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

1632 CORRESPONDENCE GASTROENTEROLOGY Vol. 132, No. 43. Laird PW. The power and the promise of DNA methylation markers.Nat Rev Cancer 2003;3:253–266.4. Muller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M,Muhlthaler M, Ofner D, Margreiter R, Widschwendter M. Methylationchanges in faecal DNA: a marker for colorectal cancerscreening? Lancet 2004;363:1283–1285.5. Chen WD, Han ZJ, Skoletsky J, Olson J, Sah J, Myeroff L, Platzer P,Lu S, Dawson D, Willis J, Pretlow TP, Lutterbaugh J, Kasturi L,Willson JK, Rao JS, Shuber A, Markowitz SD. Detection in fecalDNA of colon cancer-specific methylation of the nonexpressedvimentin gene. J Natl Cancer Inst 2005;97:1124–1132.doi:10.1053/j.gastro.2007.03.003Reply. We were pleased to learn that our recent reporton aberrant MAL methylation in colon cancer was independentlyconfirmed. 1 However, the authors appear tohave made a critical oversight in interpreting both theirown data and our study regarding distinct diagnosticcriteria for positive methylation that determine the prevalenceof “methylated” tumors.We used quantitative real-time MSP (qMSP) assay, asensitive, sequence-specific and quantitative assay. 2 Themethylation level in a given specimen was measured asthe ratio of DNA molecules demonstrating methylationat all CpG sites within primers and Taqman probe sequencesrelative to the total number of DNA moleculesmeasured by a CpG-free -actin PCR amplicon (methylationindex [MI]). 1 As described in our report, we diagnoseda specimen as methylated only when this MI exceeded0.2 in order to exclude low-level methylationevents occurring in a mere minority of cells. 1 This strictcriterion was chosen because our scope was to detectmethylation events likely to be associated with mRNAsilencing and which occur in the majority of the cells inthe specimen.The authors used qualitative MSP, a sensitive but nonquantitativemethod. 3 Qualitative MSP is limited in itsability to discriminate low-level from biologically relevant,high-level methylation. If we had applied a low MIthreshold criterion (MI 0.01) comparable to the standardqualitative MSP band visible on a gel, to our dataset,the prevalence of methylation in normal mucosae andprimary tumors would have risen to a frequency similarto the authors’ data (12% and 68%, respectively). Therefore,we agree with the authors that low-level MAL methylationmay constitute an emerging potential early detectionbiomarker for colon cancer meriting furtherinvestigation. However, we conclude that low-level methylationis not likely to influence mRNA expression and isunsuitable for studies emphasizing biologically relevantmethylation. In support of our conclusion, reexpressionof MAL mRNA was observed in 5-aza-2=-deoxycitidine(Aza-C)-treated HT29 cells, although these cells stillmaintained low-level MAL methylation after Aza-C treatment(MI 0.09). 1Finally, we consider validation with bisulfite sequencingto have been unnecessary in our qMSP study. qMSPensures highly efficient discrimination specific from nonspecificPCR products because of methylated sequencespecificdetection using TaqMan probes containingCpGs. 2 As quality controls, we confirmed that our MALqMSP-amplicondid not amplify from unconverted DNAor unmethylated DNA. Furthermore, we observed properand sensitive amplification from fully methylated positivecontrol DNA, even after 625-fold dilution.In conclusion, both our study and the authors’ own datadescribe essentially the same phenomenon, but with vastlydifferent diagnostic criteria. We would like to emphasize theimportance of appropriately designing and interpreting epigeneticstudies of cancers, in light of the distinct nature ofeach analytical method and the focus of each study.YURIKO MORIFUMIAKI SATOSTEPHEN J. MELTZERDepartment of MedicineJohns Hopkins School of MedicineBaltimore, Maryland1. Mori Y, Cai K, Cheng Y, Wang S, Paun B, Hamilton JP, Jin Z, SatoF, Berki AT, Kan T, Ito T, Mantzur C, Abraham JM, Meltzer SJ. Agenome-wide search identifies epigenetic silencing of somatostatin,tachykinin-1, and 5 other genes in colon cancer. Gastroenterology2006;131:797–808.2. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, ShibataD, Danenberg PV, Laird PW. MethyLight: a high-throughput assayto measure DNA methylation. Nucleic Acids Res 2000;28:E32.3. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specificPCR: a novel PCR assay for methylation status ofCpG islands. Proc Natl Acad Sci USA1996;93:9821–9826.doi:10.1053/j.gastro.2007.03.004AGA Position Statement of ComputedTomographic ColonographyDear Sir:We read with interest the recent American GastroenterologicalAssociation (AGA) Institute’s statement entitled“Position of the American Gastroenterological Association(AGA) Institute on Computed TomographicColonography.” 1 This statement announces that “theAGA Institute has convened a task force to develop trainingstandards for gastroenterologists’ performance of CTcolonography.” As a combined gastroenterologist (PL)and radiologist (AF) team, we believe that this initiative isseriously misguided.Interpretation of computed tomographic colonography(CTC), as currently practiced, requires the interpretingphysician to interact on a workstation with a volumetricCT dataset that usually consists of imagesacquired in both supine and prone positions. At a minimumthe colon is examined with a volume-rendered 3Dendoluminal display, synchronized with a simultaneousmultiplanar 2D display (primary 3D read), and/or a 2Dsimultaneous axial supine/prone display with 3D ofpoints of interest (primary 2D read).