Molecular strategies to define HLA haplotype loss in microdissected tumor cells

Loss of heterozygosity (LOH) of chromosome 6p21 is an important mechanism t hat generates HLA haplotype loss in various human tumors. This mechanism pr oduces non-reversible HLA-deficient tumor cells char can escape T cell immu ne responses in peptide-vaccinated cancer patients. However, the exact freq uency of this mechanism is still unknown, because contaminating stroma in s olid tumor tissues masks the tumor DNA obtained from solid samples. A micro dissection technique was applied to 4-8 mum sections of cryopreserved tumor tissues from a group of colorectal and laryngeal carcinomas. Fifteen patie nts were analyzed for the presence of LOH associated with the beta (2)-micr oglobulin gene in chromosome 15, and five patients for LOH associated with HLA genes in chromosome 6. In two cases, autologous metastasis tissue sampl es were also available. The patients were selected for showing an altered H LA class I tumor phenotype as determined by immunohistological techniques. DNA was obtained from this microdissected material and amplified in order t o detect the presence or absence of nine previously selected microsatellite markers. HLA sequence based typing (SBT) was also applied to these microdi ssected DNA samples to define the HLA genotype. Microdissection greatly imp roved the definition of LOH, with nearly 100% signal reduction in one of th e alleles, In addition, this procedure allowed us to detect beta (2)-microg lobulin LOH in rumors that expressed some HLA molecules. Our data indicate that this procedure can be successfully applied to microdissected samples f rom solid rumors, thus enhancing the power and sensitivity of LOH detection . (C) American Society for Histocompatibility and Immunogenetics, 2000. Pub lished by Elsevier Science Inc.