title: Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma.
abstract: Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology.
authors.affiliation: Department of Pathology, Division of Neuropathology, The Ohio State University College of Medicine, Columbus, Ohio, United States of America.
authors.forename: Behiye
authors.initials: B
authors.lastname: Kaya
authors.affiliation: Akdeniz University, Engineering Faculty, Computer Engineering Department, Antalya, Turkey.
authors.forename: Evgin
authors.initials: E
authors.lastname: Goceri
authors.affiliation: Department of Radiation Oncology, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Aline
authors.initials: A
authors.lastname: Becker
authors.affiliation: Department of Neurological Surgery, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Brad
authors.initials: B
authors.lastname: Elder
authors.affiliation: Division of Neuro-oncology, The Ohio State University Wexner Medical Center, Columbus, Ohio, United States of America.
authors.forename: Vinay
authors.initials: V
authors.lastname: Puduvalli
authors.affiliation: Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States of America.|William G. Lowie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Jessica
authors.initials: J
authors.lastname: Winter
authors.affiliation: Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Metin
authors.initials: M
authors.lastname: Gurcan
authors.affiliation: Department of Pathology, Division of Neuropathology, The Ohio State University College of Medicine, Columbus, Ohio, United States of America.
authors.forename: José Javier
authors.identifier: http://orcid.org/0000-0003-0680-4520
authors.initials: JJ
authors.lastname: Otero
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001932:Brain Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D018450:Disease Progression; D005260:Female; D005910:Glioma / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local; D038941:Polypyrimidine Tract-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference; D012189:Retrospective Studies; D055815:Young Adult
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C450006:PTBP1 protein, human; D038941:Polypyrimidine Tract-Binding Protein