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CONCLUSIONSWe have identified novel epigenetic changes in colorectal tumors and some are considered aspromising diagnostic biomarkers. Hypermethylation of ADAMTS1, MGMT and especiallyMAL are frequently seen in precursor lesions as well as carcinomas regardless of MSI-status,and rarely in normal mucosa, making them suitable for early detection. In addition,hypermethylation of CRABP1, MLH1, NR3C1, RUNX3 and SCGB3A1 were seen almostexclusively among MSI-carcinomas, making them suitable as identifiers of these tumors.Overall, the augmented methylation frequencies from normal mucosa, via adenomas tocarcinomas suggest that epigenetic biomarkers may be used for cancer risk assessment.For the first time we showed that NF1, a negative regulator of KRAS, may be involved incolorectal cancer. Through mutation and copy-number analyses we found that more than40% of the colorectal carcinomas had mutations in the NF1 gene, which might causealternative splicing or wrong protein folding. However, further studies are necessary to verifyany potential functional consequence.Including the NF1 data we show that alterations in one or more of the KRAS, BRAF, NF1and RASSF1A genes are found in 74% of the analyzed carcinomas, underlining theimportance of the MAPK pathway in colorectal cancerThe mutation status of one gene, RCC2, among 41 genes analyzed was shown to carryprognostic information among patients with MSI tumors. Mutation of RCC2 was associatedwith good prognosis, even with the inclusion of tumor stage in multivariate regressionanalysis. RCC2 as a prognostic marker was identified in a clinical test series and confirmed inan unselected prospective series.64
FUTURE PERSPECTIVESHigh throughput technology for detection of genetic and epigenetic cancer markers.We are planning genome-wide strategies that include the use of a next-generation sequencingsystem such as SOLiD, 454 or Solexa, opening up innumerable possibilities. This is a verypowerful high-throughput technology which allows both genetic and epigenetic genomewide analyses. One possibility is to perform chromatin immunoprecipitation (ChIP) followedby submitting the precipitated DNA fragments to genome wide sequencing analyses (SEQ),a so-called ChIP-SEQ experiment. New targets for epigenetic silencing may be found bysequencing all DNA fragments bound to e.g. proteins with a methyl binding domain (MBDs).Sequencing all DNA fragments which bind to a certain transcription factor, can be used tomap its downstream effects. The ChIP products may also be combined with microarraystudies (ChIP-on-chip) which will enable us to study different kinds of regulation at thetranscriptome level.Non-invasive early diagnostics of colorectal cancerEven though much effort is put into large-scale studies it is still of uttermost importance tohave a good pipeline for detailed methylation analyses of resulting target genes in general andespecially genes with a diagnostic potential. In order to achieve this, we are establishing twotypes of quantitative DNA methylation analyses for all new potential biomarkers, real-timePCR based methods and pyrosequencing. Such technology will be a necessary tool todetermine and optimize the detection levels of cancer-specific markers in a non-invasivematerial such as stool or blood. Potential biomarkers will be analyzed with quantitativestudies in series of normal mucosa samples, benign precursor lesions and carcinomas. If themethylation markers have high methylation frequencies in carcinomas and/or precursorlesions and are unmethylated in normal samples, a panel of suitable fecal samples will beanalyzed in a blinded manner in order to determine the sensitivity and specificity levels. Weare currently working on optimizing the fecal DNA extraction protocol in order to producemaximum yield of high quality, human DNA.65
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Novel genetic and epigenetic altera
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TABLE OF CONTENTSACKNOWLEDGEMENTS .
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ACKNOWLEDGEMENTSThe present work ha
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Prefacetechnology[3]. This new tech
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SummaryThe subgroup of carcinomas w
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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 42 and 43: Results in BriefUnivariate survival
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 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
Page 94 and 95: Journal of Translational Medicine 2
Page 105: Paper IITerje Ahlquist, Guro E Lind
Page 108 and 109: BackgroundMost cases of colorectal
Page 110 and 111: ADAMTS1 CDKN2A CRABP1 HOXA9 MAL MGM
Page 112 and 113: pseudogene, leading to a high rate
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strands. Proc Natl Acad Sci U S A 1
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concomitant absence of transcript a
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Volume 10 Number 7 July 2008 pp. 68
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682 RAS Signaling in Colorectal Car
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Table W2. Detailed Somatic Events o
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Identification of RCC2 as a prognos
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INTRODUCTIONMicrosatellite instabil
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unselected series of primary tumors
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specificity, i.e. that they only am
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On the assumption that DNA repair a
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In order to ensure that gene mutati
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Figure 2. Mutation frequency differ
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and TAF1B (0.50), ACVR2A and ASTE1
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Multivariate analysesA multivariate
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When comparing our findings of muta
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The test series included a low numb
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entering M-phase remains to be seen
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12. Duval A, Reperant M, Hamelin R
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34. Martineau-Thuillier S, Andreass
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AppendicesAppendix I:List of abbrev
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Critical Reviews TM in Oncogenesis,
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TARGET GENES OF MSI COLORECTAL CANC
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