title: Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.
abstract: BACKGROUND
  In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment.
  
  
  METHODS
  Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis.
  
  
  RESULTS
  More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method.
  
  
  CONCLUSIONS
  Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations.
authors.affiliation: Department of Orthopedics and Traumatology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Kunz
authors.affiliation: Department of Orthopedics and Traumatology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fellenberg
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Linda
authors.initials: L
authors.lastname: Moskovszky
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Sápi
authors.affiliation: 1st Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
authors.forename: Tibor
authors.initials: T
authors.lastname: Krenacs
authors.affiliation: Department of Pediatrics, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Poeschl
authors.affiliation: Department of Orthopedics and Traumatology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Lehner
authors.affiliation: Department of Orthopedics, Semmelweis University, Budapest, Hungary.
authors.forename: Miklos
authors.initials: M
authors.lastname: Szendrõi
authors.affiliation: Department of Orthopedics and Traumatology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Ewerbeck
authors.affiliation: Institute of Anatomy and Cell Biology, Department of Medical Cell Biology, University of Marburg, Marburg, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kinscherf
authors.affiliation: Department of Pediatrics, University Hospital Heidelberg, Heidelberg, Germany; Department of Translational Pulmonology, Translational Lung Research Center (TLRC), Member of the German Center for Lung, University of Heidelberg, Heidelberg, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Fritzsching
mesh_terms: D000951:Antigens, Neoplasm / Q000097:blood; D003952:Diagnostic Imaging / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D012516:Osteosarcoma / Q000276:immunology*; D059016:Tumor Microenvironment / Q000276:immunology*
chemical_list: D000951:Antigens, Neoplasm