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

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8 + T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA‐A2 peptides. To identify the reasons underlying the loss of single HLA‐A allospecificities, we searched for genomic alterations at the locus for HLA Class I α‐chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA‐A2 gene sequences as determined by polymerase chain reaction‐sequence specific primers (PCR‐SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group‐specific primer‐mixes spanning the 5′‐flanking region of the HLA‐A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA‐A2‐restricted cytotoxic T‐cell clones were performed in HLA‐A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA‐A2 negative cells. We could document the occurrence of selective HLA‐A2 deficiencies in cultured advanced‐stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA‐A gene locus. © 2002 Wiley‐Liss, Inc.