Novel genetic and epigenetic alterations in ... - Ous-research.no
Novel genetic and epigenetic alterations in ... - Ous-research.no Novel genetic and epigenetic alterations in ... - Ous-research.no
Results in BriefUnivariate survival analysis indicated that several genes could aid in discriminating good andpoor prognosis, but only mutations in RCC2 was associated with a beneficial five-yeardisease-free survival in both tumor series (P = 0.035 and 0.011, respectively). This findingwas confirmed using multivariate analyses even with the inclusion of the strongest knownpredictor of prognosis to date, tumor stage at diagnosis (P = 0.028 and 0.021, respectively) asmutations in RCC2 separated patients with a localized disease into those with poor and goodsurvival (P = 0.004)In conclusion, analysis of an (A)10 repeat in RCC2 using readily available technology refinesprognosis in a group of microsatellite instable tumors.42
DISCUSSIONFresh-frozen versus formalin embedded tissueIn the present study both fresh frozen and formalin-fixed paraffin embedded tissue sampleshave been used. Formalin fixation is an excellent way of preserving good histological detailsin tissue sections, and is for this purpose superior to frozen sections. On the other hand, thispreservation method is inferior to snap- or fresh frozen tissue for retaining high qualityDNA and RNA. One of the main hurdles is the fragmentation of DNA as well as theinhibitory effects on PCR efficiency. This means that one should aspire to design short PCRproducts,and carefully optimize the reactions. In the present thesis we only analyzed shortPCR products with fragment analysis in the formalin embedded series, and thereby thisproblem was in large overcome. However, we did experience a small decrease in the successrate between the mutation results obtained from the fresh frozen test series versus thosefrom the formalin-fixed validation series (99.9%; range 99.1-100%, and 92%; range 0-100%,respectively).Methodological considerationsDNA methylation analysesBisulfite treatmentBisulfite treatment has been around for a while[137], but it was not until the early 1990s thatthe method was used to map 5-methyl cytosine[138;139]. With this, Frommer and Clarkefound a way of converting the non-readable epigenetic information into readable geneticinformation. Under acidic pH and high bisulfite concentration, bisulfite treatment convertsunmethylated, but not methylated, cytosine into uracil in a highly specific manner[140]. Thesubsequent difference in nucleotide sequence can be exploited to determine methylationstatus by numerous PCR-based methods. Insufficient bisulfite conversion fails to convert allunmethylated cytosines to thymine, making it difficult to discriminate methylated fromunmethylated cytosines in downstream analyses. The conversion rate can be limited by43
- Page 1 and 2: Novel genetic and epigenetic altera
- Page 3 and 4: TABLE OF CONTENTSACKNOWLEDGEMENTS .
- Page 5 and 6: ACKNOWLEDGEMENTSThe present work ha
- Page 7 and 8: Prefacetechnology[3]. This new tech
- Page 10 and 11: SummaryThe subgroup of carcinomas w
- Page 12 and 13: Introduction“Epigenetic inheritan
- Page 14 and 15: Introductionamino acid change it is
- Page 16 and 17: Introductionmethylation during embr
- Page 18 and 19: IntroductionDNA is most of the time
- Page 20 and 21: IntroductionFigure 5. DNA methylati
- Page 22 and 23: IntroductionFigure 6. Incidence rat
- Page 24 and 25: IntroductionFigure 8. Tumor staging
- Page 26 and 27: Introductioninasmuch as 80% of colo
- Page 28 and 29: IntroductionInstabilities involved
- Page 30 and 31: Introductionthere seems to be a fid
- Page 32 and 33: Introductionsevere alterations are
- Page 34 and 35: Introductionpopulation-wide screeni
- Page 36 and 37: IntroductionFigure 12. Present and
- Page 38 and 39: RESULTS IN BRIEFPaper Ia. “DNA hy
- Page 40 and 41: Results in Briefinstability, and se
- Page 44 and 45: Discussionseveral factors, and full
- Page 46 and 47: Discussionlow threshold, we increas
- Page 48 and 49: DiscussionIt may seem like unnecess
- Page 50 and 51: Discussionthan 96% DHPLC do not sta
- Page 52 and 53: DiscussionFigure 13. Mutation detec
- Page 54 and 55: DiscussionClinical impact of molecu
- Page 56 and 57: Discussionmarkers with a very high
- Page 58 and 59: Discussionchromosomes in metaphase[
- Page 60 and 61: DiscussionThese examples underline
- Page 62 and 63: Discussiongenes. One is based on mu
- Page 64 and 65: CONCLUSIONSWe have identified novel
- Page 66 and 67: Future PerspectivesMolecular risk a
- Page 68 and 69: REFERENCES1. Breasted J (1930) The
- Page 70 and 71: References29. Deng G, Chen A, Pong
- Page 72 and 73: References57. Al-Sukhni W, Aronson
- Page 74 and 75: References84. Kunkel TA (1993) Nucl
- Page 76 and 77: ReferencesLeggett B, Levine J, Kim
- Page 78 and 79: References133. Lind GE, Thorstensen
- Page 80 and 81: References156. Meling GI, Lothe RA,
- Page 82 and 83: ReferencesT, Song X, Day RH, Sledzi
- Page 84 and 85: References196. Honda S, Haruta M, S
- Page 86 and 87: ORIGINAL ARTICLESAPPENDIXAppendix I
- Page 89 and 90: GASTROENTEROLOGY 2007;132:1631-1639
- Page 91: Paper IbGuro E Lind, Terje Ahlquist
Results <strong>in</strong> BriefUnivariate survival analysis <strong>in</strong>dicated that several genes could aid <strong>in</strong> discrim<strong>in</strong>at<strong>in</strong>g good <strong>and</strong>poor prog<strong>no</strong>sis, but only mutations <strong>in</strong> RCC2 was associated with a beneficial five-yeardisease-free survival <strong>in</strong> both tumor series (P = 0.035 <strong>and</strong> 0.011, respectively). This f<strong>in</strong>d<strong>in</strong>gwas confirmed us<strong>in</strong>g multivariate analyses even with the <strong>in</strong>clusion of the strongest k<strong>no</strong>wnpredictor of prog<strong>no</strong>sis to date, tumor stage at diag<strong>no</strong>sis (P = 0.028 <strong>and</strong> 0.021, respectively) asmutations <strong>in</strong> RCC2 separated patients with a localized disease <strong>in</strong>to those with poor <strong>and</strong> goodsurvival (P = 0.004)In conclusion, analysis of an (A)10 repeat <strong>in</strong> RCC2 us<strong>in</strong>g readily available tech<strong>no</strong>logy ref<strong>in</strong>esprog<strong>no</strong>sis <strong>in</strong> a group of microsatellite <strong>in</strong>stable tumors.42