Abstract
BackgroundIn 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.MethodsImmunohistochemistry 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.ResultsMore 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.ConclusionStandard 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.
Highlights
Patients with osteosarcoma still suffer from a poor prognosis with a 5 year median survival of 30–75%
We considered CD31, CD8 and FOXP3 as immunovascular biomarkers of major interest in osteosarcoma, representing intratumoral vessels (CD31), tumor attacking T-cells (CD8) and immunosuppressive or anergic T-cells (FOXP3)
Systematic evaluation of different conditions for antigen retrieval (Table 1) in serial sections revealed that enzymatic antigen retrieval in combination with polymer detection is superior to heat-induced antigen retrieval and conventional avidin-biotin complex based detection (Fig. 1A,B)
Summary
Patients with osteosarcoma still suffer from a poor prognosis with a 5 year median survival of 30–75%. In contrast to emerging quantification techniques [8,22,23,24,25,26,27], the current gold standard for quantification of tumor microenvironment relies on conventional immunohistochemical analysis of subjectively selected hot spots [8,17,28] It is even unclear if such hot spot areas can be defined in all tissues analyzed since hot spot based quantification originates from histopathological examination of carcinomas with homogeneous morphologies and abundant hot spot distribution patterns of vessels and tumor infiltrating cells [8,17,28,29]. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
