Increased PD-L1 and T-cell infiltration in the presence of HLA class I expression in metastatic high-grade osteosarcoma: a rationale for T-cell-based immunotherapy

Abstract

Introduction: Immunotherapy may be an excellent choice for treating osteosarcoma given its exceptionally high genomic instability, potentially generating neoantigens. In this study, we aim to investigate the HLA class I expression, PD-L1 and tumour-infiltrating lymphocytes in primary osteosarcomas and relapses/metastases, as well as their changes during disease progression.

Materials and methods: Tumour samples from multiple stages of the disease (pretreatment biopsies, surgical resections of primary osteosarcomas, relapses and metastases) were collected and stained for HLA-A (HCA2), HLA-B/C (HC10), β2-microglobulin and PD-L1 using immunohistochemistry on whole sections. Density and type of T-cell infiltrate were characterised by a triple immunofluorescent staining CD3-CD8-FOXP3.

Results: Overall, 85 formalin-fixed, paraffin-embedded blocks from 25 osteosarcoma patients were included. HLA class I expression was detected in 94% of osteosarcomas (strongly positive in 56%, heterogeneous in 38%) and negative or weakly positive in 6%, without differences between the stages of the disease. HLA-A expression was more frequently negative than HLA-B/C. Tumour-infiltrating lymphocytes were highly heterogeneous and mainly observed in tumour areas with expression of HLA class I. Density of T cells was significantly higher in metastases than in primary tumours and local relapses (p = 0.0003). Positive PD-L1 expression was found in 13% of primary tumours, 25% of relapses and 48% of metastases and correlated with a high T-cell infiltrate (p = 0.002).

Conclusion: An increased number of tumour-infiltrating T cells and PD-L1 expression in metastases compared with primary tumours, suggesting accessibility for T cells, could imply that osteosarcoma patients with metastatic disease may benefit from T-cell-based immunotherapy.

Keywords: HLA class I; Immunotherapy; Osteosarcoma; PD-L1; Tumour-infiltrating lymphocytes.

Fig. 1

Different HLA class I phenotypes in osteosarcoma. Representative staining patterns of HLA-A expression using immunohistochemistry (HCA2 antibody): negative/weak expression with endothelial cells as positive internal controls (a), heterogeneous expression with both negative and positive regions (b) and diffuse positive expression (c). Scale bars 50 μm. Negative and heterogeneous expression was observed more frequently for HLA-A compared to HLA-B/C (d). The final HLA class I expression status was determined according to the positivity of at least one of the heavy chains stainings (either HCA2, HC10 or both), dependent to the β2-microglobulin positivity score (e)

Fig. 2

Characterisation of T-cell density and subtypes in osteosarcoma. Representative images for tumour-infiltrating lymphocytes in primary tumour (a) and the associated local relapse (b) and metastasis (c). Triple immunofluorescent staining using anti-CD3 (red), anti-CD8 (blue) and anti-FOXP3 (green) was used to identify CD3⁺CD8⁻, CD3⁺CD8⁺ and CD3⁺CD8⁻FOXP3⁺ T cells. Density of T-cell infiltration was higher in metastatic lesions (d), but the proportion of CD3⁺CD8⁻ and CD3⁺CD8⁺ T cells was comparable with primary tumours (e). CD3⁺CD8⁻FOXP3⁺ T cells were found more frequently in local relapses

Fig. 3

High PD-L1 expression in metastatic osteosarcoma lesions. Representative images for PD-L1 immunostaining in an osteosarcoma patient. PD-L1 was negative in the primary tumour (a) while a membranous expression was detected on isolated cells (mainly macrophages) in the local relapse (b) and was more diffuse and observed on both osteosarcoma cells and immune cells in the lung metastasis (c). Scale bars 50 μm. PD-L1 expression was observed more frequently in metastatic osteosarcoma lesions, compared to primary tumours and local relapses (d). T-cell infiltration was higher in PD-L1 positive tumours (e, f)

Fig. 4

Correlation between HLA class I status, total T cells and patient survival. Kaplan–Meier survival curves for disease-free survival and overall survival according to HLA class I status (a, b) and density of T-cell infiltration (c, d) of the primary tumour. Overall survival from the time of diagnosis of metastatic disease according to HLA class I status (e) and T-cell infiltration (f) of the first metastatic lesion. p value obtained by log-rank test