Epigenetic modifications of the genome such as the methylation of cytosine in accumulations of CpG dinucleotides in the so-called CpG islands or the acetylation of histone proteins are frequent events in many tumour entities. If these modifications are located within the promoter sequence of a gene, this usually leads to a switch-off of gene expression and thus to a loss of gene function. Since tumours particularly affect genes that code for cell cycle regulation, DNA repair mechanisms, apoptosis, angiogenesis and metastasis, tumourigenesis, tumour progression and aggressiveness are attributed to the loss of one or more of these genes through epigenetic promoter modification, in addition to other events such as mutations and translocations.
In this project, the status of promoter methylation is determined in a selection of carcinomas from the lung, breast, colon, pancreas, bladder, liver and prostate as well as in a selection of tumours of mesenchymal origin, e.g. lipo-, fibro-, chondro- and Ewingssarcomas and in Hodgkin and non-Hodgkin lymphomas using pyrosequencing and bisulphite sequencing. The tissue bank of the Department of Pathology at the University of Ulm with its extensive, cryo-asserved and formalin-fixed tissue collection with the corresponding histopathological assessment is available for these examinations. For each tumour entity, 20-30 cases are selected in which the tumour proportion in the tissue sample is as high as possible (> 80%) and clinical data are available in addition to the histological findings. In tumours such as Hodgkin's lymphoma, which contain a high proportion of non-tumour tissue, the tumour cells are enriched using laser microdissection.
To determine the degree of methylation of the promoter region of the genes, the tumour DNA is treated with sodium bisulphite. In this reaction, cytosine is deaminated to uracil while 5-methyl-cytosine is inert in this reaction. In a subsequent amplification of this promoter region by PCR and primer pairs matching the converted DNA, each original cytosine appears as thymidine, while 5-methyl-cytosine presents as cytosine. By sequencing the PCR product, the degree of methylation and the position of the methylated CpG dinucleotides in the promoter sequence of the respective gene can now be precisely determined. Due to the large number of sequencings, the new technology of pyro-sequencing instead of classical sequencing is the method of choice for this study.
We hope that this study will reveal a neoplasia-dependent, epigenetically determined signature of inactivated genes, which will enable us to make statements about the prognosis of tumour diseases and provide information on the use of new therapeutic methods.