title: Reliable quantification of protein expression and cellular localization in histological sections.
abstract: In targeted therapy, patient tumors are analyzed for aberrant activations of core cancer pathways, monitored based on biomarker expression, to ensure efficient treatment. Thus, diagnosis and therapeutic decisions are often based on the status of biomarkers determined by immunohistochemistry in combination with other clinical parameters. Standard evaluation of cancer specimen by immunohistochemistry is frequently impeded by its dependence on subjective interpretation, showing considerable intra- and inter-observer variability. To make treatment decisions more reliable, automated image analysis is an attractive possibility to reproducibly quantify biomarker expression in patient tissue samples. We tested whether image analysis could detect subtle differences in protein expression levels. Gene dosage effects generate well-graded expression patterns for most gene-products, which vary by a factor of two between wildtype and haploinsufficient cells lacking one allele. We used conditional mouse models with deletion of the transcription factors Stat5ab in the liver as well Junb deletion in a T-cell lymphoma model. We quantified the expression of total or activated STAT5AB or JUNB protein in normal (Stat5ab+/+ or JunB+/+), hemizygous (Stat5ab+/Δ or JunB+/Δ) or knockout (Stat5abΔ/Δ or JunBΔ/Δ) settings. Image analysis was able to accurately detect hemizygosity at the protein level. Moreover, nuclear signals were distinguished from cytoplasmic expression and translocation of the transcription factors from the cytoplasm to the nucleus was reliably detected and quantified using image analysis. We demonstrate that image analysis supported pathologists to score nuclear STAT5AB expression levels in immunohistologically stained human hepatocellular patient samples and decreased inter-observer variability.
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Schlederer
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Mueller
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Haybaeck
authors.affiliation: Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Heider
authors.affiliation: Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Nicole
authors.initials: N
authors.lastname: Huttary
authors.affiliation: Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
authors.forename: Margit
authors.initials: M
authors.lastname: Rosner
authors.affiliation: Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Hengstschläger
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria; Unit for Translational Methods in Cancer Research University of Veterinary Medicine Vienna (Vetmeduni Vienna), Vienna, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Moriggl
authors.affiliation: Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Dolznig
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria; Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria; Unit of Pathology of Laboratory Animals, University of Veterinary Medicine Vienna (Vetmeduni Vienna), Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Kenner
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D018345:Mice, Knockout; D015203:Reproducibility of Results; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; C584097:JunB protein, mouse; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; C494096:Stat5b protein, mouse; D014157:Transcription Factors