Novel genetic and epigenetic alterations in ... - Ous-research.no

unselected series of primary tumors collected between 1987 and 1989 in the South-Eastregion of Norway.[11] Forty-six Swedish samples and six Danish samples were acquired viacollaborators. These samples include no clinical data and are included to analyze mutationfrequencies of the genes included. All clinico-pathological associations are thereforeperformed on the Norwegian tumors.Hypothesis testing validation seriesAn unselected prospective series of ~950 colorectal tumor samples were collected between1993 and 2003 at Aker University Hospital in Oslo. Samples were formalin-fixed andembedded in paraffin. All tumors were subject to a formal resection and extensive clinicaldata for each patient was kept. MSI status was determined for all right-sided and one third ofthe left-sided colonic tumors, yielding 108 MSI-positive tumors which were included in thestudy. No rectal tumors were included.Determination of MSIMSI status was determined using the consensus guidelines given by the National CancerInstitute (BAT25, BAT26, D2S123, D5S346 and D17S250).[6] The Bethesda markerprotocol had to be optimized both for fresh-frozen tissue and for DNA from formalin-fixedsamples. The fresh frozen samples were analyzed in a pentaplex PCR using 37ng DNAtemplate in a 10μl reaction volume consisting of 1 x Multiplex PCR Mastermix (containingbuffer, 1.5mM MgCl 2 , nucleotides, and enzyme, QIAGEN GmbH, Hilden, Germany),primers and water (see Supplementary table for primer and PCR details), while the paraffin5