|Institution:||University of Oslo|
|Full text PDF:||https://www.duo.uio.no/handle/10852/11448
Testicular germ cell tumours are the most frequent type of cancer in young adult Caucasian males. Yet, it is one of the neoplastic diseases with the best prognosis and outcome, making it an ideal model for curative disease. Germ cell tumours are hypothesised to originate from early primordial germ cells or foetal gonocytes. Cells derived from the histological subtype embryonal carcinoma have been shown to closely resemble embryonic stem cells, derived from the inner cell mass of the early blastocyst, and are considered their malignant counterpart. Here, gene expression data from six embryonic stem cell lines and five embryonal carcinoma cell lines previously generated using the Affymetrix Human Exon 1.0 ST microarray were used to generate a list of gene candidates for transcript variation specific to embryonal carcinomas. These candidates were evaluated using in silico tools and databases, before choosing six of them for validation with PCR-based methods. Of these, ASH2L was found to use an alternative promoter and starting exon more frequently in the malignant embryonal carcinoma cell lines compared to the embryonic stem cell lines. When expanded to a larger series of clinical samples, we found that the ASH2L alternative start exon was significantly more frequently employed in undifferentiated elements of testicular germ cell tumours. In addition, we also identified that ETV5, originally a candidate for expression variation at the whole-gene level, exhibits frequent overexpression of its 3’ end at the mRNA level in three out of the five embryonal carcinoma cell lines. When expanded to clinical samples, we also identified several seminoma samples which have the same upregulation of 3’ mRNA levels. We hypothesise that these events are caused by alternative promoter usage and that they reflect early pathogenesis and pluripotency of testicular germ cell tumours. Additionally, genes identified from the exon microarray data exhibiting differential expression at the whole-gene level between embryonal carcinoma and embryonic stem cells were validated, to see if increased transcription correlates with upregulation at the protein level. By in situ analysis of a tissue microarray containing 510 testicular tissue cores, we were able to validate NANOG as a potent biomarker for testicular germ cell tumours. The homeobox protein showed abundant expression also in other histological subtypes and at various cellular localisations, where nuclear staining was found to be the most prominent. Nuclear staining was found to be correlated with the degree of differentiation, thus confirming NANOG as a potent marker for pluripotency.