Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of Metastatic Ovarian Carcinoma
2005; Elsevier BV; Volume: 4; Issue: 4 Linguagem: Inglês
10.1074/mcp.t500003-mcp200
ISSN1535-9484
AutoresKatherine M. Sheehan, Valerie Calvert, Elaine W. Kay, Yiling Lu, David A. Fishman, Virginia Espina, Joy Aquino, Runa Speer, Robyn P. Araujo, Gordon B. Mills, Lance A. Liotta, Emanuel F. Petricoin, Julia Wulfkuhle,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoCancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge. Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge. Major discovery efforts brought about by advances in genomic and proteomic technologies have resulted in many new potential drug targets. Most of these new targets are proteins involved in cellular signaling, and a number of array-based technologies are being developed to assess and validate these candidates (1.Liotta L.A. 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Protein microarrays can be used to profile the working state of cellular signal pathways in a manner not possible with gene microarrays since post-translational modifications cannot be accurately portrayed by global gene expression patterns alone (3.Lal S.P. Christopherson R.I. dos Remedios C.G. Antibody arrays: an embryonic but rapidly growing technology.Drug Discov. Today. 2002; 7: S143-S149Google Scholar, 7.Hunter T. Signaling—2000 and beyond.Cell. 2000; 100: 113-127Google Scholar, 8.Blume-Jensen P. Hunter T. Oncogenic kinase signalling.Nature. 2001; 411: 355-365Google Scholar, 9.Celis J.E. Gromov P. Proteomics in translational cancer research: toward an integrated approach.Cancer Cell. 2003; 3: 9-15Google Scholar, 10.Jeong H. Tombor B. Albert R. Oltvai Z.N. Barabasi A.L. The large-scale organization of metabolic networks.Nature. 2000; 407: 651-654Google Scholar, 11.Charboneau L. 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Because most new drug targets for cancer and many other diseases are signaling-related, a proteomic approach that can elucidate ongoing post-translational phosphorylation events now makes it possible to generate a diagnostic portrait, based on the activity of the drug targets themselves, of who will respond to a particular therapy and who will not. Thus, providing clinicians with knowledge of which pathways are active in a patient's tumor will enable them to specifically apply targeted therapy. The technology may also be used to monitor total and phosphorylated proteins over time, before and after treatment, or between disease and non-disease states, allowing us to infer the activity levels of the proteins in a particular pathway in real time (21.Liotta L.A. Kohn E.C. Petricoin E.F. Clinical proteomics: personalized molecular medicine.J. Am. Med. Assoc. 2001; 286: 2211-2214Google Scholar, 22.Petricoin E.F. Zoon K.C. Kohn E.C. Barrett J.C. Liotta L.A. 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Proteomic approaches to molecular network analysis may provide more valuable clinical information than just "response" analysis and is an enabling tool for true patient-tailored therapy.PROTEIN MICROARRAY FORMATSProtein microarray formats can be divided into two major classes: forward phase arrays and reverse phase arrays (RPAs). 1The abbreviation used is: RPA, reverse phase array. In the forward phase array format, the analyte(s) of interest is captured from the solution phase by a capture molecule, usually an antibody, that is immobilized on a substratum and acts as bait molecule (1.Liotta L.A. Espina V. Mehta A.I. Calvert V. Rosenblatt K. Geho D. Munson P.J. Young L. Wulfkuhle J. Petricoin III, E.F. Protein microarrays: meeting analytical challenges for clinical applications.Cancer Cell. 2003; 3: 317-325Google Scholar, 2.Pavlickova P. Schneider E.M. Hug H. Advances in recombinant antibody microarrays.Clin. Chim. Acta. 2004; 343: 17-35Google Scholar) (Fig. 1). In a forward phase array, each spot contains one type of immobilized antibody or bait protein. Each array is incubated with one test sample such as a cellular lysate or serum sample representing a specific treatment condition, and multiple analytes from that sample are measured simultaneously. In contrast, the RPA format immobilizes an individual complex test sample in each array spot such that an array is comprised of hundreds of different patient samples or cellular lysates. In the RPA format, each array is incubated with one detection protein (i.e. antibody), and a single analyte end point is measured and directly compared across multiple samples (17.Paweletz C.P. Charboneau L. Bichsel V.E. Simone N.L. Chen T. Gillespie J.W. Emmert-Buck M.R. Roth M.J. Petricoin III, E.F. Liotta L.A. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.Oncogene. 2001; 20: 1981-1989Google Scholar, 24.Zha H. Raffeld M. Charboneau L. Pittaluga S. Kwak L.W. Petricoin III, E. Liotta L.A. Jaffe E.S. Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray.Lab. Investig. 2004; 84: 235-244Google Scholar, 26.Grubb R.L. Calvert V.S. Wulfkuhle J.D. Paweletz C.P. Linehan W.M. Phillips J.L. Chuaqui R. Valasco A. Gillespie J. Emmert-Buck M. Liotta L.A. Petricoin E.F. Signal pathway profiling of prostate cancer using reverse phase protein microarrays.Proteomics. 2003; 3: 2142-2146Google Scholar, 27.Wulfkuhle J.D. Aquino J.A. Calvert V.S. Fishman D.A. Coukos G. Liotta L.A. Petricoin III, E.F. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays.Proteomics. 2003; 3: 2085-2090Google Scholar, 28.Nishizuka S. Chen S.-T. Gwadry F.G. Alexander J. Major S.M. Scherf U. Reinhold W.C. Waltham M. Charboneau L. Young L. Bussey K.J. Kim S. Lababidi S. Lee J.K. Pittaluga S. Scudiero D.A. Sausville E.A. Munson P.J. Petricoin III, E.F. Liotta L.A. Hewitt S.M. Raffeld M. Weinstein J.N. Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.Cancer Res. 2003; 63: 5243-5250Google Scholar, 29.Nishizuka S. Charboneau L. Young L. Major S. Reinhold W.C. Waltham M. Kouros-Mehr H. Bussey K.J. Lee J.K. Espina V. Munson P.J. Petricoin III, E. Liotta L.A. Weinstein J.N. Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14229-14234Google Scholar) (Fig. 1). Probing multiple arrays spotted with the same lysate concomitantly with different phosphospecific antibodies provides the effect of generating a multiplex readout. Efforts are ongoing in our laboratory to multiplex the arrays even further through the use of dual color infrared dye-labeled antibodies as well as quantum dots. Using these technologies, it is hoped that multiple analytes can be measured on the same spot on the same array (30.Calvert V.S. Tang Y. Boveia V. Wulfkuhle J. Schutz-Geschwender A. Olive D.M. Liotta L.A. Petricoin E.F. Development of multiplexed protein profiling and detection using near infrared detection of reverse phase protein microarrays.Clin. Prot. 2004; 1: 81-89Google Scholar, 31.Geho D.H. Lahar N. Ferrari M. Petricoin E.F. Liotta L.A. Opportunities for nanotechnology-based innovation in tissue proteomics.Biomed. Microdevices. 2004; 6: 231-239Google Scholar). The utility of reverse phase protein microarrays lies in their ability to provide a map of known cell signaling proteins. Identification of critical nodes, or interactions, within the network is a potential starting point for drug development and/or the design of individual therapy regimens (21.Liotta L.A. Kohn E.C. Petricoin E.F. Clinical proteomics: personalized molecular medicine.J. Am. Med. Assoc. 2001; 286: 2211-2214Google Scholar, 22.Petricoin E.F. Zoon K.C. Kohn E.C. Barrett J.C. Liotta L.A. Clinical proteomics: translating benchside promise into bedside reality.Nat. Rev. Drug Discov. 2002; 1: 683-695Google Scholar). The array format is also amenable to extremely sensitive analyte detection (Fig. 2) with detection levels approaching attogram amounts of a given protein and variances of less than 10% (1.Liotta L.A. Espina V. Mehta A.I. Calvert V. Rosenblatt K. Geho D. Munson P.J. Young L. Wulfkuhle J. Petricoin III, E.F. Protein microarrays: meeting analytical challenges for clinical applications.Cancer Cell. 2003; 3: 317-325Google Scholar, 32.Espina V. Woodhouse E.C. Wulfkuhle J. Asmussen H.D. Petricoin III, E.F. Liotta L.A. Protein microarray detection strategies: focus on direct detection methods.J. Immunol. Methods. 2004; 290: 121-133Google Scholar). Detection ranges could be substantially lower in a complex mixture such as a cellular lysate; however, the sensitivity of the RPAs is such that low abundance phosphorylated isoforms can still be measured from a spotted lysate amount of less than 10 cell equivalents. This level of sensitivity combined with analytical robustness is critical if the starting input material is only a few hundred cells from a biopsy specimen.Fig. 2Example of sensitivity and reproducibility analysis of the reverse phase protein microarrays. The first spot of A1 and C1 is 45 pg, and the first spot of B1 and D1 is 38 pg. Rows A and C are duplicates from dilutions for recombinant active Akt protein. Rows B and D are duplicates from dilution for phospho-Akt (Ser-473) peptide. In each grid, the sample was diluted 2-fold from the first spot. Each dilution was spotted in duplicates. From columns 1 to 10, each first spot is 2-fold diluted from the previous one. For example, the first spot of grid A1 is 45 pg, the first spot of grid A2 is 22.5 pg, and the first spot of grid A3 is 11.25 pg. The array was stained with phospho-Akt antibody (1:250 dilution).View Large Image Figure ViewerDownload (PPT)The reverse phase protein array has demonstrated a unique ability to analyze signaling pathways using small numbers of cultured cells or cells isolated by laser capture microdissection from human tissue procured during clinical trials (17.Paweletz C.P. Charboneau L. Bichsel V.E. Simone N.L. Chen T. Gillespie J.W. Emmert-Buck M.R. Roth M.J. Petricoin III, E.F. Liotta L.A. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.Oncogene. 2001; 20: 1981-1989Google Scholar, 24.Zha H. Raffeld M. Charboneau L. Pittaluga S. Kwak L.W. Petricoin III, E. Liotta L.A. Jaffe E.S. Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray.Lab. Investig. 2004; 84: 235-244Google Scholar, 26.Grubb R.L. Calvert V.S. Wulfkuhle J.D. Paweletz C.P. Linehan W.M. Phillips J.L. Chuaqui R. Valasco A. Gillespie J. Emmert-Buck M. Liotta L.A. Petricoin E.F. Signal pathway profiling of prostate cancer using reverse phase protein microarrays.Proteomics. 2003; 3: 2142-2146Google Scholar, 27.Wulfkuhle J.D. Aquino J.A. Calvert V.S. Fishman D.A. Coukos G. Liotta L.A. Petricoin III, E.F. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays.Proteomics. 2003; 3: 2085-2090Google Scholar). Using this approach, microdissected pure cell populations are taken from human biopsy specimens, and a protein lysate is arrayed onto nitrocellulose-coated slides (Fig. 3). Key technological components of this method offer unique advantages over tissue arrays (33.Torhorst J. Bucher C. Kononen J. Haas P. Zuber M. Kochli O.R. Mross F. Dieterich H. Moch H. Mihatsch M. Kallioniemi O.-P. Sauter G. Tissue microarrays for rapid linking of molecular changes to clinical endpoints.Am. J. Pathol. 2001; 159: 2249-2256Google Scholar) or antibody arrays (34.Sreekumar A. Nyati M.K. Varambally S. Barrette T.R. Ghosh D. Lawrence T.S. Chinnaiyan A.M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.Cancer Res. 2001; 61: 7585-7593Google Scholar, 35.Knezevic V. Leethanakul C. Bichsel V.E. Worth J.M. Prabhu V.V. Gutkind J.S. Liotta L.A. Munson P.J. Petricoin III, E.F. Krizman D.B. Proteomic profiling of the cancer microenvironment by antibody arrays.Proteomics. 2001; 1: 1271-1278Google Scholar). First the RPA can use denatured lysates so that antigen retrieval, which is a large limitation for tissue arrays, is not problematic. Protein microarrays can also consist of non-denatured lysates derived directly from microdissected tissue cells so that protein-protein, protein-DNA, and/or protein-RNA complexes can be detected and characterized. Each patient sample is printed on the array in serial dilutions, providing an internal standard. When an internal reference standard of known and fixed amounts of the analyte are applied to the same array, a direct and quantitative measurement of the phosphorylated end point can be attained within the linear dynamic range of the assay. Finally RPAs do not require direct labeling of the patient sample as a readout for the assay, which provides a marked improvement in reproducibility, sensitivity, and robustness of the assay over other techniques (36.Espina V. Mehta A.I. Winters M.E. Calvert V. Wulfkuhle J. Petricoin III, E.F. Liotta L.A. Protein microarrays: molecular profiling technologies for clinical specimens.Proteomics. 2003; 3: 2091-2100Google Scholar).Fig. 3Application of reverse phase arrays in mapping molecular networks of ovarian cancer.A, using laser capture microdissection (LCM), ovarian cancer epithelial cells were isolated under direct microscopic vision from stained tissue sections leaving residual stroma on the slide. Approximately 25,000 cells were dissected for each case and lysed directly on the laser capture microdissection cap with extraction buffer. One hundred arrays were printed on nitrocellulose-coated slides. H&E, hematoxylin and eosin. B, example of an ovarian cancer reverse phase array probed for active extracellular signal-regulated kinase (ERK) signaling using a phosphospecific antibody detected with a tyramide-based avidin/biotin amplification system. Samples are printed in two columns: the left column represents primary tumors, and the right column represents metastatic lesions. Cases are printed in duplicate, five-point dilution curves to ensure the linear detection range for the antibody concentration is achieved. The sixth point represents a negative control consisting of extraction buffer alone. A positive control lysate (A431 squamous carcinoma cell line) is printed on the array for monitoring immunostaining performance. Phosphorylation-specific reference peptides are printed in a 12-point dilution curve on the bottom of the array for comparative, precise quantification of patient samples between arrays. pERK, phosphorylated extracellular signal-regulated kinase; EGF, epidermal growth factor. C, stained slides for the multiple phosphorylation-specific end points were scanned using Adobe Photoshop. Following total protein estimation with a Sypro Ruby stain, the intensity values of each antibody were normalized to total protein, and dilution curves were generated using Microvigene software. Histograms could then be generated to compare alterations in cell signaling between the primary and metastatic samples.View Large Image Figure ViewerDownload (PPT)The RPA platform has been used to explore a variety of signaling pathways involved in malignant progression and tumor biology (17.Paweletz C.P. Charboneau L. Bichsel V.E. Simone N.L. Chen T. Gillespie J.W. Emmert-Buck M.R. Roth M.J. Petricoin III, E.F. Liotta L.A. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.Oncogene. 2001; 20: 1981-1989Google Scholar, 26.Grubb R.L. Calvert V.S. Wulfkuhle J.D. Paweletz C.P. Linehan W.M. Phillips J.L. Chuaqui R. Valasco A. Gillespie J. Emmert-Buck M. Liotta L.A. Petricoin E.F. Signal pathway profiling of prostate cancer using reverse phase protein microarrays.Proteomics. 2003; 3: 2142-2146Google Scholar, 27.Wulfkuhle J.D. Aquino J.A. Calvert V.S. Fishman D.A. Coukos G. Liotta L.A. Petricoin III, E.F. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays.Proteomics. 2003; 3: 2085-2090Google Scholar, 28.Nishizuka S. Chen S.-T. Gwadry F.G. Alexander J. Major S.M. Scherf U. Reinhold W.C. Waltham M. Charboneau L. Young L. Bussey K.J. Kim S. Lababidi S. Lee J.K. Pittaluga S. Scudiero D.A. Sausville E.A. Munson P.J. Petricoin III, E.F. Liotta L.A. Hewitt S.M. Raffeld M. Weinstein J.N. Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.Cancer Res. 2003; 63: 5243-5250Google Scholar, 29.Nishizuka S. Charboneau L. Young L. Major S. Reinhold W.C. Waltham M. Kouros-Mehr H. Bussey K.J. Lee J.K. Espina V. Munson P.J. Petricoin III, E. Liotta L.A. Weinstein J.N. Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14229-14234Google Scholar, 37.Celis J.E. Moreira J.M. Gromova I. Cabezon T. Ralfkiaer U. Guldberg P. Straten P.T. Mouridsen H. Friis E. Holm D. Rank F. Gromov P. Towards discovery-driven translational research in breast cancer.FEBS J. 2005; 272: 2-15Google Scholar). For example, in a study of prostate tissue, pathway profiling of microdissected cells from normal, stroma, and prostate tumors revealed the preliminary finding that activation of protein kinase C α is down-modulated in prostate cancer progression (26.Grubb R.L. Calvert V.S. Wulfkuhle J.D. Paweletz C.P. Linehan W.M. Phillips J.L. Chuaqui R. Valasco A. Gillespie J. Emmert-Buck M. Liotta L.A. Petricoin E.F. Signal pathway profiling of prostate cancer using reverse phase protein microarrays.Proteomics. 2003; 3: 2142-2146Google Scholar). If validated, this finding could have profound effects on the rationale behind some current therapies (38.Tolcher A.W. Reyno L. Venner P.M. Ernst S.D. Moore M. Geary R.S. Chi K. Hall S. Walsh W. Dorr A. Eisenhauer E. A randomized phase II and pharmacokinetic study of the antisense oligonucleotides ISIS 3521 and ISIS 5132 inpatients with hormone-refractory prostate cancer.Clin. Cancer Res. 2002; 8: 2530-2535Google Scholar) and illustrates the importance of proteomic technology coupled to signal pathway profiling in providing new and unexpected insights into cellular processes.MAPPING MOLECULAR NETWORKS IN EPITHELIAL OVARIAN CANCERWith these potentials in mind, we are elucidating the value of RPAs in several types of human cancer tissues to gain insights into potential novel therapeutic strategies. In particular, epithelial ovarian cancer represents a clinical challenge for which much remains to be discovered. Of all gynecological cancers, it carries the worst prognosis primarily due to the late stage at presentation. For the patient with advanced disease, cytoreductive surgery and cytotoxic chemotherapy with taxane and platinum compounds will produce an initial response in the majority of patients, but ultimately most will experience relapse or develop drug resistance and consequently die of their disease (39.Agarwal R. Kaye S.B. Ovarian cancer: strategies for overcoming resistance to chemotherapy.Nat. Rev. Cancer. 2003; 3: 502-516Google Scholar).As a result of poor outcome and inability to detect disease confined to the organ, a lot of emphasis has been directed at identifying new disease biomarkers, indicators of response to therapy, and novel treatment options for patients with advanced or refractory disease. Newer chemotherapeutic agents including topoisomerase I inhibitors and taxane analogues may offer scope for defeating resistant neoplastic cells (40.See H.T. Kavanagh J.J. Novel agents in epithelial ovarian cancer.Cancer Investig. 2004; 22: 29-44Google Scholar). Furthermore our increasing knowledge of the molecular biology of ovarian cancer coupled with advances in global expression profiling has led to the development of novel targeted therapies including monoclonal antibodies, small molecule inhibitors, gene therapy, selective hormonal agents, and cytokines (40.See H.T. Kavanagh J.J. Novel agents in epithelial ovarian cancer.Cancer Investig. 2004; 22: 29-44Google Scholar). For epithelial ovarian cancer, much emphasis has been placed on developing agents that block epidermal growth factor receptor signaling with either monoclonal antibody (cetuximab) (41.Mendelsohn J. Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer.J. Clin. Oncol. 2003; 21: 2787-2799Google Scholar) or small molecule inhibitors of the receptor tyrosine kinase (gefitinib) (42.Sewell J.M. Macleod K.G. Ritchie A. Smyth J.F. Langdon S.P. Targeting the EGF receptor in ovarian cancer with the tyrosine kinase inhibitor ZD 1839 ("Iressa").Br. J. Cancer. 2002; 86: 456-462Google Scholar). Other innovative agents undergoing trials that specifically target signal transduction pathways directly associated with tumor growth and progression include bevacizumab (Avastatin), a monoclonal antibody that inhibits vascular endothelial growth factor receptor (43.Ferrara, N. (2002) Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 10–14Google Scholar), and imatinib mesylate (Gleevec), a small molecule inhibitor of three tyrosine kinases, Bcr-Abl, c-Kit, and platelet-derived growth factor receptor (44.Sattler M. Salgia R. Targeting c-Kit mutations: basic science to novel therapies.Leukoc. Res. 2004; 28: S11-S20Google Scholar). However, as a consequence of the heterogeneous nature of ovarian cancer, the efficacy of specific cancer agents will invariably only suit a subset of patients and probably only at a particular stage of their disease. Given the multitude of molecular defects in ovarian cancer, it is therefore necessary to profile multiple signal transduction pathways simultaneously and define a carcinogenic "fingerprint" specific to the patient. Novel agents can then be selectively applied either alone or in combination with other novel or existing treatments.Our laboratories have a significant interest in defining and tailoring combinations of new specific drug targets for patients with ovarian and other cancers using the reverse phase arrays to characterize the activated state of cellular signaling pathways in human patient biopsy material. At a molecular level, the process of growth, invasion, and migration of neoplastic cells is driven by a substantial number of integrated and interconnecting pathways that can be quantitatively and sensitively detected in human tissue lysates using protein microarray methodology (45.Liotta L.A. Kohn E.C. The microenvironment of the tumour-host interface.Nature. 2001; 411: 375-379Google Scholar). In a previous study, ovarian cancer epithelial cells were microdissected by laser capture (46.Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Google Scholar), and the activation state of prosurvival and mitogenic signaling pathways was evaluated to assess the profiles in primary tumors at different stages of disease progression (27.Wulfkuhle J.D. Aquino J.A. Calvert V.S. Fishman D.A. Coukos G. Liotta L.A. Petricoin III, E.F. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays.Proteomics. 2003; 3: 2085-2090Google Scholar). While the levels of phosphorylated extracellular signal-regulated kinase 1/2 were higher in advanced stage tumors and in those with endometrioid morphology in conjunction with Akt, expression levels tended to be more patient-specific rather than stage-specifi
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