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.
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.