CHROMOSOME 6p LOSS OF HETEROZYGOSITY (LOH) ANALYSIS ON MICRO-DISSECTED TUMOUR CELLS IN HNSCC WITH DOWNREGULATED HLA CLASS I EXPRESSION.
M Verdaasdonk, M Feenstra, A van der Zwan, D van Wichen, P Slootweg, J van den Tweel, R de Weger and M Tilanus. Dept. of Pathology, University Medical Center, Utrecht, the Netherlands

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Downregulated HLA class I expression is correlated with allelic loss at 6p21.3which is the
location of the Human Leukocyte Antigen (HLA) coding sequences in head and neck
carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at
6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 Megabase HLA region. In the current report, the detection threshold (25%) was addressed in tumours containing marginal loss by micro-dissection of tumour cells. LOH was investigated on microdissected tumour cells and this confirmed that assigned loss at a threshold of 25% reduction in ratio is accurate. In addition we describe high-density microsatellite analysis of chromosome 6p21.3 in HNSCC with downregulated HLA class I expression. Seven additional markers were selected for their genetic distances to individual HLA class I and TAP genes. The aim of the study was to further investigate genetic alterations at 6p21.3 and to pin-point allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B and -C loci and TAP genes. To identify loss of the entire chromosome 6 the 6q located marker "D6S311" was included. Appropriate HLA-LOH analysis requires carefully selected markers covering the 6p21.3 region. Important criteria for these markers are the number of alleles, percentage of heterozygosity and the location relative to HLA and TAP genes. Previously we proposed a panel of 5 microsatellite markers covering the 6p21.3 region: i.e. D6S291, D6S273, D6S265, D6S105 and D6S276. From the present study we conclude that in addition to these markers, application of markers in close proximity to HLA class I and TAP genes is required to accurately identify HLA loss in tumours.