The E48 antigen is a successfully explored molecular marker for the diagnosis and therapy of HNSCC. The applicability of E48 as an HNSCC-associated antigen, however, is restricted due to its heterogeneous expression in 30% of tumors; and identification of additional target antigens is therefore desired. E48 belongs to the Ly-6 antigen family, comprising a group of highly homologous, low m.w., GPIanchored surface proteins, of which some show tissue-restricted expression patterns. To identify novel human HNSCC-associated Ly-6 members with squamous cell-associated expression patterns, we performed comprehensive gene-screening consisting of BLAST searches within GenBank databases, followed by expression analysis. Using this approach, the Ly-6K gene could be annotated as a novel member of the human Ly-6 family. Expression of the human Ly-6 genes E48, Ly-6K, PSCA, GML, RIG-E, G6C and Ly-6H was prescreened by qualitative RT-PCR and subsequently analyzed by quantitative RT-PCR in normal keratinocytes, HNSCC cell lines, normal mucosa, HNSCC tumors as well as normal peripheral blood and bone marrow cells. PSCA was highly expressed in normal mucosa, but 100-fold decreased expression was seen in HNSCC. For Ly-6H, GML and G6C, no or very low expression was observed in keratinocytes and HNSCC. Expression of RIG-E was high in normal and malignant squamous cells and in peripheral blood and bone marrow cells, thus limiting its applicability as an HNSCC-associated marker. In contrast, besides the E48 gene, the Ly-6K gene also appeared to be selectively expressed in HNSCC and normal squamous cells. Moreover, expression of Ly-6K was shown in HNSCC cell lines, in which no E48 expression could be detected. These data justify further evaluation of Ly-6K as potential target antigen for the diagnosis and therapy of HNSCC.

Loss of heterozygosity (LOH) plays an important role in the expression of recessive mutations in mammalian cells. To gain insight into the rate and mechanisms of LOH the autosomal HLA‐A gene was used as a model system. Spontaneous HLA‐A2 mutants originated with a rate of respectively 4.1 × 10−6 and 6.9 × 10−6 per cell per generation in TK6 and WI‐L2‐NS, two isogenic lymphoblastoid cell lines which differ in TP53 status. The rate of loss of HLA‐A2 is 10–50 times higher compared to the mutation rate of the X‐linked HPRT gene. The homozygous TP53 mutation in WI‐L2‐NS had no effect on the rate of HLA‐A2 loss or the spectrum of these mutations. Microsatellite analysis of most of the HLA‐A2 mutants (84%) showed LOH for multiple markers on chromosome arm 6p telomeric of a recombination breakpoint, LOH for all 6p markers, or LOH for markers on both the 6p‐ and 6q‐arms. Cytogenetic analysis showed that these mechanisms gave mutant cells which harbored two intact chromosomes 6 and which were indistinguishable from non‐mutant cells. Therefore, loss of HLA‐A2 is mainly caused by somatic recombination (33–50%) or chromosome loss with duplication of the remaining chromosome (34–40%). These findings correspond to the mechanisms behind loss of the wild‐type RB1 allele in retinoblastoma and suggest that both somatic recombination and chromosome loss followed by duplication contribute to tumorigenesis. Genes Chromosomes Cancer 21:30–38, 1998. © 1998 Wiley‐Liss, Inc.

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A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%-49% for various donors. During culturing ex vivo, HLA-A(-) cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus. Up to 90% of the ex vivo arisen HLA-A2(-) cell population showed LOH of multiple 6p markers, and 50% had lost heterozygosity at 6q. This ex vivo spectrum resembles that found in HLA-A2 mutants obtained from lymphoblastoid cells. The HLA-A2 mutants present in vivo may reflect only a small fraction of the mutants that can be detected ex vivo. In normal lymphocytes, in vivo only mitotic recombination appears to be sustained, indicating the importance of this mechanism for tumor initiation in normal cells. Although mutations resulting in LOH at both chromosome 6 arms were shown to result in nonviable cells in normal lymphocytes, they have been shown to result in viable mutants in lymphoblastoid cells. We hypothesize that these types of mutations also occur in vivo but only survive in cells that already harbor a mutated genetic background. In light of the high rate at which these types of mutations occur, they may contribute to cancer progression.

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