Mutation screening of DNA isolated from formalin fixed paraffin embedded tumor blocks
Master thesis
Permanent lenke
http://hdl.handle.net/11250/226975Utgivelsesdato
2014-12-11Metadata
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- Master's theses (IHA) [318]
Sammendrag
Colorectal carcinoma is one of the leading cancers in Norway. Molecular profiling of the different stages of colorectal cancers is still in demand as molecular markers to stratify patients for correct treatment response is lacking. This study was carried out to evaluate capillary electrophoresis based Sanger sequencing and fragment analysis on the ability to detect somatic TP53 mutations in fresh frozen- and formalin fixed and paraffin waxed embedded (FFPE) tissue from Dukes’ stage B and C colon adenocarcinomas. A small subset of samples was also subjected to ion semiconductor sequencing on the Ion Torrent PGMTM – ion semiconductor sequencing. These methods demonstrated that FFPE tissue performed well with the fragment analysis assay and on Ion Torrent PGMTM, while the same samples required additional purification steps for Sanger sequencing. Sanger sequencing revealed the highest number of mutations, and deleted nucleotide composition that were difficult to interpret with Sanger sequencing were successfully verified on the Ion Torrent PGMTM. Fragment analysis failed to detect all the mutations found by Sanger sequencing. The fragment analysis assay can detect mutations at 7 nucleotide positions (SNPs) located in exon 5, 7 and 8, of the TP53 DNA binding domain. DNA was isolated from 37 surgically removed fresh frozen colon tumors and 40 surgically removed FFPE preserved tumors to perform TP53 mutation analysis. Univariate, Kaplan-Meier analyses and the log-rank test for TP53 and Dukes’ stage parameters revealed differences in survival amongst Dukes’ stage status, with no such difference for TP53 status. Most mutations were evaluated as inactive mutants, and the mutation spectra corresponded well to data published in TP53 databases. The quest for new clinically approved biomarkers continues as cancer genome studies reveals an ever growing repertoire of mutations previously assumed insignificant in carcinogenesis.