Higher Frequency of Selective Losses of HLA-A and -B Allospecificities in Metastasis Than in Primary Melanoma Lesions

Abstract

Expression of HLA class I molecules is essential for the recognition of tumor cells by CD8+ T cells. In this study, 48 bioptic samples of 42 patients in all stages of melanoma were investigated after short-time cultivation of tumor cells. To confirm melanocytic origin of cultured cells, samples were screened for mRNA expression of melanoma markers gp100, tyrosinase, MAGE-3, MelanA, and MUC18 by reverse transcriptase-polymerase chain reaction. Surface expression of specific HLA-A and -B allospecificities on melanoma cells were analyzed with a standard microcytotoxicity assay after stimulation with interferon (IFN)-alpha and compared with the background found in peripheral blood mononuclear cells from the corresponding patients. Immunohistochemistry and flow cytometry confirmed specific losses in cases where the appropriate monoclonal antibodies were available. The level of expression of HLA-I, HLA-II, and intercellular adhesion molecule 1 antigens on melanoma cells cultured in the presence or absence of IFN-alpha and IFN-gamma was determined cytofluorometrically. All cell cultures tested were found to be positive for one or more melanocytic markers by reverse transcriptase-polymerase chain reaction. The specific HLA-I alleles on the cultured cells were detectable in 45 of 48 samples. In 11 cases a specific loss of one HLA-I allele was observed (2 x A2, B7, B8, B18, 4XB44, B47, B49). Ten of these samples were derived from locoregional lymphnode metastases or from distant metastatic tumors. Only one sample from a primary melanoma showed a specific loss of HLA-I (B47). IFN-alpha upregulated expression of HLA-I up to 4-fold. IFN-gamma enhanced the appearance of HLA-II up to 35-fold and the expression of intercellular adhesion molecule 1 up to 40-fold. Selective loss of HLA-I allospecificities might be a major step in tumor progression.

Loss of single HLA Class I allospecificities in melanoma cells due to selective genomic abbreviations

“…23 In contrast to Giacomini et al 40 we frequently detected downregulation of single HLA allospecificities even in low-passage melanoma cell cultures. Regulatory effects on the RNA level or selective post-translational mechanisms might be the reason for the observed phenotypical loss of a single HLA Class I allospecificity in most samples analyzed so far.…”

“…MM cells were stimulated for 2 days with 500 U/ml of IFN-␣ to upregulate expression of HLA Class I molecules before analyzing them with the same test, as described earlier. 23 Detected losses of HLA Class I allospecificities were confirmed by flow cytometry and immunohistochemistry on sections from the corresponding tumor lesions when the appropriate monoclonal antibodies were available. Comparability of the melanoma cell cultures with the corresponding original tumor has been proven by similar antigen expression profiles, as documented previously.…”

“…23 Out of this group 5 melanoma cell cultures derived from 3 patients were investigated more closely (Table I). Two melanoma cell cultures gained special interest because they exhibit a phenotypic loss of the HLA-A2 allospecificity that was not due to transcriptional downregulation.…”

“…Comparability of the melanoma cell cultures with the corresponding original tumor has been proven by similar antigen expression profiles, as documented previously. 23 …”

“…We have shown previously that losses of single HLA-A or -B allospecificities occur preferentially in advanced stages of melanoma. 23 The observed increased frequency of HLA-B downregulation might reflect reduced transcription associated with overexpression of c-myc oncogene. 24 In 2 of our patients, however, the HLA Class I loss appeared of genomic origin and could not be explained by transcriptional downregulation.…”