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Oncoimmunology. 2017 Aug 24;6(9):e1331193. doi: 10.1080/2162402X.2017.1331193. eCollection 2017.

CD163-positive tumor-associated macrophages and CD8-positive cytotoxic lymphocytes are powerful diagnostic markers for the therapeutic stratification of osteosarcoma patients: An immunohistochemical analysis of the biopsies fromthe French OS2006 phase 3 trial.

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Department of Pathology, IUCT-Oncopole, CHU of Toulouse and University of Toulouse, Pharmacology and Structural Biology Institute, Toulouse, France.
Biostatistics Unit, Claudius Regaud Institut, IUCT-Oncopole, Toulouse, France.
Department of Pathology, CHU la Timone, Marseille, France.
Department of Pathology, CHRU Lille, Lille, France.
Department of Pathology, Curie Institut, Paris, France.
Department of Pathology, CHU Nancy, Nancy, France.
AP-HP, Hôpital Cochin, Service de Pathologie and Université Paris Descartes, Paris, France.
Pediatric Oncology Department, Hautepierre, Strasbourg, France.
Department of Pathology, CHU Tours, Tours, France.
INSERM UMR1238, Université de Nantes, Nantes, France.
Department of Children and Adolescents Oncology, Gustave Roussy Cancer Campus, Villejuif, France.
Medical School of Rangueil, University Paul Sabatier, Toulouse, France.
Department of Pediatric Orthopedic Surgery, Necker Hospital, Paris, France.
Department of Orthopedic Surgery, CHU Nantes, Nantes, France.
Unicancer, Paris, France.
Biostatistics Unit, Gustave Roussy Cancer Campus, Villejuif, France.
Department of Medical Oncology, Curie Institut, Paris, France.


The French phase 3 trial (OS 2006) testing zoledronic acid, an osteoclast inhibitor, with chemotherapy and surgery did not improve the outcome of patients with osteosarcoma (OS). To understand this unexpected result, the presence of infiltrating immune cells was investigated in 124 pre-therapeutic biopsies of patients enrolled in the trial. The percentage of CD68/CD163 tumor-infiltrating macrophages (TAMs), CD8+ lymphocytes, osteoclasts, and the PD1/PDL-1 checkpoint were assessed by immunohistochemistry. M1/M2 macrophage polarization was characterized by pSTAT1/CMAF staining. The expression of these biomarkers was correlated with clinical outcome. No statistical correlations were found with response to chemotherapy. High CD163 levels (>50% of cells per core; 43.8% of patients) were associated with CMAF nuclear expression and significantly correlated with better overall survival (p = 0.0025) and longer metastasis progression-free survival (MPFS, p = 0.0315) independently of metastatic status (p = 0.002). Only a trend was observed for patients with high CD68-positive cells (p = 0.0582). CD8+ staining was positive in >50% of cases with a median staining of 1%. Lower CD8+ levels were associated with metastatic disease at diagnosis and the presence of CD8-positive cells significantly correlated with improved overall survival in zoledronate-treated patients (p = 0.0415). PD1/PDL-1 staining was negative in >80% of cases and was not correlated with outcome. Finally, CD163-positive TAMs and CD8 positive cells are crucial prognostic biomarkers in OS, whereas PD1/PDL-1 checkpoint plays a minor role. For the first time, we described a correlation between CD8 positive cells and survival in zoledronate-treated patients. The immunohistochemical analysis of the microenvironment in biopsies may represent a novel tool for therapeutic stratification.


CD163; CD8; Osteosarcoma; PD1/PDL-1 Checkpoint; macrophages

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