MOLECULAR APPROACHES FOR THE DETECTION OF VARIABLE HLA EXPRESSION IN TUMOURS.
          Manita Feenstra, Karen Duran, Ilonka Stuy, Dick van Wichen, Roel de Weger, Marcel Tilanus. Department of Pathology, University Hospital, Utrecht, The Netherlands.
          HLA class I and B2m expression was analysed on cryostat sections of 84 head and neck squamous cell carcinomas (HNSCC) using monomorphic and locus specific monoclonal antibodies. None of the tumours tested showed a total loss of HLA class I and B2m when analysed with the monomorphic W6/32 (HLA class I determinant) and anti-B2m Mabs. Whereas weak expression was found in 8 tumours (10%). When analysed with locus-specific antibodies (HCA2 and HC10, anti-HLA-A and anti-HLA-B/C, respectively) 37 tumours (44%) showed a loss, weak or heterogeneous expression of one or both of these loci. These tumours were selected for further analysis. 8 tumours showing a weak expression for HLA class I and B2m were analysed for mutations in either allele of the B2m gene by sequencing based mutation analysis. No mutations have been detected in the B2m gene. It can be concluded that mutations in B2m is not a frequent event in solid HNSCC tumours. Subsequently the tumours with aberrant HLA class I expression are analysed for Loss of Heterozygosity (LOH) using 5 microsatellite markers within the HLA coding sequence and 2 microsatellite markers surrounding the B2m gene. In 10 tumours aberrant HLA class I and B2m expression, as was determined by immuno-histochemistry, correlated with deletions of (parts of) the HLA loci and/or deletion of one B2m allele. These prelimary results indicate that LOH is an important mechanism by which tumour cells down-regulate HLA class I expression and LOH studies can be used as a marker for HLA class I loss. Due to the limited specificity of the allele-and locus specific anti-sera used in studies on HLA expression, the exact frequency of HLA loss in tumours is not known. Application of molecular biological techniques, i.e. LOH studies, mutation analysis of HLA alleles and of factors involved in antigen presentation, and characterization of mRNA expression (regulation) are challenging alternatives to the complexity of immuno-histochemical approaches.