Microsatellite analysis of microdissected tumor cells and 6p high density microsatellite analysis in head and neck squamous cell carcinomas with down-regulated human leukocyte antigen class I expression

Down-regulated human leukocyte antigen (HLA) class I expression is frequent ly correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previ ously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at l east one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold ( 25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing: marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-re gulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic lo ss to individual HLA class I genes, using additional markers closely locate d to the HLA-A, -B, and -C loci and the,transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite ma rkers and subsequent fluorescent detection is a rapid and sensitive techniq ue to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples a nd samples containing a small amount of tumor cells is facilitated by laser -assisted microdissection of tumor cells. In addition, we showed that accur ate HLA LOH analysis requires application of microsatellite markers in clos e proximity to HLA class I and TAP genes.