Prognostic significance of HLA class I expression in osteosarcoma defined by anti-pan HLA class I monoclonal antibody, EMR8-5

Abstract

With the goal of establishing efficacious peptide-based immunotherapy for patients with bone and soft tissue sarcomas, we previously identified the cytotoxic T lymphocyte-defined osteosarcoma antigenic gene Papillomavirus binding factor. The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas. Seventy-four formalin-fixed paraffin-embedded specimens of various bone and soft tissue sarcomas, including 33 osteosarcomas, were stained with the anti-HLA class I monoclonal antibody EMR8-5, which we recently generated. The expression of HLA class I was lost or downregulated in 46 of these specimens (62%). With respect to osteosarcoma, loss or downregulation of HLA class I expression was seen in 13 (52%) of 25 primary tumors and seven (88%) of eight metastatic tumors. In six of 11 HLA class I-negative osteosarcoma specimens, the expression of beta-2 microglobulin was also lost. Subsequently the prognostic significance of HLA class I expression was analyzed in 21 patients with osteosarcoma who had completed multidrug neoadjuvant chemotherapy and undergone adequate surgery. Patients with osteosarcoma highly expressing HLA class I showed significantly better overall and event-free survival than those with HLA class I-negative osteosarcoma. In contrast, such prognostic significance of HLA class I expression was not found in 15 patients with malignant fibrous histiocytoma of soft tissue. These findings suggest that the class I-restricted cytotoxic T lymphocyte pathway plays a major role in immune surveillance of patients with osteosarcoma.

Figure 1

Immunohistochemical grading of tumor specimens. Representative sections of osteosarcoma specimens stained with the anti‐HLA class I monoclonal antibody EMR8‐5 are shown (original magnification, ×200). ‘Negative’ indicates that less than 5% of tumor cells were stained positively. ‘Low’ indicates a positive tumor cell number from 5 to 50%. ‘High’ indicates a positive tumor cell number of over 50%. As seen typically in the low‐grade section, endothelial cells were stained positively with EMR8‐5, which was used as an internal positive control. The same immunohistochemical grading was used for sections stained with the antibody against anti‐β‐2 microglobulin.

Figure 2

Grading of T‐cell infiltration. Representative sections of osteosarcoma specimens stained with an anti‐CD8 monoclonal antibody are shown (original magnification, ×200). Infiltration of T cells into tumor tissues was evaluated by semiquantitative scoring on a scale of + (scattered or mild infiltration), ++ (moderate infiltration) and +++ (diffuse infiltration).

Figure 3

Expression of HLA class I in primary and metastatic osteosarcoma. Sections of the primary tumor and the lung metastatic tumor obtained from the same patients were stained with our anti‐HLA class I monoclonal antibody (original magnification, ×200). The numbers in the figure correspond to patient numbers in Table 2.

Figure 4

Expression of HLA class I and β‐2 microglobulin mRNA in osteosarcoma. The expression of HLA class I and β‐2 microglobulin mRNA was examined in five fresh frozen osteosarcoma specimens. U20S is an osteosarcoma cell line used as a positive control. Immunohistochemical status of HLA class I expression was low in patient 12, high in patient 15 and negative in patients 11, 17 and 20. G3PDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 5

Expression of β‐2 microglobulin in HLA class I‐negative osteosarcoma. Sections of HLA class I‐negative primary osteosarcoma were stained with the anti‐β‐2 microglobulin antibody (original magnification, ×200). In patients 2 and 20, the anti‐β‐2 microglobulin antibody reacted highly with their specimens. In contrast, in patient 21 the reactivity was graded as negative for both the anti‐HLA class I monoclonal antibody and the anti‐β‐2 microglobulin antibody.

Figure 6

Overall survival of 21 patients with osteosarcoma stratified by HLA class I status. Overall survival was estimated using Kaplan–Meier plots. The date that the histological diagnosis was made was used as time 0. There was a significant difference between patients with osteosarcoma highly expressing HLA class I and those with negative expression of HLA class I (P = 0.032).

Figure 7

Event‐free survival of 21 patients with osteosarcoma stratified by HLA class I status. Event‐free survival was estimated using Kaplan–Meier plots. The date that the histological diagnosis was made was used as time 0. Osteosarcomas with high expression of HLA class I had a significantly better prognosis than those with negative expression (P = 0.0014).

Figure 8

Overall survival of 15 patients with malignant fibrous histiocytoma of soft tissue stratified by HLA class I status. Overall survival was estimated using Kaplan–Meier plots. The date that the histological diagnosis was made was used as time 0.