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 BRIEFPaper Ia. “DNA hypermethylation of MAL: a promising diagnostic biomarker for colorectal tumors”In a previous study we had identified a short list of candidate epigenetic markers from agenome wide screen[131]. In this commentary to Gastroenterology we reported the initialmethylation results for MAL, seemingly a highly promising biomarker for early detection ofcolorectal tumors. MAL was frequently methylated both in colorectal carcinomas (83%; 40of 48 carcinomas) and adenomas (73%; 43 of 59 adenomas) as assessed by methylationspecificpolymerase chain reaction (MSP). Hypermethylation of this gene promoter washighly cancer specific as only 11% (2/18) of normal mucosa taken in distance from theprimary tumor and 4% (1/23) of normal mucosa from cancer-free individuals weremethylated. Methylation frequency in normal mucosa was significantly lower compared toboth adenomas and carcinomas (P < 0.0001 for both). These findings was in contrast to arecently published article in the same journal, in which MAL was reported to be methylatedin only 6% of colorectal carcinomas[132]. The authors criticized our results in thecommentary and we were not allowed to respond to the response. Thus, the detaileddiscussion is included in the full length paper (Ib).Paper Ib. “Hypermethylated MAL gene – a silent marker of early colon tumorigenesis”In the present study, we have compared the promoter methylation status of MAL in normalcolorectal mucosa samples with benign and malignant colorectal tumors. The article presentsthe detailed study of MAL and includes technical and biological validation experiments. Themethylation frequencies were slightly altered from Ia due to addition of more samples.Eighty percent (49/61) of the carcinomas, 71% (45/63) of the adenomas, 10% (2/21) of thenormal mucosa from cancer patients, and 4% (1/23) of the normal mucosa from non-cancerindividuals harbored hypermethylation of the MAL promoter. RNA expression levels ofMAL were determined for 46 cell lines from various cancer types, and showed thatmethylation of MAL was associated with reduced transcriptional activity (P = 0.041). Whentreating the cell lines with the demethylating agents 5-aza-2’deoxycytidine and trichostatin-A,the cells responded with increased RNA expression, as expected if the transcriptionalrepression is caused by promoter hypermethylation. The in situ protein expression of MAL38
Results in Briefwas determined by use of a tissue micro array. Among 231 scorable CRC tissue cores, 198were negative for MAL staining. In conclusion, it seems like MAL is inactivated by othermechanisms in addition to hypermethylation as even unmethylated carcinomas showed noprotein expression, while positive staining for MAL was seen in all the normal colon mucosacontrol samples. We speculate that the hypermethylation of MAL may act as a “geneexpression seal” ensuring that the gene remains in an inactive state.These findings show that hypermethylated MAL is suitable as a diagnostic marker for earlycolorectal tumorigenesis with a potentially high sensitivity and specificity, the latter calculatedbetween tumors and normal samples. We also show that the low 6% of methylation detectedby the Johns Hopkins group was due to suboptimal primer design.Paper II. “Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumorsidentify early onset markers”In this study we analyzed and compared the methylation status of a selected set of markersthat potentially could discriminate between non-malignant and malignant tissue from thelarge bowel. The methylation status of eleven genes (ADAMTS1, CDKN2A, CRABP1,HOXA9, MAL, MGMT, MLH1, NR3C1, PTEN, RUNX3, and SCGB3A1) was determinedby MSP in 154 tissue samples including normal mucosa (20 non-cancerous samples; 18normal samples from cancer individuals), adenomas (n=63), and carcinomas (n=52) of thecolorectum. The reliability of the MSP scorings was tested by quantitative MSP analysis in ablinded manner for one example gene (MGMT), and the results were in perfect concordancewith the MSP data. Part of the results were previously published[131;133], but was includedhere in order to analyze co-variance between the different genes. We saw a stepwise,significant increase in methylation frequencies as the mean number of methylated genes persample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa fromcancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas (P < 0.0001). This increase inmethylation from benign to malignant lesions was also evident at the individual gene levelfor ADAMTS1, CDKN2A, CRABP1, MLH1, NR3C1, RUNX3, and SCGB3A1. We alsoreport that PTEN is unmethylated in all carcinomas and is not subject to inactivation byhypermethylation in CRC. Hypermethylation of CRABP1, MLH1, NR3C1, RUNX3, andSCGB3A1 were seen almost exclusively in proximal carcinomas with microsatellite39
- 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 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 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
RESULTS IN BRIEFPaper Ia. “DNA hypermethylation of MAL: a promis<strong>in</strong>g diag<strong>no</strong>stic biomarker for colorectal tumors”In a previous study we had identified a short list of c<strong>and</strong>idate epi<strong>genetic</strong> markers from age<strong>no</strong>me wide screen[131]. In this commentary to Gastroenterology we reported the <strong>in</strong>itialmethylation results for MAL, seem<strong>in</strong>gly a highly promis<strong>in</strong>g biomarker for early detection ofcolorectal tumors. MAL was frequently methylated both <strong>in</strong> colorectal carc<strong>in</strong>omas (83%; 40of 48 carc<strong>in</strong>omas) <strong>and</strong> ade<strong>no</strong>mas (73%; 43 of 59 ade<strong>no</strong>mas) as assessed by methylationspecificpolymerase cha<strong>in</strong> reaction (MSP). Hypermethylation of this gene promoter washighly cancer specific as only 11% (2/18) of <strong>no</strong>rmal mucosa taken <strong>in</strong> distance from theprimary tumor <strong>and</strong> 4% (1/23) of <strong>no</strong>rmal mucosa from cancer-free <strong>in</strong>dividuals weremethylated. Methylation frequency <strong>in</strong> <strong>no</strong>rmal mucosa was significantly lower compared toboth ade<strong>no</strong>mas <strong>and</strong> carc<strong>in</strong>omas (P < 0.0001 for both). These f<strong>in</strong>d<strong>in</strong>gs was <strong>in</strong> contrast to arecently published article <strong>in</strong> the same journal, <strong>in</strong> which MAL was reported to be methylated<strong>in</strong> only 6% of colorectal carc<strong>in</strong>omas[132]. The authors criticized our results <strong>in</strong> thecommentary <strong>and</strong> we were <strong>no</strong>t allowed to respond to the response. Thus, the detaileddiscussion is <strong>in</strong>cluded <strong>in</strong> the full length paper (Ib).Paper Ib. “Hypermethylated MAL gene – a silent marker of early colon tumorigenesis”In the present study, we have compared the promoter methylation status of MAL <strong>in</strong> <strong>no</strong>rmalcolorectal mucosa samples with benign <strong>and</strong> malignant colorectal tumors. The article presentsthe detailed study of MAL <strong>and</strong> <strong>in</strong>cludes technical <strong>and</strong> biological validation experiments. Themethylation frequencies were slightly altered from Ia due to addition of more samples.Eighty percent (49/61) of the carc<strong>in</strong>omas, 71% (45/63) of the ade<strong>no</strong>mas, 10% (2/21) of the<strong>no</strong>rmal mucosa from cancer patients, <strong>and</strong> 4% (1/23) of the <strong>no</strong>rmal mucosa from <strong>no</strong>n-cancer<strong>in</strong>dividuals harbored hypermethylation of the MAL promoter. RNA expression levels ofMAL were determ<strong>in</strong>ed for 46 cell l<strong>in</strong>es from various cancer types, <strong>and</strong> showed thatmethylation of MAL was associated with reduced transcriptional activity (P = 0.041). Whentreat<strong>in</strong>g the cell l<strong>in</strong>es with the demethylat<strong>in</strong>g agents 5-aza-2’deoxycytid<strong>in</strong>e <strong>and</strong> trichostat<strong>in</strong>-A,the cells responded with <strong>in</strong>creased RNA expression, as expected if the transcriptionalrepression is caused by promoter hypermethylation. The <strong>in</strong> situ prote<strong>in</strong> expression of MAL38
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