[Show abstract][Hide abstract] ABSTRACT: Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5' untranslated region (UTR) through the 3'-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5' and 3'-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.
[Show abstract][Hide abstract] ABSTRACT: The heterodimeric TAP (transporter associated with antigen processing) complex plays a key role in immune surveillance. By forming the portal between the cytoplasm and the endoplasmic reticulum, it enables cells to present antigenic peptides by human leukocyte antigen class I antigens to cytotoxic T cells, so infected and malignant cells can be eradicated. Because the nature of the peptide determines whether an immune response is evoked, peptide selective transport by TAP may influence immune surveillance. Currently, seven TAP1 and four TAP2 alleles are known. Each may have its own effect on peptide transport. In this study, we investigated whether TAP1 and TAP2 alleles are associated with the development of head and neck squamous-cell carcinoma (HNSCC). We developed a high-throughput SNaPshot™ assay to determine the frequencies of the TAP1 and TAP2 alleles in 79 Dutch control subjects and 94 patients with HNSCC. Strikingly, all control and HNSCC samples contained a TAP1*0101 allele, with the exception of one tumor patient. The most frequent alleles were TAP1*0101 (88.2%) and TAP2*0101 (81.2%). No significant difference was found between control subjects and patients with HNSCC. Here, we report the TAP1 and TAP2 allele frequencies in the Dutch population, the development of a high-throughput TAP typing technique, and a new TAP1*0501 allele.
[Show abstract][Hide abstract] ABSTRACT: Human Leukocyte Antigen (HLA) class I molecules are essential for tumor cell recognition by cytotoxic T cells of the adaptive immune system. Loss of HLA expression provides tumor cells with an escape mechanism to evade the immune system and thus immune therapy. Therefore, HLA loss, and in particular loss of heterozygosity (LOH), is frequently studied in tumors using microsatellite marker LOH analysis. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in nine head and neck squamous cell carcinomas (HNSCCs) using fluorescence in situ hybridization (FISH). These tumors were selected from 53 HNSCCs based upon the HLA class I immunohistochemical staining and LOH data. FISH analysis showed that only two tumors with LOH and one without LOH indeed had loss and a normal chromosome 6 copy number, respectively. Strikingly, for the remaining six tumors, LOH analysis did not reflect the genome HLA copy number. We demonstrated that LOH analysis cannot distinguish loss from gain and that the HLA region is not homogeneously affected within a tumor. Tumor heterogeneity and complex aneuploidy in tumors hinder a straightforward interpretation of microsatellite marker analysis. For immune therapy strategies in cancer patients, knowledge of the HLA expression on tumor cells is essential, to which LOH analysis has a limited contribution.
[Show abstract][Hide abstract] ABSTRACT: In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.