • Phillips D.
      • Dupuis B.
      • Parker R.L.
      HER-2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization.
      ). The Dako HercepTest is performed with the same antibody as used in our study for the assessment of HER2 levels with RPPA. Based on the κ estimates obtained in our study, the agreement between IHC and RPPA data in the initial set can be considered as very good (κ = 0.899), and that of the validation set as good (κ = 0.643). However, the true validation took place when the model parameters trained on the initial set were applied to predict the HER2 score using the RPPA intensities from the truly independent validation set (κ = 0.403), which confirmed the validity of the approach. If we had chosen to use set 1 as the validation set and set 2 as the prediction set, we could report a much better level of prediction, resulting in an agreement of κ = 0.702. This again emphasizes the need for well-characterized samples obtained by means of good standardized protocols.
      Another assay for the detection of HER2 amplification and overexpression is based on the FISH technique (
      • Seelig S.
      Fluorescence in situ hybridization versus immunohistochemistry: importance of clinical outcome.
      ,
      • Lal P.
      • Salazar P.A.
      • Hudis C.A.
      • Ladanyi M.
      • Chen B.
      HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring.
      ,
      • Owens M.A.
      • Horten B.C.
      • Da Silva M.M.
      HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues.
      ,
      • Press M.F.
      • Sauter G.
      • Bernstein L.
      • Villalobos I.E.
      • Mirlacher M.
      • Zhou J.Y.
      • Wardeh R.
      • Li Y.T.
      • Guzman R.
      • Ma Y.
      • Sullivan-Halley J.
      • Santiago A.
      • Park J.M.
      • Riva A.
      • Slamon D.J.
      Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials.
      ,
      • Tubbs R.R.
      • Hicks D.G.
      • Cook J.
      • Downs-Kelly E.
      • Pettay J.
      • Hartke M.B.
      • Hood L.
      • Neelon R.
      • Myles J.
      • Budd G.T.
      • Moore H.C.
      • Andresen S.
      • Crowe J.P.
      Fluorescence in situ hybridization (FISH) as primary methodology for the assessment of HER2 status in adenocarcinoma of the breast: a single institution experience.
      ,
      • Ross J.S.
      • Symmans W.F.
      • Pusztai L.
      • Hortobagyi G.N.
      Standardizing slide-based assays in breast cancer: hormone receptors, HER2, and sentinel lymph nodes.
      ). FISH is considered more objective and reproducible (
      • Tubbs R.R.
      • Hicks D.G.
      • Cook J.
      • Downs-Kelly E.
      • Pettay J.
      • Hartke M.B.
      • Hood L.
      • Neelon R.
      • Myles J.
      • Budd G.T.
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      • Crowe J.P.
      Fluorescence in situ hybridization (FISH) as primary methodology for the assessment of HER2 status in adenocarcinoma of the breast: a single institution experience.
      ,
      • Dendukuri N.
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      Testing for HER2-positive breast cancer: a systematic review and cost-effectiveness analysis.
      ,
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      • Paik S.
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      ,
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      ,
      • Perez E.A.
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      ,
      • Hicks D.G.
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      ). We therefore also tested the initial breast cancer sample set through FISH for HER2 positivity, and again good concordance between RPPA and FISH was demonstrated. RPPA analysis of FFPE breast cancer tissue could have allowed differentiation between HER2 negative and positive samples.
      Based on the aforementioned results, we assume that HER2 can quantitatively be extracted out of FFPE tissue samples and that the RPPA analysis of these samples delivers biologically meaningful results. This outcome is supported by a recent study by Wulfkuhle et al. (
      • Wulfkuhle J.D.
      • Berg D.
      • Wolff C.
      • Langer R.
      • Tran K.
      • Illi J.
      • Espina V.
      • Pierobon M.
      • Deng J.
      • DeMichele A.
      • Walch A.
      • Bronger H.
      • Becker I.
      • Waldhör C.
      • Höfler H.
      • Esserman L
      • I-SPY 1 TRIAL Investigators
      • Liotta L.A.
      • Becker K.-F.
      • Petricoin E.F.
      Molecular analysis of HER2 breast cancer by functional protein pathway activation mapping.
      ) showing that RPPA measurements of total HER2 protein can efficiently discriminate dichotomized HER2 status (negative: HER2 IHC 0 or 1+ and 2+/FISH; positive: HER2 IHC 2+/FISH+ and IHC 3+) as determined by IHC or FISH measurements, or both, in the same tissue.
      In a second study on NSCLC, we further demonstrated the validity of RPPA profiling of FFPE tissue by confirming two markers, cytokeratin 5 and napsin A, known to be differentially expressed between two NSCLC subtypes (AC and SCC) (
      • Mukhopadhyay S.
      • Katzenstein A.L.
      Subclassification of non-small cell lung carcinomas lacking morphological differentiation on biopsy specimens: utility of an immunohistochemical panel containing TTF-1, napsin A, p63, and CK5/6.
      ). In an analysis of more than 150 proteins, increased expression of PAK2 in SCC relative to AC was shown and was orthogonally verified by means of Western blot analysis.
      The p21-activated kinases (PAKs) are a family of serine/threonine protein kinases that are activated in response to extracellular signals (through both GTPase-dependent and -independent mechanisms) and regulate a wide variety of cellular processes including cell morphogenesis, motility, apoptosis, and cell cycle regulation. In epithelial cancer cells, the expression of PAKs promotes migration and increases anchorage-independent growth (
      • Kumar R.
      • Vadlamudi R.K.
      Emerging functions of p21-activated kinases in human cancer cells.
      ). Increased expression of PAK1 was found to be significantly associated with the malignant progression of human colorectal carcinoma (
      • Carter J.H.
      • Douglass L.E.
      • Deddens J.A.
      • Colligan B.M.
      • Bhatt T.R.
      • Pemberton J.O.
      • Konicek S.
      • Hom J.
      • Marshall M.
      • Graff J.R.
      Pak-1 expression increases with progression of colorectal carcinomas to metastasis.
      ) and with tamoxifen resistance in hormone-receptor-positive breast tumor from premenopausal patients (
      • Holm C.
      • Rayala S.
      • Jirström K.
      • Stål O.
      • Kumar R.
      • Landberg G.
      Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients.
      ). Interestingly, strong PAK1 expression was described as prevalent in SCC (
      • Ong C.C.
      • Jubb A.M.
      • Haverty P.M.
      • Zhou W.
      • Tran V.
      • Truong T.
      • Turley H.
      • O'Brien T.
      • Vucic D.
      • Harris A.L.
      • Belvin M.
      • Friedman L.S.
      • Blackwood E.M.
      • Koeppen H.
      • Hoeflich K.P.
      Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells.
      ). PAK2 might play a similar role in tumorigenesis. However, the relationship between PAK2 activity and cell survival appears to be complex. PAK2 is unique among the PAKs in that it is also activated through proteolytic cleavage by caspases or caspase-like proteases. Whereas the activation of PAK2 by Rac or CDC42, as with PAK1, promotes cell survival by phosphorylating Bad and Bcl-2 (
      • Jakobi R.
      • Moertl E.
      • Koeppel M.A.
      p21-activated protein kinase gamma-PAK suppresses programmed cell death of BALB3T3 fibroblasts.
      ), apoptotic stimuli such as DNA damage lead to proteolytic cleavage of PAK2, generating a p34 fragment. The activated p34 fragment leads to extensive membrane blebbing, cytoplasmic shrinkage, and apoptosis (
      • Rudel T.
      • Bokoch G.M.
      Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.
      ,
      • Jakobi R.
      • McCarthy C.C.
      • Koeppel M.A.
      • Stringer D.K.
      Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal degradation.
      ). This strongly suggests that PAK2 could be helpful for the diagnosis and/or identification of new treatment targets of SCC.
      Through two separate studies on breast cancer and NSCLC, we were able to demonstrate that RPPA analysis of FFPE tissue lysates gives rise to biologically meaningful data. FFPE is the fixation of choice in the clinical routine for preserving human tissue samples. Our findings encourage the application of RPPA in different tissues and disease contexts for detecting differences in protein expression and activation levels that could be beneficial for different purposes such as understanding disease mechanisms, the identification of new targets for drug development, distinction among tumor subtypes for diagnosis, or the stratification of patients for therapy regimens. Overall, the outcome of our study demonstrates the usefulness of RPPA as a screening tool for the selection of candidate proteins for the above-mentioned purposes.

      Acknowledgments

      We thank Y. Heubach and P. Graemmel for excellent technical assistance.

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