title: CD8+/FOXP3+-ratio in osteosarcoma microenvironment separates survivors from non-survivors: a multicenter validated retrospective study.
abstract: Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8+T-cells to FOXP3+T-cells (CD8+/FOXP3+-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004-2012). Highly standardized immunohistochemistry of CD8+and FOXP3+, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8+/FOXP3+-ratios above the median (3.08) compared to patients with lower CD8+/FOXP3+-ratios (p= 0.000001). No patients with a CD8+/FOXP3+-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival (p= 0.001) in patients with CD8+/FOXP3+-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8+/FOXP3+-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.
authors.affiliation: Department of Translational Pulmonology; Translational Lung Research Center (TLRC); Member of the German Center for Lung; University of Heidelberg , Heidelberg, Germany ; Divison of Pediatric Pulmonology & Allergy and Cystic Fibrosis Center; Department of Pediatric Oncology, Hematology; Immunology and Pulmonology; University of Heidelberg , Heidelberg, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Fritzsching
authors.affiliation: Department of Orthopedics and Traumatology; University Hospital Heidelberg , Heidelberg, Germany.
authors.forename: Joerg
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: Pathology Department; Instituto Valenciano de Oncologia , Valencia, Spain.
authors.forename: Isidro
authors.initials: I
authors.lastname: Machado
authors.affiliation: Division of Neonatology; Department of Pediatrics; University of 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 Pathology; Valencia University Medical School , Valencia, Spain.
authors.forename: Antonio Llombart
authors.initials: AL
authors.lastname: Bosch
authors.affiliation: Center for Orthopedics and Traumatology; Hospital Bielefeld , Germany.
authors.forename: Ludger
authors.initials: L
authors.lastname: Bernd
authors.affiliation: 2nd Department of Pediatrics; Semmelweis University , Budapest, Hungary.
authors.forename: Monika
authors.initials: M
authors.lastname: Csóka
authors.affiliation: Department of General Pathology; Institute of Pathology; University Hospital Heidelberg , Germany.
authors.forename: Gunhild
authors.initials: G
authors.lastname: Mechtersheimer
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 Orthopedics and Traumatology; University Hospital Heidelberg , Heidelberg, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Kunz
keywords: CD8+ T cells; CD8/FOXP3-ratio; CONSORT, consolidated standard of reporting trials; COSS, Cooperative German–Austrian–Swiss Osteosarcoma Study Group; EORTC, the European Organization for Research and Treatment of Cancer; FFPE, formalin-fixed and paraffin embedded; IDO, Indoleamine 2, 3-dioxygenase; MAP, methotrexate-cisplatin-doxorubicin; PD-L1, Programmed-death-Ligand-1; REMARK, reporting recommendations for tumor marker prognostic studies; TIL, tumor infiltrating lymphocyte; TNFα, tumornecrosis factor-α; TNM-I, tumor, nodes, metastases-immuno; Treg, regulatory Tcell.; immunoscore; osteosarcoma; regulatory T cells; tumor microenvironment