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PLoS One. 2014 Mar 3;9(3):e90727. doi: 10.1371/journal.pone.0090727. eCollection 2014.

Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

Author information

1
Department of Orthopedics and Traumatology, University Hospital Heidelberg, Heidelberg, Germany.
2
1st Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University, Budapest, Hungary.
3
Department of Pediatrics, University Hospital Heidelberg, Heidelberg, Germany.
4
Department of Orthopedics, Semmelweis University, Budapest, Hungary.
5
Institute of Anatomy and Cell Biology, Department of Medical Cell Biology, University of Marburg, Marburg, Germany.
6
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.

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.

CONCLUSION:

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.

PMID:
24594971
PMCID:
PMC3940945
DOI:
10.1371/journal.pone.0090727
[Indexed for MEDLINE]
Free PMC Article
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