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- Materials and Methods
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 β-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. (Cancer Sci 2006; 97: 1374–1380)
Bone and soft tissue sarcomas are malignant neoplasms of mesenchymal origin. Despite the relatively low prevalence of these tumors, the aggressive clinical behavior of especially high-grade sarcomas gives rise to considerable concerns about their management.(1,2) As exemplified by osteosarcoma, which is the most common primary sarcoma of bone, 30–40% of cases are still refractory to current multimodality treatments including definitive surgery and neoadjuvant chemotherapy.(1,3,4) This emphasizes the need for alternative treatments including immunotherapy.
Immunotherapeutic trials for bone and soft tissue sarcomas were initially conducted in patients with osteosarcoma during the 1970s.(5,6) As a result of several technological advances in tumor immunology following these studies, immunotherapeutic trials again started recently for various bone and soft tissue sarcomas in a more modern fashion.(7–10) In these studies, dendritic cells(7) and antigenic peptides(8,10) have been used as vaccines. Nevertheless, only a few tumors showed decreases in their tumor burden in response to these vaccines,(7,8) suggesting the presence of escape mechanisms from immune surveillance in patients with bone and soft tissue sarcomas.
It is known that tumor cells can lose HLA class I molecules from the cell surface, thereby escaping from recognition of tumor antigens by CD8+ T cells.(11–13) Such loss or downregulation of HLA class I molecules has been demonstrated in malignant melanoma(14–17) and a variety of carcinomas, including those arising from the breast,(18–22) lung,(23,24) stomach,(25) colon,(26–28) bladder(29) and ovary.(30) In contrast, few studies have so far addressed HLA class I expression in bone and soft tissue sarcoma tissues.(31,32)
We have recently generated a monoclonal antibody (mAb) against HLA class I molecules that can be used for formalin-fixed, paraffin-embedded specimens (Torigoe T, Asanuma H, Shimozawa K, Nakazawa E, Tamura Y, Hirohashi Y, Kitamura H, Honma I, Tsuruma T, Tsukamoto T, Hirata K, Hasegawa T and Sato N, submitted for publication, 2006). Using this mAb, we examined the expression profiles of HLA class I molecules immunohistochemically in various bone and soft tissue sarcomas, and analyzed the prognostic significance of HLA class I expression, focusing on osteosarcoma.
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- Materials and Methods
The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas with a view toward future immunotherapeutic trials using peptide vaccines derived from papillomavirus binding factor, which we defined as an osteosarcoma antigen.(37) The anti-HLA class I mAb used in the present study, EMR8-5, recognizes the common epitope of HLA-A, B and C molecules in formalin-fixed, paraffin-embedded tissue sections. EMR8-5 appears suitable for immunohistochemical analysis of osteosarcoma specimens as they often exhibit osteoblastic features that make production of cryostat sections problematic.
There are two other monoclonal anti-HLA class I antibodies, HC10 and HCA2, that have been well characterized and can be used for the immunostaining of formalin-fixed tissue specimens. However, HC10 barely reacts with HLA-A allele proteins, and HCA2 reacts to some HLA-A allele proteins, but not to most HLA-B or C allele proteins.(38,39)
We found that the expression of HLA class I was lost or downregulated in 47 (63%) of 74 formalin-fixed paraffin-embedded specimens of various bone and soft tissue sarcomas. Previously, Mechtersheimer et al. examined 82 frozen specimens of various sarcomas using the anti-HLA class I mAb W6/32.(31) They reported loss or downregulation of HLA class I expression in 28 specimens (34%). With respect to osteosarcoma, Mechtersheimer et al. evaluated four specimens and none of them showed downregulation of HLA class I expression.(31) In the present study, the frequency of loss or downregulation of HLA class I expression was 88% (7/8) in the metastatic tumors, which was higher than in the primary tumors (52%, 13/25). In addition, patients with osteosarcoma exhibiting high expression of HLA class I showed significantly better overall and event-free survival than did 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 who undergo multidrug chemotherapy protocols. At the same time, they imply that the loss or downregulation of HLA class I expression may become a substantial obstacle in immunotherapeutic approaches for patients with osteosarcoma.
Although the sample size was very small, the expression of HLA class I was negative in two of two synovial sarcoma specimens in the present study, and was downregulated in four of five specimens in the study by Mechtersheimer et al.(31) The propensity of synovial sarcoma cells to lose HLA class I may have also served as an obstacle for immunotherapeutic trials such as the one we undertook using an SYT–SSX fusion gene-derived peptide vaccine.(10)
Apart from sarcomas, the loss or downregulation of HLA class I molecules occurred in 33% of head and neck cancers, 54% of breast cancers, 31% of lung cancers, 32% of renal cancers, 43% of colon cancers, 55% of cervix cancers, 70% of prostate cancers and 51% of melanomas.(11) Such loss or downregulation of HLA class I has been associated with disease progression of malignant melanoma(14) and ovary carcinoma,(30) and with poor prognosis in colorectal cancer.(28) In contrast, some studies of malignant melanoma have shown a lack of prognostic significance of HLA class I expression.(15,16) It should also be noted that total loss of HLA class I has been proposed as an indicator of good prognosis in breast cancer(21) and non-small cell lung cancer.(24)
There are several mechanisms proposed to explain abnormal HLA class I phenotypes: (i) structural alterations of the genes that encode HLA class I antigen subunits; (ii) impaired transcriptional activity of these genes; (iii) deregulation of antigen-processing machinery components responsible for functional HLA class I expression such as β-2 microglobulin and transporters associated with antigen processing (TAP-1 and TAP-2);(40) and (iv) degradation of HLA class I proteins by proteasomes. We analyzed these possibilities by examining the expression of HLA class I mRNA and also the expression of β-2 microglobulin protein and mRNA (Table 3). HLA class I mRNA was detectable in all five osteosarcoma tissues examined, including three tissues with negative cell surface expression of HLA class I proteins. β-2 microglobulin protein was negative in one of five osteosarcoma tissues with negative cell surface expression of HLA class I proteins. In this particular case, deregulation of β-2 microglobulin is likely attributed to the loss of HLA class I expression. The remaining cases may be explained by the possibility that β-2 microglobulin has non-functional mutations, as has been reported in malignant melanoma.(41) However, we found no mutations in the entire open reading frame of β-2 microglobulin mRNA in five osteosarcoma tissues examined, including three HLA class I negative osteosarcomas (Tsukahara T, unpublished observation, 2006). Other possibilities, such as deregulation of TAP and the involvement of proteasomes, remain to be elucidated by further experiments including western blotting.
The higher frequency of loss of HLA class I expression seen in metastatic osteosarcomas (88%) than in primary osteosarcomas (52%) can be explained by the following mechanisms: (i) osteosarcoma cells with loss of HLA class I expression metastasized selectively; (ii) the expression of HLA class I was lost during the process of metastasis or at the metastatic sites; and (iii) the group of osteosarcoma cases with loss of HLA class I expression showed higher frequency of metastasis. In this regard, the expression status of HLA class I did not alter or decrease in the metastatic lesion compared with that in the primary lesion in four patients. Therefore we assume that the regulatory mechanism of HLA class I expression does not differ between the primary lesion and the metastatic lesion. Primary osteosarcomas with loss of HLA class I have a propensity to metastasize, which eventually leads to poor survival of patients, as seen in the present study.
Al-Batran et al. reported a significant correlation between the expression of HLA class I and the infiltration of T cells in stage IV melanoma tissues.(17) With respect to osteosarcoma, Trieb et al. examined the expression of HLA-DR and infiltration of T cells.(42) Although T cells infiltrated 33 of 35 tumor specimens(42) there was no signification correlation between T-cell infiltration and HLA-DR expression in osteosarcoma. In the present study, moderate or diffuse infiltration of CD8+ T cells was found mostly in osteosarcoma tissues with high HLA class I expression. However, the relationship between HLA class I expression and infiltration of CD8+ T cells was not statistically significant.
In conclusion, we showed for the first time that the status of HLA class I expression affected the overall survival and event-free survival of patients with osteosarcoma. Because of the small sample size of the present analysis, larger studies need to be conducted to verify the significance of HLA class I molecules for prognosis and the application of T cell-based immunotherapy for patients with bone and soft tissue sarcomas.