Investigation of HLA class I downregulation in breast cancer by RT-PCR

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. We have previously reported the biochemical and immunohistochemical analysis of HLA total class I (W6/32 mAb), alpha-chain (Q1/28,TP25.99 mAbs) and beta(2)-microglobulin (Namb-1 mAb) subunits expression in 25 primary breast carcinomas. This study at protein level resulted in the observation of three different HLA class I expression patterns by both techniques: high, low, and absent downregulation patterns. To better characterize the HLA class I antigens downregulation we extended such analysis also at RNA level by RT-PCR using HLA-A, HLA-B, HLA-C, and beta(2)-microglobulin specific primers either in breast cancer or normal tissues derived from the same patient. None (100%) of the alpha-chain genes analyzed in patient tumor tissues showed significant reduction of expression. In 10 patients out of 25 (40%) the beta(2)-microglobulin gene showed complete loss of expression compared with the corresponding normal tissue counterpart, which showed a constitutive expression, whereas in 2 patients (12.5%) its expression was comparable with the normal counterpart. Sequence analysis at genomic level revealed no defects affecting beta(2)-microglobulin gene in those patients showing lack of expression. Also TAP1 and TAP2 genes expression were investigated in order to confirm or exclude involvement of the MHC class I molecules assembling machinery. The RT-PCR approach mainly confirmed our beta(2)-microglobulin biochemical analysis indicating that in breast cancer specimens it is possible to address the HLA class I gene downregulation as a phenomenon occurring at post-transcriptional level mainly affecting the beta(2)-microglobulin gene expression.