Characterization of the immune escape phenotype of human gastric cancers with and without high-frequency...

Gastric cancers with and without high-frequency microsatellite instability (MSI-H) represent distinctive pathways of carcinogenesis. The aim of this study was to clarify if human leukocyte antigen (HLA) class I antigen subunits and antigen processing machinery (APM) components are differentially downregulated in these two groups of tumours. Using reverse transcription PCR (RT-PCR), loss of heterozygosity (LOH) analysis, methylation-specific PCR (MSP), DNA sequencing, immunohistochemistry, and flow cytometry, we analysed expression and/or alteration of HLA class I antigen subunits and APM components, including low molecular weight polypeptide proteasome subunit (LMP)2, LMP7, LMP10, transporter associated with antigen processing (TAP)1, TAP2, tapasin, proteasome activator (PA) 28alpha, and PA28beta in two stage-matched panels of 30 MSI-H and 30 microsatellite stable (MSS) gastric cancers. Mutations at coding microsatellites (cMS) located within beta2-microglobulin (beta2m) and genes encoding APM components, including endoplasmic reticulum (ER) chaperone protein genes, such as calnexin, SEC63, SEC31, and P4HB (p55), were also analysed. HLA class Ia transcripts were totally downregulated in 18.3% of cancer cases. Locus-specific downexpression of HLA-A, -B, and -C was detected in 41.7%, 45.0%, and 31.7% of cases. Loss of HLA-A was significantly more frequent in MSI-H cancers. The LOH ratios of the HLA-A, -B, and -C loci microsatellite markers were relatively low: 5/32 (15.6%) for D6S306, 4/32 (12.5%) for D6S258, 4/33 (12.1%) for D6S273, and 4/30 (13.3%) for D6S1666. Methylation of HLA-A, -B, and -C was detected in 38.3%, 40.0%, and 28.3% of cases. A significant association between methylation and reduction in expression was observed in gastric cancer tissues. Mutations at cMS of beta2m and APM components were detected in 3.3-46.7% of MSI-H cancers but in none of MSS cancers. These data show that gastric cancers have various defects in HLA class I antigen subunits and APM components and that the MSI phenotype is associated with frequent HLA-A inactivation and frameshift mutations of the beta2m and APM genes.